ENGINEERED IMMUNE CELLS WITH MULTIPLEX AND LOCALIZED ACTIVITY

Abstract

The present disclosure provides engineered immune cells and methods for their creation and use. The immune cells comprise activating and blocking receptors, and exhibit multiplex and localized activity.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to engineered immune cells that have an enhanced safety profile and large therapeutic window.

SEQUENCE LISTING

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled A2TH-005-01US-Sequence-Listing.txt, created on Sep. 20, 2021 and is 404 kilobytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.

BACKGROUND

[0003] Approximately 1.8 million people per year are diagnosed with a form of cancer in the United States. Similarly, it is estimated that 23.5 million Americans suffer from an autoimmune disease, almost all of which decrease life expectancy. Despite continual advances in treatment, education, and detection, there are over 600,000 deaths per year attributed to cancer in the U.S., while autoimmune diseases remain a leading cause of death among patients under the age 65.

[0004] Engineered immune cells have been touted as potentially effective treatments for a variety of severe conditions like cancer, viral infections, auto-immune ailments, and organ transplant rejection. These immune cells, whether chimeric antigen receptor (CAR)-engineered cells or T cell receptor (TCR)-engineered cells, often show efficacious results in vitro. However, in vivo, these results are rarely duplicated. Often, these treatments show a lack of efficacy in vivo and/or produce such severe side effects, that they cannot be used as therapeutics. Thus, despite decades of consistent research, only two CAR T cell therapies have received FDA approval--Kymriah.TM. for acute lymphoblastic leukemia and Yescarta.TM. for diffuse large B-cell lymphoma.

SUMMARY OF THE INVENTION

[0005] The present disclosure provides engineered immune cells that comprise two types of modular, engineered ligand binding receptors caused to be expressed on the surface of the cells. The first of these receptors is an activating receptor, which is designed to activate when bound to a cognate ligand on the surface of another cell, causing it to trigger an activating signal. The immune cells are engineered such that when the strength of the activating signal crosses a threshold, it causes a cytotoxic response by the immune cell, killing the cell expressing the cognate ligand. The second of these receptors is a blocking receptor, which when bound to a cognate blocking ligand on the surface of a non-target cell, is designed to activate and trigger a blocking signal. The blocking signal blocks the activating signal, which prevents the cytotoxic response against the non-target cell.

[0006] Generally, the activating receptors are designed to bind to cognate activating ligands that are expressed on both target cells, such as tumor cells, and non-target cells. The blocking ligands may be expressed only by non-target cells, or expressed at lower levels by target cells compared to non-target cells. In this way, when the engineered immune cells contact target cells, the activating receptors bind to the activating ligands, which leads to the cytotoxic response. In contrast, when the engineered immune cells contact non-target cells, the blocking receptors bind to the blocking ligands, blocking the cytotoxic response. This designed scheme provides the general means by which the engineered immune cells safely kill target cells, while limiting effects on non-target cells. However, the immune cells of the present disclosure have been engineered to provide several other advantageous features that expand their therapeutic window and efficacy, while limiting deleterious effects.

[0007] One of these advantageous features is that, when activated, the blocking receptors have been designed to reduce cell surface expression of the activating receptors. Thus, as the immune cells circulate to areas of a patient's body lacking target cells, the levels of activating receptors expressed on the surface of the cell are reduced. The receptors can be configured such that this reduction is reversible upon the activating receptors binding to their cognate ligands in the absence of the blocking ligands. This intentionally lowers the likelihood that a cytotoxic response will be triggered in the absence of an appropriate target, which enhances the safety profile of the immune cells.

[0008] Further, by engineering the immune cells to reduce the expression of activating receptors in the absence of an appropriate target cell, the immune cells are less likely to exhibit chronic activation and/or ligand-independent tonic signaling. As a result, the immune cells of the present disclosure are designed to limit immune cell exhaustion, differentiation, and activation-induced immune cell death, while concurrently exhibiting high generation and persistence.

[0009] A further advantage of the engineered immune cells of the present disclosure is that, when they contact target and/or non-target cells, the activating and blocking receptors are designed to diffuse into regions on the immune cell surface proximate to the target and/or non-target cells. The receptors form micro-clusters in these regions. In micro-clusters proximate to non-target cells, the blocking receptors bind to cognate ligands on the proximate non-target cells. The receptors can be configured such that cross-talk between the receptors causes a localized reduction in surface expression of the activating receptors, recruits more blocking receptors to the micro-cluster, and prevents breakup of the micro-cluster. This leads to a localized signal that blocks cytotoxic effects on the non-target cell.

[0010] In contrast, when the engineered immune cells contact target cells, activating receptors in micro-clusters proximate to the target cells are activated. This leads to a localized signal that, when it passes a threshold, triggers a cytotoxic response by the immune cell that kills the proximate target cells. The immune cells and receptors can be configured such that binding of the activating receptors to their cognate ligands may also locally reverse any reduced surface expression of the activating receptor. This ensures a sufficiently strong activating signal to trigger the cytotoxic response on the proximate target cell.

[0011] The immune cells of the present disclosure can form these aforementioned micro-clusters when simultaneously contacting both target and non-target cells. This ensures an appropriate, localized response that kills target cells, while minimizing deleterious effects on non-target cells.

[0012] The immune cells of the present disclosure also feature blocking receptors that are engineered to produce a ligand-dependent signal that dominates and blocks the activating signal from the activating receptors. This ensures that the immune cells can be configured to possess a strong safety profile with a wide therapeutic window.

[0013] Moreover, in some methods and systems of the disclosure, the engineered immune cells can be produced based on the levels of blocking and activating ligands expressed by non-target cells. Because the blocking receptors can be tuned to have a signal that dominates initial contact with a non-target cells, a sufficiently safe immune cell can be produced, without relying on a large surplus of blocking receptors expressed as compared to activating receptors. Further, the ability of the blocking receptors to reduce the surface expression of the activating receptors ensures that this level of safety increases in the presence of non-target cells.

[0014] Another advantage conferred by the immune cells of the present disclosure is that receptors can be produced using modular receptor components. Thus, the immune cells can be readily engineered to have receptor pairs that target desired ligands expressed on target and non-target cells. Moreover, the modular receptor components can be used and interchanged to tune or adjust the relative signal strengths of each receptor type. This ensures that an engineered immune cell's receptors provide a sufficiently strong activation signal, which can be adequately blocked to prevent non-target effects. A surprising discovery is that this modular nature extends to both chimeric antigen receptors (CAR) and T cell receptors (TCR). Not only are CARs and TCRs of the present disclosure able to interact with each other, but parts of CARs and TCRs can be interchanged to produce customized receptors and cells.

[0015] The relative signal strength and activity of each receptor type can also be modulated based on cross-talk between receptors. A surprising feature of the present disclosure is, not only that cross-talk can impact signal strength and activity, but that the impact of this cross-talk can change depending on the distance between pairs of blocking and activating receptors. As the distance between a blocking receptor and activating receptor decreases, the impact of this cross-talk increases. Thus, the present disclosure provides engineered immune cells configured to express receptors such that they are proximate to one another to ensure optimal interaction and strong cross-talk.

[0016] The receptors may be designed, for example, with physiochemical properties that ensure the receptors have a desired spacing. This spacing may ensure a maximum level of cross-talk between receptors and/or ensure that the receptors do not diffuse close enough to, for instance, invert the blocking receptor signal. The receptors can be engineered, for example, to have opposing charges or steric hindrances to prevent them from moving too close to one another. Alternatively, or in addition, the immune cells may be engineered to have receptors that are covalently linked to achieve a desired spacing. For example, a rigid covalent linker between the receptors can hold the receptors at a desired spacing from one another. The rigid linker concurrently keeps the receptors close enough to ensure cross-talk while maintain adequate spacing to prevent the blocking receptor from, for instance, inverting or becoming ligand-independent.

[0017] Another feature of the present disclosure is that the blocking receptor can be designed using interchangeable hinges that connect an extracellular ligand binding domain to a transmembrane domain and/or an intracellular domain. The hinges can be designed to have different lengths and flexibilities. The length and flexibility of a hinge can be used to tune the strength of the blocking signal. Longer and/or more flexible hinges can be used to increase the strength of the blocking receptor's signal or surface expression. In contrast, the blocking receptor can be engineered with shorter and/or more rigid hinges to decrease the strength of the blocking receptor's signal or surface expression. The blocking receptor can be configured to use a hinge selected from a group of hinges that have a known impact on the half maximal concentration (EC.sub.50) of the activating ligand for the activating receptor to cause the immune cell to trigger a cytotoxic response. This allows pairs of blocking and activating receptors to be chosen or engineered to exhibit a desired level of activation/inhibition.

[0018] Thus, the present disclosure provides engineered immune cells, and methods for reliably producing them, with a large therapeutic window, i.e., cells with a large range between their minimum effective dose and maximum tolerated dose. The cells possess target-sensitive receptors that produce an activation signal sufficient to trigger cytotoxic effects when encountering target cells, while concurrently producing minimal non-target effects. The engineered immune cells of the present disclosure also exhibit low exhaustion, differentiation, tonic signaling, and activation-induced immune cell death, and other features consistent with effective in vitro and in vivo function.

[0019] In one aspect, the present disclosure provides an engineered immune cell that includes an activating receptor expressed on a surface of the engineered immune cell. Binding of the activating receptor to an activating ligand on a target cell promotes a cytotoxic response by the engineered immune cell. The immune cell also includes a blocking receptor expressed on the surface of the engineered immune cell. Binding of the blocking receptor to a blocking ligand on a target cell causes the engineered immune cell to exhibit reduced surface expression of the activating receptor. High exogenous IL-2 may overcome this level of regulation, though the activation/blockade is still enforced by other features of intracellular signaling of the activator and blocker receptors.

[0020] Binding of the blocking receptor to the blocking ligand on the target cell may also cause the blocking receptor to trigger an inhibitory signal that blocks the activating signal, thereby preventing the cytotoxic response by the immune cell. The engineered immune may have an inhibitory signal dominates and blocks the activating signal.

[0021] The reduced surface expression of the activating receptor of the cells of the present disclosure may be reversible. The reduced surface expression of the activating receptor may reverse upon the engineered immune cell binding to the activating ligand on a target cell in the absence of the blocking ligand. The reduced surface expression of the activating receptor may be localized to a region of the engineered immune cell surface proximal to the blocking receptor. When a plurality of the blocking receptor binds to a plurality of the blocking ligand, the reduced surface expression may be localized to regions of the engineered immune cell surface proximal to blocking receptors.

[0022] When the immune cell encounters a target cell having both the blocking and activating ligands, a plurality of activating and blocking receptors diffuse into a region on the of the immune cell surface proximal to the target cell and form a micro-cluster. In the micro-cluster, binding of blocking receptors to the blocking ligands causes the engineered immune cell to exhibit reduced surface expression of the activating receptor in the micro-cluster.

[0023] In certain immune cells of the disclosure, the blocking receptor cannot bind to the blocking ligand until the activating receptor binds to the activating ligand.

[0024] The present disclosure also provides method for treating a cancer using the immune cells of the disclosure. In certain methods of the disclosure, the method includes providing an engineered immune cell to a patient, wherein the engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell. In certain methods, when the engineered immune cell encounters a tumor cell of the patient, the activating receptor binds to an activating ligand on the tumor cell while the blocking receptor remains unbound. This promotes a cytotoxic response by the engineered immune cell that results in a cytotoxic effect on the tumor cell. When the engineered immune cell encounters a normal cell of the patient, the blocking receptor binds to a blocking ligand on the normal cell and causes the engineered immune cell to exhibit reduced surface expression of the activating receptor. This causes a signal from the blocking receptor to dominate a signal from the activating receptor, which prevents the cytotoxic response by the engineered immune cell.

[0025] In certain methods, the reduced surface expression of the activating receptor is temporary. The reduced surface expression may be reversible. The reduced surface expression may be reversed upon the engineered immune cell binding to the first ligand on a tumor cell.

[0026] In certain methods of the disclosure, the reduced surface expression of the activating receptor may be localized to a region of the engineered immune cell surface proximal to the blocking receptor bound to the blocking ligand on the normal cell. A plurality of the blocking receptor may bind to a plurality of the blocking ligand on the normal cell, and the reduced surface expression may be localized to the region of the engineered immune cell surface proximal to the plurality of the blocking receptor.

[0027] A further aspect of the disclosure are methods of producing an engineered immune cell with activating and blocking receptors. The methods of the disclosure may include, producing an engineered immune cell that expresses activating receptors and blocking receptors based on a ratio of a quantity of an activating ligand to a quantity of a blocking ligand that are expressed in non-tumor cells of a patient.

[0028] In certain methods, a tumor cell of a patient expresses the activating ligand and does not express the blocking ligand.

[0029] In certain methods of the disclosure, binding of the activating receptors to the activating ligands triggers an activating signal that promotes a cytotoxic response by the engineered immune cell. Additionally, binding of the blocking receptors to blocking ligands on a non-tumor cell may cause the blocking receptors to trigger an inhibitory signal that blocks the activating signal.

[0030] In certain methods of the disclosure, the engineered immune cell expresses the blocking and activating receptors at a ratio based on the ratio of the quantity of the activating ligand to the quantity of the blocking ligand that are expressed in the non-tumor cells of the patient.

[0031] In some methods, the inhibitory signal of one of the blocking receptors dominates and blocks the activating signal of one of the activating receptors.

[0032] In certain methods, the ratio of the blocking receptors to the activating receptors is less than 1. The ratio of the blocking receptors to the activating receptors, needed to achieve a blocking signal to provide a certain level of blocking for the activating signal, may be inversely proportional to the quantity of the activating ligand expressed on non-tumor cells of the patient. In certain methods, when the immune cell contacts a non-tumor cell of the patient the blocking receptors bind to blocking ligands on the non-tumor cell and reversibly increase the ratio of blocking receptors to activating receptors expressed by the immune cell.

[0033] In certain methods of the disclosure, each blocking receptor comprises a ligand binding domain (LBD), a hinge, transmembrane domain, and intracellular domain (ICD), and the LBD, hinge, and ICD have a known effect on the strength of the inhibitory signal. Each activating receptor may comprise a ligand binding domain (LBD), a hinge, transmembrane domain, and the LBD has a known effect on the activation signal.

[0034] The present disclosure also provides a method of producing an engineered immune cell that includes obtaining a sample from a patient comprising target and non-target cells; performing an assay to determine a ratio of a quantity of an activating ligand to a quantity of a blocking ligand expressed on the non-target cells; and producing an engineered immune cell that expresses activating receptors and blocking receptors based on the determined ratio.

[0035] In certain methods, the target cells express the activating ligand and do not express the blocking ligand.

[0036] In some methods, binding of the activating receptors to the activating ligands triggers an activating signal that promotes a cytotoxic response by the engineered immune cell; and

[0037] binding of the blocking receptors to blocking ligands on a non-target cell causes the blocking receptors to trigger an inhibitory signal that blocks the activating signal.

[0038] In some methods, the engineered immune cell expresses the blocking and activating receptors at a ratio based on the ratio of the quantity of the activating ligand to the quantity of the blocking ligand that are expressed in the non-target cells of the patient.

[0039] In certain methods, the inhibitory signal of one of the blocking receptors dominates and blocks the activating signal of one of the activating receptors. The ratio of the blocking receptors to the activating receptors is less than 1 in certain methods. The ratio of the blocking receptors to the activating receptors may be inversely proportional to the quantity of the activating ligand expressed on non-target cells of the patient. In some methods, when the immune cell contacts a non-target cell of the patient the blocking receptors bind to blocking ligands on the non-target cell and reversibly increases the ratio of blocking receptors to activating receptors expressed by the immune cell.

[0040] In certain methods of the disclosure, each blocking receptor comprises a ligand binding domain (LBD), a hinge, transmembrane domain, and intracellular domain (ICD), and the LBD, hinge, and ICD have a known effect on the strength of the inhibitory signal. Each activating receptor may comprise a ligand binding domain (LBD), a hinge, transmembrane domain, and the LBD has a known effect on the activation signal.

[0041] In a further aspect, the present disclosure provides engineered immune cells with activating and blocking receptors that exhibit cross-talk between receptors. Thus, the present disclosure provides an engineered immune cell with an activating receptor that triggers a cytotoxic signal that promotes a cytotoxic response of the engineered immune cell when the activating receptor binds a first ligand of a target cell; a blocking receptor that sends an interfering signal that inhibits the cytotoxic response of the engineered immune cell when the blocking receptor binds a second ligand of the target cell, wherein cross-talk between the activating receptor and the blocking receptor affects an activation threshold for the cytotoxic response.

[0042] In certain immune cells, in the absence of the first and second ligands, the effect of the cross-talk on the activation threshold is minimized and/or reduced. The effect of the cross-talk on the activation threshold may increase with proximity of the activating receptor to the blocking receptor.

[0043] In certain immune cells of the disclosure, the activating receptor and blocking receptor are covalently linked together, or have physicochemical properties favoring interaction with one another such that the receptors are proximal to one another.

[0044] In some immune cells of the disclosure, when the blocking receptor binds to the second ligand, the cross-talk between the blocking and activating receptors causes the immune cell to exhibit reduced surface expression of the activating receptor.

[0045] An immune cell of the disclosure may include a plurality of the activating and blocking receptors, and when the immune cell contacts a target cell the plurality of the activating and blocking receptors diffuses into a region on the surface of the immune cell proximal to the target cell and forms a micro-cluster in which the effect of the cross-talk on the activation threshold is localized.

[0046] In some immune cells of the disclosure, cross-talk between the activating receptor and the blocking receptor prevents the blocking receptor from binding to the second ligand until the activating receptor binds to the first ligand.

[0047] The present disclosure also provides methods for treating cancer using the immune cells of the present disclosure. Certain methods may include providing an engineered immune cell to a patient, wherein the engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell. The activating receptor may trigger a cytotoxic signal that promotes a cytotoxic response of the engineered immune cell when the activating receptor binds a first ligand of a target cell; and the blocking receptor may send an interfering signal that inhibits the cytotoxic response of the engineered immune cell when the blocking receptor binds a second ligand of the target cell, wherein cross-talk between the activating receptor and the blocking receptor affects an activation threshold for the cytotoxic response.

[0048] In certain methods, the absence of the second ligand, the effect of the cross-talk on the activation threshold is minimized and/or reduced. The effect of the cross-talk on the activation threshold may increase with proximity of the activating receptor to the blocking receptor.

[0049] In certain methods, the activating receptor and blocking receptor are linked together or have physicochemical properties favoring interaction with one another, such that the receptors are proximal to one another.

[0050] In certain methods, when the blocking receptor binds to the second ligand, the cross-talk between the blocking and activating receptors causes the immune cell to exhibit reduced surface expression of the activating receptor. The immune cell may include a plurality of the activating and blocking receptors, and when the immune cell contacts a target cell the plurality of the activating and blocking receptors diffuses into a region on the surface of the immune cell proximal to the target cell and forms a micro-cluster in which the effect of the cross-talk on the activation threshold is localized.

[0051] In methods of the disclosure, the cross-talk between the activating receptor and the blocking receptor may prevent the blocking receptor from binding to the second ligand until the activating receptor binds to the first ligand.

[0052] The present disclosure also provides methods of producing engineered immune cells as disclosed herein. Certain methods include, determining an amount of cross-talk between an activating receptor and a blocking receptor for an engineered immune cell, wherein the amount of cross-talk between the activating receptor and the blocking receptor affects an activation threshold for the cytotoxic response; and producing an engineered immune cell that expresses different concentrations of activating receptors and blocking receptors based on the determined amount of cross-talk between the activating receptor and the blocking receptor.

[0053] In some methods for producing immune cells, in the absence of cognate ligands for the activating and blocking receptors, the amount of the cross-talk is minimized and/or reduced. The methods may include producing an engineered immune cell that expresses different concentrations of activating receptors and blocking receptors is further based on a ratio of a quantity of an activating ligand to a quantity of a blocking ligand that are expressed in non-tumor cells of a sample.

[0054] The cross-talk between the activating receptor and the blocking receptor may prevent the blocking receptor from binding to the blocking ligand until the activating receptor binds to the activating ligand. In certain methods, an amount of the cross-talk between the activating receptor and blocking receptor increases with proximity of the activating receptor to the blocking receptor.

[0055] Methods include producing immune cells where the activating receptor and blocking receptor may be covalently linked, or have physicochemical properties favoring interaction with one another such that the receptors are proximal to one another.

[0056] In a further aspect, the present disclosure provides engineered immune cells with activating and blocking receptors in which the blocking receptor provides an inhibitory signal that dominates the activation signal from the activating receptor.

[0057] Thus, the present disclosure includes an engineered immune cell with an activating receptor on the surface of the engineered immune cell, wherein binding of the activating receptor to a first ligand on a target cell causes the activating receptor to trigger an activating signal that promotes a cytotoxic response by the engineered immune cell; and a blocking receptor on the surface of the immune cell, wherein binding of the blocking receptor to a second ligand on a target cell causes the blocking receptor to trigger an inhibitory signal stronger than the activating signal such that the inhibitory signal dominates and blocks the activating signal from the activating receptor, thereby preventing a localized cytotoxic response by the engineered immune cell.

[0058] In certain immune cells of the disclosure, binding of the blocking receptor to the second ligand may cause the engineered immune cell to exhibit reduced surface expression of the activating receptor. The reduced surface expression may be reversible.

[0059] The immune cells may include a plurality of activating and blocking receptors and the ratio of the blocking receptors to the activating receptors expressed by the immune cells is less than or equal to 1.

[0060] In certain immune cells of the disclosure, the blocking receptor does not bind to the second ligand until the activating receptor binds to the activating ligand.

[0061] In certain cells, the inhibitory signal may be localized to a region of the engineered immune cell surface adjacent to the blocking receptor. Similarly, the activation signal may be localized to a region of the engineered immune cell surface adjacent to the activating receptor.

[0062] When the immune cells of the disclosure encounter a target cell having both the first and second ligands, a plurality of activating and blocking receptors may diffuse into a region on the of the immune cell surface proximal to the target cell and form a micro-cluster in which the blocking receptors prevent the localized cytotoxic response by the engineered immune cells. Binding of the blocking receptors in the micro-cluster to the second target antigen may prevent breakup of the micro-cluster. When the immune cells simultaneously contact a second target cell having the first ligand and lacking the second ligand, a second plurality of the activating receptors may diffuse into a second region on the surface of the immune cells proximal to the second target cell and form a second micro-cluster that promotes the localized cytotoxic response by the engineered immune cells that results in a cytotoxic effect on the second target cell.

[0063] The present disclosure also provides methods for treating cancer using the immune cells of the present disclosure. The methods include a method in which an engineered immune cell is provided to a patient, wherein the engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell, wherein: when the engineered immune cell encounters a tumor cell, the activating receptor binds to a first ligand on the tumor cell and the activating receptor triggers an activating signal in the engineered immune cell that promotes a cytotoxic response by the engineered immune cell that results in a cytotoxic effect on the tumor cell; and when the engineered immune cell encounters a normal cell, the activating receptor binds to the first ligand on the normal cell and the blocking receptor binds to a second ligand on the normal cell, wherein the activating receptor triggers an activating signal in the engineered immune cell and the blocking receptor triggers an inhibitory signal in the engineered immune cell that is stronger than the activating signal such that the inhibitory signal dominates and blocks the activating signal from the activating receptor, thereby preventing a localized cytotoxic response by the engineered immune cell.

[0064] In some methods, binding of the blocking receptor to the second ligand causes the engineered immune cell to exhibit reduced surface expression of the activating receptor. The reduced surface expression may be reversible.

[0065] In methods of the disclosure, the immune cell may express different concentrations of the activating and blocking receptors based on a ratio of a quantity of the first ligand to a quantity of a second ligand expressed in a normal cell of the patient. The ratio of the concentration of blocking receptors expressed to activating receptors expressed may be less than or equal to 1.

[0066] In certain methods, when the immune cell encounters at least one tumor cell, a first plurality of the activating receptors diffuses into a first region on the surface of the immune cell proximal to the tumor cell and forms a first micro-cluster that promotes the localized cytotoxic response by the immune cell that results in a cytotoxic effect on the tumor cell. When the immune cell simultaneously encounters a normal cell, a plurality of the activating and blocking receptors may diffuse into a second region on the surface of the immune cell proximal to the normal cell and form a second micro-cluster causing the inhibitory signal from the blocking receptors to dominate the activating signal from the activating receptors in the second micro-cluster preventing the localized cytotoxic response by the engineered immune cell on the normal cell. Binding of the blocking receptors in the second micro-cluster to the second ligand may prevent breakup of the second micro-cluster.

[0067] In some methods of the disclosure, the blocking receptor does not bind to the second ligand until the activating receptor binds to the activating ligand.

[0068] Some methods include cross-talk between the activating receptor and the blocking receptor that affects an activation threshold for the localize cytotoxic response.

[0069] In a further aspect, the present disclosure provides engineered immune cells with activating and blocking receptors that have multiplex and localized activity. An immune cell of the disclosure may include activating and blocking receptors on a surface of the cell. When the engineered immune cell encounters a tumor cell and a healthy cell a first region of the activating and blocking receptors forms proximal to the healthy cell and blocking receptors in the first region inhibit cytotoxic effects on the healthy cell, while, simultaneously, a second region of the activating and blocking receptors forms proximal to the tumor cell and promotes a cytotoxic response by the engineered immune cell that exhibits cytotoxic effects on the tumor cell.

[0070] The activating and blocking receptors in the first region may bind to cognate activating and blocking ligands on the healthy cell, and the activating receptors in the second region may bind to cognate activating ligands on the tumor cell. The activating and blocking receptors may form a first micro-cluster in the first region, and the activating and blocking receptors may form a second micro-cluster in the second region.

[0071] Binding of the blocking receptors in the first micro-cluster to the cognate blocking ligands on the healthy cell may cause the engineered immune cell to exhibit reduced surface expression of the activating receptor in the first micro-cluster. Binding of the blocking receptors in the first micro-cluster to the cognate blocking ligands on the healthy cell may prevent breakup of the first micro-cluster.

[0072] The immune cell may express different concentrations of the activating and blocking receptors based on a ratio of a quantity of the activating ligand to a quantity of the blocking ligand expressed in a healthy cell. The ratio of the concentration of blocking receptors to activating receptors expressed by the immune cell may be less than or equal to 1.

[0073] In certain immune cells of the disclosure, the blocking receptors do not bind to the cognate blocking ligands until the activating receptors bind to cognate activating ligands.

[0074] In some immune cells of the disclosure, the cytotoxic response by the engineered immune cell that exhibits cytotoxic effects on the tumor cell is localized to the second region. The localized cytotoxic response does not exhibit cytotoxic effects on the healthy cell.

[0075] The present disclosure also provides methods for treating cancer using the immune cells of the disclosure. A method for treating cancer may include providing an engineered immune cell to a patient, the engineered immune cell comprising activating and blocking cell-surface receptors. When the engineered immune cell encounters a tumor cell and a healthy cell of the patient, a first set of the activating and blocking receptors collect into a first cell-surface region of the engineered immune cell proximal to the healthy cell in which the blocking receptors inhibit cytotoxic effects of the engineered immune cell on the healthy cell. Simultaneously, a second set of the activating and blocking receptors collect into a second cell-surface region of the engineered immune cell proximal to the tumor cell in which the activating receptors promote a cytotoxic response by the engineered immune cell that kills the tumor cell.

[0076] The activating and blocking receptors in the first cell-surface region may bind to cognate activating and blocking ligands on the healthy cell, and the activating receptors in the second cell-surface region may bind to cognate activating ligands on the tumor cell.

[0077] The activating and blocking receptors may form a first micro-cluster on the first cell-surface region, and the activating and blocking receptors may form a second micro-cluster on the second cell-surface region.

[0078] Binding of the blocking receptors in the first micro-cluster to the cognate blocking ligands on the healthy cell may cause the engineered immune cell to exhibit reduced surface expression of the activating receptor in the first micro-cluster. Binding of the blocking receptors in the first micro-cluster to the cognate blocking ligands on the healthy cell may prevent breakup of the first micro-cluster.

[0079] In some methods of the disclosure, the immune cell expresses different concentrations of the activating and blocking receptors based on a ratio of a quantity of the activating ligand to a quantity of a blocking ligand expressed in a healthy cell. The ratio of the concentration of blocking receptors to activating receptors expressed by the immune cell may be less than or equal to 1.

[0080] In certain methods, blocking receptors do not bind to the cognate blocking ligands until the activating receptors bind to the cognate activating ligands.

[0081] In certain methods of the disclosure, the cytotoxic response is localized to the second cell-surface region. The localized cytotoxic response does not exhibit cytotoxic effects on the healthy cell.

[0082] In a further aspect, the present disclosure provides immune cells that have activating and blocking receptors that form micro-clusters on the surface of the immune cells.

[0083] An engineered immune cell of the disclosure may include activating and blocking receptors on a surface of the engineered immune cell, wherein: when the engineered immune cell encounters a tumor cell, a first plurality of the activating receptors diffuse into a first region on the surface of the engineered immune cell and form a first micro-cluster proximal to the tumor cell that promotes a cytotoxic response by the engineered immune cell that results in cytotoxic effects on the tumor cell; and when the engineered immune cell encounters a normal cell, a second plurality of the activating and blocking receptors diffuse into a second region on the surface of the engineered immune cell and form a second micro-cluster proximal to the normal cell, wherein the blocking receptors in the second micro-cluster inhibit cytotoxic effects on the normal cell.

[0084] The activating receptors in the first micro-cluster may bind to cognate activating ligands on the tumor cell, and the activating and blocking receptors in the second micro-cluster may bind to cognate activating and blocking ligands on the normal cell. When the immune cell encounters a normal cell, the second micro-cluster may mediate formation of a complementary cluster of ligands on the normal cell. Binding of the blocking receptors in the second micro-cluster to the cognate blocking ligands on the normal cell may cause the engineered immune cell to exhibit reduced surface expression of the activating receptor in the second micro-cluster. Binding of the blocking receptors in the second micro-cluster to the cognate blocking ligands on the normal cell may prevent breakup of the second micro-cluster.

[0085] In some immune cells of the disclosure, when the immune cell simultaneously contacts a normal cell and a tumor cell, the first plurality of the activating receptors diffuse into the first region and form the first micro-cluster proximal to the tumor cell that promotes the cytotoxic response by the engineered immune cell that results in cytotoxic effects on the tumor cell; and the second plurality of the activating and blocking receptors diffuse into the second region and form the second micro-cluster proximal to the normal cell in which the blocking receptors inhibit cytotoxic effects on the normal cell. Expression of the activating receptor in the second micro-cluster may be reduced after the second micro-cluster forms.

[0086] The immune cell may express different concentrations of the activating and blocking receptors based on a ratio of a quantity of the activating ligand to a quantity of a blocking ligand expressed in a normal cell of a patient. The ratio of the concentration of blocking receptors expressed to activating receptors expressed is less than or equal to 1.

[0087] In some immune cells of the disclosure, the blocking receptors do not bind to the cognate blocking ligands until the activating receptors bind to the cognate activating ligands.

[0088] The present disclosure provides methods for treating cancer using the immune cells disclosed herein. A method for treating cancer may include providing an engineered immune cell to a patient, the engineered immune cell comprising activating and blocking cell-surface receptors.

[0089] When the engineered immune cell encounters a normal cell, a first plurality of the activating and blocking receptors collect into a micro-cluster within a region of the cell-surface of the engineered immune cell proximal to the normal cell, wherein binding of one of the blocking receptors in the micro-cluster to a blocking ligand on the normal cell inhibits breakup of the micro-cluster, and wherein the engineered immune cell kills tumor cells that exhibit an activating ligand bound by the activating receptor and do not exhibit the blocking ligand such that the blocking receptor remains unbound.

[0090] When the immune cell simultaneously contacts a normal cell and a tumor cell, a first plurality of the activating receptors may diffuse into the first region and form the first micro-cluster proximal to the tumor cell that promotes the cytotoxic response by the engineered immune cell that results in cytotoxic effects on the tumor cell, and a second plurality of the activating and blocking receptors may diffuse into the second region and form the second micro-cluster proximal to the normal cell, wherein binding of one of the blocking receptors in the second micro-cluster to a first ligand on the normal cell inhibits breakup of the micro-cluster.

[0091] Binding of the blocking receptors in the second micro-cluster to the first ligands on the normal cell inhibits the cytotoxic effects on the normal cell. The cytotoxic effects on the tumor cell may be localized proximal to the first micro-cluster.

[0092] Binding of the blocking receptor to the first ligand on the normal cell may cause a plurality of the first ligand on the normal cell to diffuse into a region proximal to the immune cell and form a complementary micro-cluster. Binding of the blocking receptors in the micro-cluster to the first ligand on the normal cell may cause the engineered immune cell to exhibit reduced surface expression of the activating receptor in the micro-cluster. The reduced surface expression may be reversible.

[0093] The immune cell may express different concentrations of the activating and blocking receptors based on a ratio of a quantity of the first ligand to a quantity of the second ligand expressed on a normal cell. The ratio of the concentration of blocking receptors to activating receptors expressed by the immune cell may be less than or equal to 1.

[0094] In certain methods of the disclosure, the blocking receptors in the micro-cluster do not bind to the first ligands until the activating receptors bind to the second ligands on the normal cell.

[0095] In a further aspect, the present disclosure provides engineered immune cells and methods of making and using them wherein the immune cells comprise a hinge that modulates an effect of the blocking signal and/or receptors.

[0096] Thus, the present disclosure provides a method of producing an engineered immune cell, the method including, causing an immune cell to express cell surface activating receptors and blocking receptors, wherein the blocking receptors comprise a selected hinge. Binding of the activating receptors to activating ligands on a target cell triggers an activating signal that promotes a cytotoxic response by the immune cell. Binding of the blocking receptors to blocking ligands on a non-target cell causes the blocking receptors to trigger a blocking signal that inhibits the activating signal. The selected hinge is selected to modulate an effect of the blocking signal on the activating signal.

[0097] In certain methods, the selected hinge comprises a peptide having a certain length, and the length of the peptide modulates the effect of the blocking signal on the activating signal. The effect of the blocking signal on the activating signal may be an increased inhibition of the activating signal. The increased inhibition of the activating signal may increase a half maximal effective concentration (EC.sub.50) of the activating ligand for the activating receptors to promote the cytotoxic response.

[0098] In certain methods and immune cells of the disclosure, cross-talk and/or structure function interactions between the hinge of the blocking receptor and the activating receptor further impart different signal strengths for the blocking receptor.

[0099] The effect of the blocking signal on the activating signal may be a decreased inhibition of the activating signal, and the length of the peptide is less than about 24 amino acids.

[0100] In certain aspects of the disclosure, the engineered immune cell is caused to express the blocking and activating receptors at a ratio, and the ratio blocking receptors to activating receptors expressed is decreased as the length of the peptide of the selected hinge is increased. The peptide may further have a degree of flexibility, and the peptide's length and degree of flexibility modulate the effect of the blocking signal on the activating signal.

[0101] The selected hinge may be selected from hinges that each have a known effect on the EC.sub.50 of the activating ligand for the activating receptors to promote the cytotoxic response. The length of the peptide of each hinge that may have a known effect on the EC.sub.50 of the activating ligand is between 10 and 64 amino acids in length. The peptide of the selected hinge may be at least 24 amino acids in length. The peptide of the selected hinge may be at least 64 amino acids in length. In certain aspects, the hinge having a peptide of 64 amino acids in length causes at least a fifty-fold increase in the EC.sub.50 relative to a hinge having a peptide of 10 amino acid in length. In certain aspects, the hinges that each have a known effect on the EC50 comprise hinges 2B1, 2B1 truncated, PD-1, CTLA4, BTLA, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396 and SEQ ID NO: 397.

[0102] In certain aspects of the disclosure, the length of the peptide increases surface expression of the blocking receptor.

[0103] In certain aspects, the hinge peptide is derived from leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1).

[0104] In certain aspects of the disclosure, the hinge comprises a peptide having a degree of flexibility, and the peptide's degree of flexibility modulates the effect of the blocking signal on the activating signal. The peptide may be a flexible peptide and the effect of the blocking signal on the activating signal is an increased inhibition of the activating signal. The flexible peptide may comprise glycine-glutamine repeats and/or glycine-serine repeats.

[0105] In certain aspects, the hinge comprises a rigid peptide, and the peptide and the effect of the blocking signal on the activating signal is reduced inhibition of the activating signal. The rigid peptide may include, for example, an alpha-helix, repeats of (XP) where X is any amino acid, and/or repeats consisting of alanine, glutamic acid, and lysine.

[0106] In certain aspects, the present disclosure provides engineered immune cells, and methods of making and using the same, wherein activating and blocking receptors are spaced apart by at least an average minimum distance on the immune cell surface.

[0107] Thus, in certain aspects, the disclosure provides a method of producing an engineered immune cell, the method including, causing an immune cell to express cell surface activating and blocking receptors. Binding of the activating receptor to an activating ligand on a target cell triggers an activating signal that promotes a cytotoxic response by the engineered immune cell. Binding of the blocking receptor to a blocking ligand on a non-target cell causes the blocking receptor to trigger a blocking signal that inhibits the activating signal. Wherein the receptors remain spaced apart by at least an average minimum distance on the immune cell surface.

[0108] The blocking signal of a blocking receptor may invert to an activating signal when a blocking receptor is spaced at a distance less than the average minimum distance from the activating receptor.

[0109] In certain aspects, the method also includes determining the distance less than the average minimum distance at which the blocking signal inverts.

[0110] In certain methods, the average minimum distance is about 100-1000 angstroms. In certain methods, the average minimum distance is about between about 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 angstroms. In certain methods, the average minimum distance is greater or equal to 200 angstroms. In certain methods, the average minimum distance is about 200 angstroms.

[0111] In certain methods, each receptor has a ligand binding domain (LBD), a hinge, a transmembrane domain, and an intracellular domain (ICD).

[0112] Certain methods further include covalently or non-covalently linking the receptors via a spacer such that the receptors are separated by a known spacing. The spacer may comprise a C- or N-terminal fusion. The receptors may be linked to the spacer via the LBD or ICD of each receptor. The receptors may be linked to the spacer at their respective hinge. The spacer may comprise one or more moieties that allow non-covalent binding of the receptors at their respective hinge. The spacer may comprise, for example, two moieties, that are independently fused to the LBD, ICD, or hinge of each receptor. The receptors may be linked via a spacer that comprises a non-covalent interacting motif that mediates protein-protein interaction, such as leucine zipper. The receptors may be covalently attached via the spacer, and the spacer may comprise a cleavable linker such as a disulfide linker.

[0113] In certain aspects, the receptors are linked via a spacer that comprises a rigid peptide linker. The rigid peptide may include, for example, an alpha-helix, repeats of XP where X is any amino acid, and/or repeats consisting of alanine, glutamic acid, and lysine.

[0114] In certain aspects, the receptors have physiochemical properties that prevent the receptors from being spaced at a distance less than the average minimum distance. The physiochemical properties may include, for example, opposite charges engineered by design on the receptor sequences, leading to attraction, compared to neutral or similar charges. The physiochemical properties may also or alternatively include, for example, steric effects, non-covalent interactions, and/or van der Waals interactions.

[0115] In certain aspects, the present disclosure includes an engineered immune cell comprising a cell surface activating receptor and a cell surface blocking receptor, wherein each of the cell surface activating receptor and the cell surface blocking receptor comprise physiochemical properties that prevent the cell surface activating receptor and the cell surface blocking receptor from being spaced at a distance less than an average minimum distance.

[0116] In certain aspects, the average minimum distance is about 100-1000 angstroms. In certain methods, the average minimum distance is about 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 angstroms. In certain methods, the average minimum distance is greater or equal to 200 angstroms. In certain methods, the average minimum distance is about 200 angstroms.

[0117] In certain aspects, the receptors have physiochemical properties that prevent the receptors from being spaced at a distance less than the average minimum distance. The physiochemical properties may include, for example, opposite charges engineered by design on the receptor sequences, leading to attraction, compared to neutral or similar charges. The physiochemical properties may also or alternatively include, for example, steric effects, non-covalent interactions, and/or van der Waals interactions.

[0118] In a further aspect, the present disclosure provides an engineered immune cell comprising a cell surface activating receptor, a cell surface blocking receptor, and a spacer operably associated with the cell surface activating receptor and the cell surface blocking receptor, wherein the spacer is configured to maintain the cell surface activating receptor and the cell surface blocking receptor spaced apart by at least an average minimum distance on the immune cell surface.

[0119] In certain aspects, the average minimum distance is about 100-1000 angstroms. In certain methods, the average minimum distance is about 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 angstroms. In certain methods, the average minimum distance is greater or equal to 200 angstroms. In certain methods, the average minimum distance is about 200 angstroms.

[0120] In certain aspects, each receptor of the engineered immune cell has a ligand binding domain (LBD), a hinge, a transmembrane domain, and an intracellular domain (ICD).

[0121] The spacer may covalently or non-covalently link the receptors such that the receptors are separated by a known spacing. The spacer may comprise a C- or N-terminal fusion. The receptors may be linked to the spacer via the LBD or ICD of each receptor. The receptors may be linked to the spacer at their respective hinge. The spacer may comprise one or more moieties that allow non-covalent binding of the receptors at their respective hinge. The spacer may comprise, for example, two moieties that are independently fused to the LBD, ICD, or hinge of each receptor. The receptors may be linked via a spacer that comprises a non-covalent interacting motif that mediates protein-protein interaction, such as leucine zipper. The receptors may be covalently attached via the spacer, and the spacer may comprise a cleavable linker such as a disulfide linker.

[0122] In certain aspects, the receptors are linked via a spacer that comprises a rigid peptide linker. The rigid peptide may include, for example, an alpha-helix, repeats of XP where X is any amino acid, and/or repeats consisting of alanine, glutamic acid, and lysine.

[0123] In a further aspect, the disclosure provides a method for treating cancer that includes providing an engineered immune cell to a patient, wherein the engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell. The activating receptor triggers a cytotoxic signal that promotes a cytotoxic response of the engineered immune cell when the activating receptor binds a first ligand of a target cell. The blocking receptor sends an interfering signal that inhibits the cytotoxic response of the engineered immune cell when the blocking receptor binds a second ligand of the target cell. The receptors remain spaced apart by at least an average minimum distance on the immune cell surface.

[0124] In certain methods, the average minimum distance is about 100-1000 angstroms. In certain methods, the average minimum distance is about 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 angstroms. In certain methods, the average minimum distance is greater or equal to 200 angstroms. In certain methods, the average minimum distance is about 200 angstroms.

[0125] In certain methods, the receptors possess physiochemical properties that prevent the cell surface activating receptor and the cell surface blocking receptor from being spaced at a distance less than an average minimum distance.

[0126] In certain aspects, the receptors have physiochemical properties that prevent the receptors from being spaced at a distance less than the average minimum distance. The physiochemical properties may include, for example, opposite charges engineered by design on the receptor sequences, leading to attraction, compared to neutral or similar charges. The physiochemical properties may also or alternatively include, for example, steric effects, non-covalent interactions, and/or van der Waals interactions.

[0127] In certain aspects, the immune cell includes a spacer operably associated with the cell surface activating receptor and the cell surface blocking receptor, wherein the spacer is configured to maintain the cell surface activating receptor and the cell surface blocking receptor spaced apart by at least an average minimum distance on the immune cell surface.

[0128] In certain methods, each receptor has a ligand binding domain (LBD), a hinge, a transmembrane domain, and an intracellular domain (ICD).

[0129] The spacer may covalently or non-covalently link the receptors such that the receptors are separated by a known spacing. The spacer may comprise a C- or N-terminal fusion. The receptors may be linked to the spacer via the LBD or ICD of each receptor. The receptors may be linked to the spacer at their respective hinge. The spacer may comprise one or more moieties that allow non-covalent binding of the receptors at their respective hinge. The spacer may comprise, for example, two moieties that are independently fused to the LBD, ICD, or hinge of each receptor. The receptors may be linked via a spacer that comprises a non-covalent interacting motif that mediates protein-protein interaction, such as leucine zipper. The receptors may be covalently attached via the spacer, and the spacer may comprise a cleavable linker such as a disulfide linker.

BRIEF DESCRIPTION OF THE DRAWINGS

[0130] FIG. 1 shows a schematic of the immune cells expressing activating and blocking receptors.

[0131] FIGS. 2-3 show reduced surface expression of activating receptors and reversibility of reduced surface expression of activating receptors.

[0132] FIGS. 4-6 provide experimental results showing reduced expression of activating receptors.

[0133] FIG. 7 provides experimental results showing reduced expression of activating receptors.

[0134] FIG. 8 shows a schematic for an experiment to show the reversibility of reduced expression of activating receptors.

[0135] FIGS. 9-12 provide experimental results showing reversibly reduced expression of activating receptors.

[0136] FIGS. 13-16 provide experimental results showing reversibly reduced expression of activating receptors.

[0137] FIGS. 17-19 shows experimental results indicating that the blocking receptor does not undergo reduced surface expression in an appreciable amount in the presence of non-target cells.

[0138] FIG. 20 shows a schematic of micro-clustering.

[0139] FIG. 21 shows a schematic of multiplex and localized signaling by the activating and blocking receptors.

[0140] FIG. 22 provides experimental results showing that activity of the blocking receptors is ligand-dependent.

[0141] FIG. 23 provides experimental results showing that the blocking receptors cause minimum ligand-independent inhibition of the activating receptors.

[0142] FIG. 24 provides a schematic of the activating and blocking receptors.

[0143] FIG. 25 provides experimental results for the effect the hinge of the blocking receptor has on blocking strength.

[0144] FIG. 26 provides experimental results showing that the identity of the ligand binding domain of the activating receptor drives the activity of the receptor.

[0145] FIG. 27 provides experimental results showing the effect the ligand binding domain has on the blocking receptor.

[0146] FIG. 28 shows combinations of various CAR- and TCR-based activating and blocking receptors.

[0147] FIG. 29 provides experimental results showing that a CAR-based blocking receptor can inhibit a TCR-based activating receptor.

[0148] FIG. 30 provides experimental results showing that a CAR ligand binding domain can be used with a TCR activating receptor intracellular domain.

[0149] FIG. 31 shows different receptors that can be created in accordance with the present disclosure.

[0150] FIG. 32 provides experimental results showing the effect the intracellular domain has on the strength of the blocking receptor signal.

[0151] FIGS. 33-34 provide experimental results that indicate cross-talk between receptors.

[0152] FIG. 35 shows various ways to control the distance between activating and blocking receptors.

DETAILED DESCRIPTION

[0153] The present disclosure provides engineered immune cells featuring "AND NOT" Boolean logic by expressing engineered activating and blocking receptors. The cells are designed such that when the activating receptors bind to cognate activating ligands on a target cell, they produce an activating signal. If the strength of the activating signal crosses a threshold, it causes a cytotoxic response by the immune cell, killing the cell expressing the cognate ligands. The second of these receptors is a blocking receptor, which is designed to bind to a cognate blocking ligand on the surface of another cell, thereby activating the receptor and causing it to trigger a blocking signal that blocks the activating signal, which prevents the cytotoxic response. The "AND NOT" Boolean logic engineered into the immune cells of the present disclosure makes them ideal for use as therapeutic agents.

[0154] Thus, in an exemplary method of the disclosure, a patient diagnosed with a medical condition, such as cancer, is treated with engineered immune cells that target and kill the patient's cancer cells while preserving their normal, healthy cells. One or more cellular samples may be taken from the patient, such as from a blood draw or tumor biopsy. Target cells, such as tumor cells, are identified in the sample. The identified target cells are assayed to determine the levels of expression of one or more cell-surface ligands. This may include, for example, assessing RNA expression profiles for various cell-surface receptors or using antibody probes that bind to certain cell surface receptors. Assaying target cells may determine, for example, that target cells do not express a certain cell surface ligand due to a loss of heterozygosity.

[0155] Then, immune cells, such as T cells, are harvested from the patient. These cells are caused to express engineered activating and blocking receptors. The blocking receptor is designed to bind to a blocking ligand expressed on healthy cells of the patient. This blocking ligand may be chosen because it is lost from cancer cells, e.g., due to loss of heterozygosity. The activating receptor is designed to bind to an activating ligand that is expressed on both healthy cells and cancer cells of the patient.

[0156] After the engineered immune cells are proliferated, the cells are administered to the patient. The immune cells are designed such that when an immune cell encounters a cancer cell in the patient's body, the activating receptors bind to activating ligands on the cancer cell. This triggers a cytotoxic immune response by the immune cell that kills the cancer cell. When the immune cell encounters a healthy cell the activating and blocking receptors bind to activating and blocking ligands on the healthy cell. Binding of the blocking receptors to blocking ligands inhibits and blocks the cytotoxic immune response triggered by the activating receptors binding to activating ligands. In this way, the engineered immune cells are designed to limit deleterious effects on non-target cells.

[0157] FIG. 1 shows a schematic of this "AND NOT" Boolean logic in the immune cells of the present disclosure. In FIG. 1, the immune cells 103 comprise a blocking receptor 105 and an activating receptor 107. A non-target cell 109 expresses an activating ligand 111 and blocking ligand 113. When the immune cell 103 contacts the non-target cell, the activating receptor 107 binds to the activating ligand 111 triggering an activating signal 117. Concurrently, the blocking receptor 105 binds to the blocking ligand 113, triggering a blocking signal 115. As shown schematically, when the immune cell 103 contacts a non-target cell, the strength of blocking signal is greater than the activating signal 119. Binding both blocking ligands and activating ligands causes the "AND NOT" state in the immune cell. The signal from the blocking receptors blocks the activating signal. As such, the activating signal cannot pass the threshold to trigger a cytotoxic response, which prevents a deleterious effect on the non-target cell expressing both ligands.

[0158] Conversely, a target cell 121, such as a tumor cell, expresses a blocking ligand 111, but does not express an activating ligand, or expresses an activating ligand at a lower level compared to the non-target cell 109. Thus, when the immune cell 103 contacts the target cell 121, the activating receptor 107 binds to the activating ligand 111, triggering an activating signal 117. As shown schematically, when the immune cell 103 contacts a non-target cell, the strength of the activating signal is greater than the blocking signal 123. When the strength of the activating signal crosses an activation threshold, the immune cell produces a cytotoxic response 125 that kills the target cell 121.

[0159] Generally, the cognate antigens chosen for the activating receptors are expressed on both target cells, such as tumor cells, and non-target cells. The selected blocking ligands are expressed only by non-target cells, or expressed at lower levels by target cells compared to non-target cells. In this way, when the engineered immune cells contact target cells, the activating receptors bind to the activating ligands, which leads to the cytotoxic response. In contrast, when the engineered immune cells contact non-target cells, the blocking and activating receptors bind to their cognate blocking and activating ligands. This completes the "AND NOT" Boolean logic, thereby blocking the cytotoxic response. This scheme provides the general means by which the engineered immune cells safely kill target cells while limiting effects on non-target cells.

[0160] Engineered immune cells have been used as cancer therapies, such as immunotherapies. Traditionally, engineered immune cells have been designed to target molecular targets such as neo-antigens. Neo-antigens are a class of somatic mutant proteins that are mutated during somatic growth of tumors. They provide ideal targets for immune cell therapies because they comprise variants not found on non-target, healthy cells of a patient. However, very few cancers express neo-antigens. Thus, different targets must be pursued to treat most types of cancer using engineered immune cells. However, in prior immune cell therapies that lacked the blocking receptors of the present disclosure, when the immune cells targeted antigens expressed by healthy and non-health cells, severe adverse effects arose due to non-target activity. In cancer immunology, this phenomenon is known as on-target, off-tumor recognition.

[0161] The engineered immune cells and receptors of the present disclosure provide greater flexibility in choice of molecular target. The efficacy of the blocking receptor ensures that non-target effects are limited. Thus, the immune cells of the present disclosure can be designed to target widely expressed, cell surface molecules as the activating ligand. Exemplary ligands include a cell adhesion molecule, a cell-cell signaling molecule, an extracellular domain, a molecule involved in chemotaxis, a glycoprotein, a G protein-coupled receptor, a transmembrane, a receptor for a neurotransmitter or a voltage gated ion channel.

[0162] Activating receptors of the present disclosure may be configured to target activating ligands that are encoded by genes with essential cellular functions. Advantageously, this can prevent antigen escape, increasing the long-term efficacy of the engineered immune cells as a therapeutic. By selecting activating ligands encoded by genes with essential cellular functions, loss or escape of the ligand, such as through aneuploidy in cancer cells, is less likely. Thus, the activating ligand may be encoded by a gene that is haploinsufficient, i.e., loss of copies of the gene encoding the ligand are not tolerated by the cell and lead to cell death, or a disadvantageous mutant phenotype. In fact, the engineered immune cells of the present disclosure may be designed to target activating ligands expressed on all cells of patient.

[0163] Advantageously, because the immune cells of the present disclosure can be engineered to use widely-expressed activating ligands, several problems can be avoided. For example, prior engineered immune cells often targeted minimally expressed antigens, such as certain neo-antigens. Thus, to ensure an adequate activating signal, prior immune cells were engineered with activating receptors that had very high expression or affinities for their activating ligands.

[0164] However, merely increasing the density or affinity of receptors is inadequate to ensure efficacy. High receptor affinity can lead to proportionally severe, toxic effects on non-target cells. It can also hinder an engineered immune cell from disassociating from a target cell, which limits the ability of the immune cell to subsequently bind to and kill other target cells. Further, high affinity can cause receptors to be continually activated. This chronic activation can lead to immune cell exhaustion, reduced generation and persistence, increasing differentiation to undesired phenotypes, and activation-induced immune cell death. Increasing density can lead to similar effects through ligand-independent, tonic signaling.

[0165] Use of widely expressed activating ligands, made safe through the use of a blocking receptor, allows the engineered immune cells of the present disclosure to avoid these potential issues.

[0166] The blocking receptor can be designed to bind to a cell surface molecule not expressed on the surface of the target cell, or expressed at sufficiently low levels on a target cell. Thus, where the engineered immune cells are used to treat cancer, the blocking ligand may be chosen based on the loss of heterozygosity (LOH) of the target cancer cells, i.e., the cancer cells no longer express the ligand due to a loss of genetic material from one of the homologous chromosomes. Exemplary genes whose expression is frequently lost in cancer cells, for example due to LOH, include, HLA class I alleles, minor histocompatibility antigens (MiHAs), and Y chromosome genes (in males where the homologous chromosome is the X chromosome).

[0167] As will be discussed, the immune cells of the present disclosure possess several features that leverage the general nature of the "AND NOT" Boolean logic, to provide effective, target-specific effects while minimizing deleterious non-target effects.

[0168] Reduced Activator Expression

[0169] Surprisingly, the engineered immune cells of the present disclosure, which express activating and blocking receptors, can be designed to exhibit reduced surface expression of activating receptors when they contact non-target cells.

[0170] This ability to reduce surface expression is shown schematically in FIG. 2. When an engineered immune cell contacts non-target cells, the blocking receptors and activating receptors bind to their cognate activating ligands and blocking ligands expressed on the non-target cell. As explained, this causes the blocking signal to inhibit the activating signal. Further, activation of the blocking receptors causes reduced surface expression of the activating receptors. The activating receptors may be internalized, such that they are no longer on the surface of the immune cell and able to interact with activating ligands. As a result, the threshold to trigger a cytotoxic response by the immune cell is raised. Thus, the immune cells may temporarily exhibit a reduced propensity to kill cells, which can increase the therapeutic window of the cells.

[0171] As shown in FIG. 3, when the immune cell contacts a target cell, this reduced surface expression of the activating receptor does not occur and/or is reversed. Thus, when the immune cell encounters a target cell, the activating receptors bind to activating ligands. This provides the activating signal, but also causes the immune cell to reverse the reduced surface expression of the activating receptors. When the activating receptors are expressed on the surface of the immune cell at higher numbers, the activation threshold to trigger the cytotoxic response is reduced. Thus, immune cells in contact with target cells may temporarily exhibit an increased propensity to kill cells.

[0172] In certain immune cells of the disclosure, the reduced and/or regained expression of the activating receptor can be localized to a region of the immune cell surface proximate to a target or non-target cell. Thus, returning to FIG. 3, when the immune cell contacts a non-target cells, reduction of the activating receptors occurs in regions 303 proximate to the non-target cells. This can desensitize the immune cell in the regions 303 proximate to each non-target cell, which raises the activation threshold to trigger the cytotoxic response. Similarly, the immune cell can contact a target cell, and reduced surface expression of the activating receptor is reversed and/or does not occur in a region(s) 305 proximate to the target cell(s). This allows the region 305 proximate to the target cell to experience a local activation signal sufficient to trigger a localized cytotoxic response.

[0173] Advantageously, these localized responses can occur as an immune cell simultaneously and/or sequentially contacts target and non-target cells. Thus, as shown in FIG. 3, the immune cells can provide an activating signal localized to a region 305 proximate to a target cell, while also modulating expression of the activating receptor to reduce the activation threshold in the same region. Simultaneously, the immune cell can provide localized blocking signals and localized, reduced expression of the blocking receptor.

[0174] Reducing surface expression of the activating receptors. when not in contact with target cells. confers several advantages to the immune cells of the present disclosure. For example, if an immune cell circulates away from target cells, such as in a tumor, the immune cell is presumably more likely to contact non-target cells. By reducing the surface expression of the activating receptor in response to a lack of target cells, the immune cell increases its activation threshold, which can temporarily reduce the propensity of the immune cell to trigger a cytotoxic response. This designed feature of the engineered immune cells acts as a "safe mode", which enhances the safety and protective effects provided by the "AND NOT" Boolean logic, and further limits deleterious effects caused by the immune cells.

[0175] Further, while in this "safe mode", fewer activating receptors are available to activate. Thus, the immune cells of the present disclosure are less likely to experience chronic activation or ligand-independent tonic signaling. As a result, the immune cells are less susceptible to exhaustion, differentiation, and activation-induced immune cell death, while concurrently exhibiting high generation and persistence.

[0176] An additional and important feature of this reduced expression is that it does not extend to the engineered blocking receptors. Only the engineered activating receptors experience appreciable amounts of reduced expression. This ensures the safety profile of the immune cells of the present disclosure is maintained.

[0177] Thus, the present disclosure provides an engineered immune cell with an activating receptor and blocking receptor expressed on a surface of the engineered immune cell, wherein binding of the activating receptor to an activating ligand on a target cell promotes a cytotoxic response by the engineered immune cell, and binding of the blocking receptor to a blocking ligand causes the engineered immune cell to exhibit reduced surface expression of the activating receptor.

[0178] The present disclosure also provides a method for treating a cancer that includes providing an engineered immune cell to a patient, wherein the engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell. When the engineered immune cell encounters a tumor cell of the patient, the activating receptor binds to an activating ligand on the tumor cell while the blocking receptor remains unbound, thereby promoting a cytotoxic response by the engineered immune cell that results in a cytotoxic effect on the tumor cell. When the engineered immune cell encounters a normal cell of the patient the blocking receptor binds to a blocking ligand on the normal cell and causes the engineered immune cell to exhibit reduced surface expression of the activating receptor, thereby causing a signal from the blocking receptor to dominate a signal from the activating receptor and prevent the cytotoxic response by the engineered immune cell.

[0179] Micro-Clusters

[0180] A further advantageous feature of the engineered immune cells disclosed herein is that the cells and receptors can be designed such that the activating and blocking receptors form micro-clusters on the surface of the immune cell. This ability to form micro-clusters provides an engineered immune cell with the ability to sense its proximity to target/non-target cells and provide an appropriate, localized response.

[0181] FIG. 20 shows a schematic of the micro-clustering behavior. When an immune cell encounters a target or non-target cell, its activating/blocking receptors bind to cognate ligands on the encountered cell(s). Cross-talk between these bound receptors and unbound receptors on the surface of the immune cell causes the unbound receptors to diffuse to a region on the immune cell surface proximate to the encountered cell(s). When the receptors diffuse into this region, they form a micro-cluster in which the ligand binding domains of the receptors locate in an activation synapse between the immune cell and the encountered cell(s).

[0182] Forming a micro-cluster with both activating and blocking receptors ensures that, when the blocking receptors are activated in the presence of an appropriate ligand on a non-target cell, the blocking signal is triggered proximate to the activation signal. This ensures that the blocking receptors can provide a localized inhibitory effect on the activation signal, thereby protecting the non-target cell. Thus, the micro-clusters enhance the "AND NOT" Boolean logic conferred by the activating and blocking receptors.

[0183] Advantageously, the engineered immune cells can be configured such that the activating and/or blocking ligands on target and/or non-target cells to experience a similar clustering effect on the surface of the encountered cell(s). Unbound activating and blocking ligands diffuse into an area on the target/non-target cell surface proximate to the immune cell, and become available for binding to a cognate receptor in the activation synapse.

[0184] Receptors are held in place on the surface of the immune cell by binding to cognate ligands in the activation synapse. This ensures that the receptors remain confined to a micro-cluster while the immune cell is in contact with a target/non-target cell. Maintaining the receptors within a micro-cluster helps assure that adequate numbers of activating and/or blocking receptors are within a region proximate to an encountered cell(s) to provide the requisite activating or blocking signal. It also increases the relative strength of both the activating and blocking receptors, which widens the therapeutic window of the immune cells of the disclosure.

[0185] The ability of the receptors to form micro-clusters also enhances the localized, reduced expression of the activating receptors when an immune cell encounters a non-target cell. By bringing activating and blocking receptors in close proximity, e.g., within the confines of a micro-cluster, the effect of cross-talk between the receptors is increased. This cross-talk leads to localized, reduced expression of the activating receptors in the micro-cluster.

[0186] Thus, the present disclosure provides an engineered immune cell comprising activating and blocking receptors on a surface of the engineered immune cell. When the engineered immune cell encounters a tumor cell, a first plurality of the activating receptors diffuse into a first region on the surface of the engineered immune cell and form a first micro-cluster proximal to the tumor cell that promotes a cytotoxic response by the engineered immune cell that results in cytotoxic effects on the tumor cell. When the engineered immune cell encounters a normal cell, a second plurality of the activating and blocking receptors diffuse into a second region on the surface of the engineered immune cell and form a second micro-cluster proximal to the normal cell, wherein the blocking receptors in the second micro-cluster inhibit cytotoxic effects on the normal cell.

[0187] The present disclosure also includes a method for treating cancer, the method comprising providing an engineered immune cell to a patient, the engineered immune cell comprising activating and blocking cell-surface receptors. When the engineered immune cell encounters a normal cell, a first plurality of the activating and blocking receptors collect into a micro-cluster within a region of the cell-surface of the engineered immune cell proximal to the normal cell. Binding of one of the blocking receptors in the micro-cluster to a blocking ligand on the normal cell inhibits breakup of the micro-cluster. The engineered immune cell kills tumor cells that exhibit an activating ligand bound by the activating receptor and do not exhibit the blocking ligand such that the blocking receptor remains unbound.

[0188] Multiplex and Localized Signaling

[0189] The engineered immune cells of the present disclosure have been designed to exhibit multiplex and localized activity. A shown in FIG. 21 an engineered immune cell 103 can simultaneously contact both target cells 121 and non-target cells 109. On regions of the immune cell 103 surface proximate to a non-target cell, the activating receptors 107 and blocking receptors 105 bind to activating ligands 111 and blocking ligands 113 on the non-target cell. As a result, a localized blocking signal inhibits a cytotoxic response 125 by the immune cell on the proximate non-target cell. Simultaneously or sequentially, the immune cell 103 can contact a target cell 121. The activating receptor 107 binds to the activating ligand 111 on the target cell 121. This causes a localized cytotoxic response 125, which may release cytotoxic granules 2103. The cytotoxic response only targets the target cell 121. The localized inhibition of the cytotoxic response in areas proximate to the non-target cells 109 protects them from an undesired immune response.

[0190] Thus, the present disclosure provides an engineered immune cell that includes activating and blocking receptors on the surface of the cell. When the engineered immune cell encounters a tumor cell and a healthy cell, a first region of the activating and blocking receptors form proximal to the healthy cell and blocking receptors in the first region inhibit cytotoxic effects on the healthy cell. Simultaneously, a second region of the activating and blocking receptors form proximal to the tumor cell and promotes a cytotoxic response by the engineered immune cell that exhibits cytotoxic effects on the tumor cell.

[0191] The present disclosure also provides a method for treating cancer that includes providing an engineered immune cell to a patient. The engineered immune cell has activating and blocking cell-surface receptors. When the engineered immune cell encounters a tumor cell and a healthy cell of the patient, a first set of the activating and blocking receptors collect into a first cell-surface region of the engineered immune cell proximal to the healthy cell in which the blocking receptors inhibit cytotoxic effects of the engineered immune cell on the healthy. Simultaneously, a second set of the activating and blocking receptors collect into a second cell-surface region of the engineered immune cell proximal to the tumor cell in which the activating receptors promote a cytotoxic response by the engineered immune cell that kills the tumor cell.

[0192] Dominant Blocking Receptors

[0193] The engineered immune cells of the present disclosure can be configured to have blocking receptors that produce a blocking signal that can overwhelm and fully inhibit the activating signal from the activating receptors.

[0194] As shown in FIG. 22, which is explained in greater detail below, the cells can be designed to express activating and blocking receptors that, when expressed at equivalent concentrations, it takes less blocking antigen relative activating antigen to inhibit the activating signal. Thus, each blocking receptor can inhibit the activating signal of one or more activating receptors. This means that the blocking signal from a single blocking receptor can dominate and inhibit the activating signal from a single activating receptor. This helps solidify the safety profile of the "AND NOT" Boolean logic used by the immune cells of the present disclosure.

[0195] As shown in FIG. 23, the blocking receptors can be engineered to provide minimal ligand-independent blocking activity on the activating receptors. As a corollary, the blocking receptors can provide overwhelmingly ligand-dependent activity.

[0196] Thus, the immune cells of the present disclosure may include blocking receptors that provide, for example, a less than 10.times. shift in the EC.sub.50 of the activating receptors when the immune cells are contacted with the activating ligand in the absence of the blocking ligand. The immune cells of the present disclosure can provide a less than 3.times. shift in the EC.sub.50 of the activating receptors when the immune cells are contacted with the activating ligand in the absence of the blocking ligand.

[0197] Since the blocking receptors of the present disclosure can provide an overwhelmingly ligand-dependent, dominate blocking signal, the levels of activating ligand and blocking ligand expressed on a non-target cell can be used to inform the appropriate levels of activating and blocking receptor expressed by the engineered immune cells of the present disclosure. The dominate blocking signal provides assurance that ligand quantity can be used as a proxy to inform the levels of activating and blocking receptors that should be expressed in order to assure sufficient inhibition. Moreover, the ligand-dependent nature of the blocking signal means that the expression of the blocking receptor will require little to no adjustment to prevent unintended increases to the EC.sub.50 of the activating receptors in the absence of the blocking ligand.

[0198] Thus, the present disclosure provides methods for producing engineered immune cells that express activating and blocking receptors based on a ratio of a quantity of activating ligands to a quantity of blocking ligands that are expressed in a normal, non-tumor cell of a patient. The activating and blocking receptors may be expressed at a ratio based upon the ratio of the quantity of activating ligands to the quantity of blocking ligands expressed by the normal cell.

[0199] The present disclosure also provides an engineered immune cell with an activating receptor on a surface of the engineered immune cell. Binding of the activating receptor to an activating ligand on a target cell causes the activating receptor to trigger an activating signal that promotes a cytotoxic response by the engineered immune cell. The cell also has a blocking receptor. Binding of the blocking receptor to a blocking ligand on a target cell causes the blocking receptor to trigger an inhibitory signal stronger than the activating signal such that the inhibitory signal dominates and blocks the activating signal from the activating receptor, thereby preventing a localized cytotoxic response by the engineered immune cell.

[0200] The disclosure further includes a method for treating cancer, the method comprising providing an engineered immune cell to a patient. The engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell. When the engineered immune cell encounters a tumor cell, the activating receptor binds to an activating ligand on the tumor cell and the activating receptor triggers an activating signal in the engineered immune cell that promotes a cytotoxic response by the engineered immune cell that results in a cytotoxic effect on the tumor cell. When the engineered immune cell encounters a normal cell, the activating receptor binds to the activating ligand on the normal cell and the blocking receptor binds to a blocking ligand on the normal cell. This leads to the activating receptor triggering an activating signal in the engineered immune cell and the blocking receptor triggering an inhibitory signal in the engineered immune cell that is stronger than the activating signal, such that the inhibitory signal dominates and blocks the activating signal from the activating receptor, thereby preventing a localized cytotoxic response by the engineered immune cell.

[0201] Modulating Activating and Blocking Signals of Receptors

[0202] The present disclosure also provides strategies for engineering receptors in a manner that modulates receptor signal strength to ensure strong activation signals and sufficient blocking signals.

[0203] FIG. 24 shows a schematic of the blocking and activating receptors of the present disclosure. In general, each type of receptor can comprise four parts, the ligand binding domain ("LBD"), the hinge ("H"), the transmembrane domain ("TM"), and the intracellular domain ("ICD"). Each of these four parts can have an impact on the structure-activity relationship of each receptor. By altering these parts, the behavior of each receptor can be finely tuned to exhibit desired activity. For example, altering these parts can cause the receptors to exhibit varying specificity and affinity for cognate ligands, strengths of activating and/or blocking signals, levels of cross-talk between receptors, and/or receptor surface expression.

[0204] The hinge is an extracellular domain between a receptor's extracellular ligand binding domain and transmembrane domain and/or intracellular domain. Surprisingly, the Inventors of the present disclosure have found that, for the activating receptor, a wide variety of hinge lengths and sequences are tolerated. Thus, changes to the activating receptor hinge can provide relatively little change to the structure activity relationship of the activating receptor. For example, changes to the hinge were shown to cause only minimal contributions to the activating receptors' EC.sub.50, baseline signaling, and maximum signaling.

[0205] In contrast, the Inventors of the present disclosure have found that modifications to the hinge can be used to modulate the activity of the blocking receptor, including increases in the surface expression of the blocking receptor and blocking signal strength. Thus, a feature of the present disclosure is that the blocking receptor can be designed using interchangeable hinges that connect an extracellular ligand binding domain to a transmembrane domain and/or an intracellular domain.

[0206] The hinges can be designed to have different lengths and flexibilities. As shown in FIG. 24, flexible hinges inure blocking receptors with a greater blocking strength compared to rigid hinges. However, a greater change to blocking strength can be provided by changing the length of the hinge. As shown in FIG. 25, lengthening a hinge from about 25 amino acids to about 35 amino acids confers a significant increase in blocker strength. This increase becomes more dramatic, as the hinge length approaches 65 amino acids in length. As also shown in FIG. 25, the relative flexibility/rigidity of a hinge also impacts the strength of a blocker. Although, this impact is reduced compared to that provided by the hinge length.

[0207] Thus, the blocking receptors can be designed with longer and/or more flexible hinges to increase the strength of the blocking receptor's signal or surface expression. In contrast, the blocking receptor can be engineered with shorter and/or more rigid hinges to decrease the strength of the blocking receptor's signal or surface expression. The blocking receptor can be configured to use a hinge selected from a group of hinges that have a known impact on the EC.sub.50 of the activating ligand for the activating receptor to cause the immune cell to trigger a cytotoxic response. This allows pairs of blocking and activating receptors to be chosen or engineered to exhibit a desired level of activation/inhibition.

[0208] Advantageously, as the activating receptor can tolerate a wide variety of hinges, the activating receptors can be engineered with hinges that interact with a blocking receptor at the structural level. Different activator hinges may provide varying levels of interaction with a specific blocker. Thus, various activator hinges can be tested with a particular blocking receptor to determine the identity of activating receptor hinges that lead to increased blocking by a particular blocking receptor. Such testing may include, for example, changing the hinge of an activating receptor and measuring the blocking receptor strength, i.e., the IC.sub.50, of a particular blocking receptor when a particular activating receptor hinge is used.

[0209] The Inventors of the present disclosure found that the identity of the ligand binding domain of the engineered activating receptors has the greatest impact on the structure activity relationship of the receptors. As shown in FIG. 26, different ligand binding domains, which all bind to the same activating ligand, provide effects on the receptors' EC.sub.50 that differ by orders of magnitude. In contrast, the identity of the hinge and/or intracellular domain provides a smaller impact on the receptors' EC.sub.50.

[0210] As with the LBD of the activating receptor, the identity of the blocking receptor LBD can have large effects on the IC.sub.50 of the engineered immune cells of the present disclosure. This is shown in FIG. 27, where several different ligand binding domains were tested for their effect on the IC.sub.50 of engineered immune cells. Interestingly, the Inventors of the present disclosure found that when a ligand binding domain was switched between an activating receptor and blocking receptor, the LBD provided a correlative effect on the IC.sub.50 and EC.sub.50 of an immune cell.

[0211] The Inventors of the present disclosure found that a wide variety of commonly used intracellular domains have relatively minimal impacts on the EC.sub.50 of the activating receptor. Conversely, the Inventors discovered that the intracellular domain of the blocking receptor can have large effects on the strength of the blocking signal. Thus, the intracellular domain of the blocking receptor can be changed to modulate the strength of the blocking signal to ensure adequate inhibition. As shown in FIG. 32, changing the intracellular domain of the blocking receptor can have wide ranging effects on the strength of the blocking signal.

[0212] Receptor Cross-Talk

[0213] The present disclosure also provides engineered immune cells in which the activity of the activating and blocking receptors is modulated via cross-talk between the receptors.

[0214] FIGS. 33-34 show the impact receptor cross-talk can have on the ability of the blocking receptor to inhibit the activation signal. Engineered immune cells were created with one of five different activating receptors. Though the activating receptors differed between the cell lines, each targeted the same activating ligand, epidermal growth factor receptor (EGFR), using a different antibody. As shown by the five graphs at the bottom in FIGS. 33-34, each of the different activating receptors provided the immune cells with equivalent abilities to kill target cells. Then, immune cells were created that had one of the five activating receptors and the same blocking receptor. Addition of the blocker caused some of the immune cells, like CT486, to exhibit a significant decrease in the cells' ability to kill target cells. The blocking receptors also provided varying effects in the ability of the immune cells to inhibit the activating signal in the presence of non-target cells.

[0215] This disparity in behavior between different activating receptors and a blocking receptor can be attributed to cross-talk between the receptors.

[0216] Thus, the present disclosure provides an engineered immune cell that includes an activating receptor that triggers a cytotoxic signal that promotes a cytotoxic response of the engineered immune cell when the activating receptor binds to an activating ligand of a target cell, a blocking receptor that sends an interfering signal that inhibits the cytotoxic response of the engineered immune cell when the blocking receptor binds a blocking ligand, and cross-talk between the activating receptor and the blocking receptor that affects an activation threshold for the cytotoxic response.

[0217] The disclosure also includes a method for treating cancer, the method includes providing an engineered immune cell to a patient. The engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell. The activating receptor triggers a cytotoxic signal that promotes a cytotoxic response of the engineered immune cell when the activating receptor binds to an activating ligand of a target cell. The blocking receptor sends an interfering signal that inhibits the cytotoxic response of the engineered immune cell when the blocking receptor binds a blocking ligand. Cross-talk between the activating receptor and the blocking receptor affects an activation threshold for the cytotoxic response.

[0218] Modulating Receptor Proximity

[0219] The Inventors of the present disclosure made the surprising discovery that the strength of the blocking signal can increase as the distance between activating and blocking receptors decreases, and that when the receptors are separated by a particular average minimum distance, the blocking signal provides a maximum inhibitory effect on an activating receptor. Thus, the present disclosure provides engineered immune cells, and methods of making using engineered immune cells, with activating and blocking receptors spaced apart by at least a minimum average distance on the immune cell surface. The present Inventors also discovered that when activating and blocking receptors are within a certain, close proximity to one another, the activation of the blocking receptor may cause the blocking receptor to invert and provide an activating signal.

[0220] In certain engineered immune cells of the present disclosure, this average minimum distance is between about 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 angstroms.

[0221] As shown in FIG. 35, the Inventors devised several strategies to ensure that the activating and blocking receptors are spaced at a distance to ensure a high blocking signal strength. For example, the receptors can be attached via a C-terminal or N-terminal bridge. Alternatively or in addition, the receptors can be designed to have substituent groups or amino acids with opposing charges to enforce spacing between receptors. Bulky substituent groups or amino acids can also be used to cause steric effects that prevent the receptors from diffusing too close to one another.

[0222] Thus, the present disclosure provides engineered immune cells with activating and blocking receptors that possess physiochemical properties that maintain an average minimum distance between the receptors on the cell surface. Physiochemical properties may include, for example, opposing charges on each of the cell surface activating receptor and the cell surface blocking receptor, non-covalent interactions, van der Walls interactions, and/or steric effects.

[0223] The present disclosure also or alternatively provides engineered immune cells that have a spacer operably associated with an activating and blocking receptor on the cell surface that is configured to maintain an average minimum distance between the receptors on the cell surface. The spacer may covalently or non-covalently link the activating and blocking receptors. The spacer may include C- or N-terminal fusion that links the receptors. The spacer may alternatively or in addition include two moieties that allow non-covalent binding between the LBD, ICD, and/or hinge of each receptor. The spacer may also or alternatively include a non-covalent interacting motif that mediates protein-protein interaction, such as a leucine zipper.

[0224] The distance between the activating and blocking receptors may be controlled by using a spacer that includes a linker. Any linker may be used, and many fusion protein linker formats are known. For example, the linker may be flexible or rigid. Non-limiting examples of rigid and flexible linkers are provided in Chen et al. (Adv Drug Deliv Rev. 2013; 65(10):1357-1369).

[0225] Non-limiting exemplary rigid linkers include alpha helix-forming linkers with the sequence of (EAAAK).sub.n and (EAAAK).sub.nA, wherein n=1-10. Another exemplary rigid linker is a proline rich linker having the sequence (XP).sub.n where X is any amino acid and is preferably selected from A, G, and E and n=1-10, and glycine-serine linkers with a high ratio of serine to glycine.

[0226] The ligand binding domains described herein may be linked to each other in a random or specified order. The ligand binding domains described herein may be linked to each other in any orientation of N to C terminus.

[0227] Optionally, a short oligo- or polypeptide linker, for example, between 2 and 40 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between the domains. The linker is a peptide of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 amino acid residues.

[0228] Non-limiting examples of amino acids found in linkers include Gly, Ser, Glu, Gin, Ala, Leu, Iso, Lys, Arg, Pro, and the like.

[0229] The linker may be [(Gly).sub.n1Ser]n2, where n1 and n2 may be any number (e.g. n1 and n2 may independently be 1, 2, 4, 5, 6, 7, 8, 9, 10 or more than 10). The linker may be flexible polypeptide linker that is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Ser), (Gly-Gly-Gly-Ser), or (Gly-Gly-Gly-Gly-Ser) which can be repeated n times, where n is a positive integer equal to or greater than 1. For example, n-1, n-2, n-3, n-4, n-5, n-6, n-7, n-8, n=9 and n=10. The linker may include multiple repeats of (Gly Gly Ser), (Gly Ser) or (Gly Gly Gly Ser). Also included within the scope of the invention are linkers described in WO2012/138475 (incorporated herein by reference). In some embodiments, the flexible polypeptide linkers include, but are not limited to, GGS, GGGGS (SEQ ID NO: 226), GGGGS GGGGS (SEQ ID NO: 227), GGGGS GGGGS GGGGS (SEQ ID NO: 228), GGGGS GGGGS GGGGS GG (SEQ ID NO: 229) or GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 230). In some embodiments, the linkers include multiple repeats of (Gly Gly Ser), (Gly Ser) or (Gly Gly Gly Ser (SEQ ID NO: 231)).

[0230] The linker sequence may comprise a long linker (LL) sequence. The long linker sequence may comprise GGGGS, repeated four times. Such a linker may be used to link intracellular domains in a TCR alpha fusion protein of the disclosure. The long linker sequence may comprise GGGGS, repeated three times. The linker sequence may comprise a short linker (SL) sequence. The short linker sequence may comprise GGGGS. A glycine-serine doublet can be used as a suitable linker. Alternatively, domains are fused directly to each other via peptide bonds without use of a linker.

[0231] By reducing the G:S ratio in a Gly-Ser linker, the linker can be made more rigid.

[0232] The strength of the blocking signal may be the strongest when the activating and blocking receptors are separated by a distance of 0-1000 angstroms. The strength of the blocking signal may be the strongest when the activating and blocking receptors are separated by a distance of 0-50 angstroms, 50-100 angstroms, 100-200 angstroms, 200-300 angstroms, 300-400 angstroms, 400-500 angstroms, 500-600 angstroms, 600-700 angstroms, 700-800 angstroms, 800-900 angstroms, or 900-1000 angstroms. Preferably, the distance is about 200 angstroms.

[0233] Thus, the present disclosure provides an engineered immune cell with an activating receptor on the cell surface that triggers a cytotoxic signal that promotes a cytotoxic response of the engineered immune cell when the activating receptors binds to a first ligand on a target cell; and a blocking receptor on the cell surface that sends an interfering signal that inhibits the cytotoxic response of the engineered immune cell when the blocking receptor binds a second ligand of the target cell. Proximity of the blocking receptor to the activating receptor effects an activation threshold for the cytotoxic response, and the activating and blocking receptors physiochemical properties favoring interaction with one another, such that the receptors are spaced apart at an average distance on the immune cell surface.

[0234] The present disclosure also provides a method for treating cancer that includes providing an engineered immune cell to a patient, wherein the engineered immune cell comprises an activating receptor and a blocking receptor, each expressed on a surface of the engineered immune cell. The activating receptor triggers a cytotoxic signal that promotes a cytotoxic response of the engineered immune cell when the activating receptor binds a first ligand of a target cell, and the blocking receptor sends an interfering signal that inhibits the cytotoxic response of the engineered immune cell when the blocking receptor binds a second ligand of the target cell. Proximity of the blocking receptor to the activating receptor affects an activation threshold for the cytotoxic response, and the activating and blocking receptors physiochemical properties favoring interaction with one another, such that the receptors are spaced apart at an average distance on the immune cell surface.

[0235] The present disclosure also provides a method of producing an engineered immune cell that includes producing an engineered immune cell that expresses activating receptors and blocking receptors based on a determined distance between the receptors, wherein an activation threshold for a cytotoxic response by the immune cell is maximized when the receptors are separated on the cell surface by the determined average distance.

[0236] Receptor Types

[0237] The present disclosure provides immune cells comprising activating and blocking receptors, which may independently comprise a chimeric antigen receptor (CAR) a T cell receptor (TCR) or a combination of components from CARs or TCRs.

[0238] As shown in FIG. 28, the immune cells of the present disclosure can use receptors that comprise various combinations of TCRs and CARs. For example, as shown in FIG. 29, both a blocking CAR and blocking TCR can effectively inhibit the activation signal of a TCR-based activating receptor.

[0239] Moreover, the receptors of the present disclosure can effectively use components of both CARs and TCRs to achieve desired receptor activity.

[0240] As shown in FIG. 30, the ligand binding domain of a CAR activating receptor can be used with the intracellular domain of a TCR activating receptor, and still provide a target-specific activation signal.

[0241] As shown in FIG. 31, the various components of TCRs and CARs can be interchanged to provide receptors with activities beyond blocking and activating receptors. For example, the components can be used to create Inverter TCRs, Super TCRs, Parasitic TCRs, and Molecular Integrators.

[0242] In some embodiments, one or more of the blocking receptor and activating receptor comprise a CAR. All CAR architectures are envisaged as within the scope of the instant disclosure.

[0243] The CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo. Exemplary hinges can be isolated or derived from IgD and CD8 domains, for example IgG1, CD8.alpha., or CD28, such as those disclosed by the Inventors of the present disclosure in PCT International Application No. PCT/US2020/045250 and PCT/US2021/030149, which are incorporated herein by reference in their entirety.

[0244] For example, exemplary hinges used in the receptors disclosed herein, which are isolated or derived from CDSa or CD28 include a CDSa hinge comprising an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NOS: 1 or 3 or encoded by SEQ ID NO: 4.

[0245] The CARs of the present disclosure can be designed to comprise a transmembrane domain that is fused to the hinge of the CAR. The transmembrane domain may be naturally associated with one of the domains of the CAR, such as the hinge or intracellular domain. For example, a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0246] The transmembrane domain may be derived either from a natural or synthetic source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions may be isolated or derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4.

[0247] Alternatively, the transmembrane domain may be synthetic, in which case it can comprise predominantly hydrophobic residues such as leucine and valine. Certain transmembrane domains may comprise a triplet of phenylalanine, tryptophan and valine found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. The CARs may comprise a CD28 transmembrane domain or an IL-2Rbeta transmembrane domain, such as those disclosed by the present Inventors in PCT International Application No. PCT/US2020/045250 and PCT/US2021/030149, incorporated herein by reference.

[0248] For example, the CD28 transmembrane domain may comprise an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 5. The CD28 transmembrane domain may be encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 6. An exemplary IL-2R beta transmembrane domain as disclosed here may comprise an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 7. In some aspects, an exemplary IL-2Rbeta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 8.

[0249] The intracellular signaling domains of CARs used as parts of the activating or blocking receptors are responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. Effector functions of a regulatory T cell, for example, include the suppression or downregulation of induction or proliferation of effector T cells. Thus, the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function.

[0250] While usually an entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired functions of CAR-T cells of the instant disclosure. The term intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the effector function signal.

[0251] Examples of intracellular signaling domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. In certain receptors of the disclosure, the intracellular activation domain ensures that there is T cell receptor (TCR) signaling necessary to activate the effector functions of the CAR-T cell.

[0252] The CAR intracellular domains of the instant disclosure may comprise at least one cytoplasmic activation domain. The at least one cytoplasmic activation domain can be a CD247 molecule (CD3) activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX-activating protein of 12 kDa (DAP12) activation domain, such as those disclosed by the present inventors in PCT International Application No. PCT/US2020/045250 and PCT/US2021/030149, which are incorporated by reference.

[0253] For example, the CD3z activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 9 and/or encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 10.

[0254] It is known that signals generated through a TCR alone can be insufficient for full activation of a T cell, and that a secondary or co-stimulatory signal may be also required. Thus, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0255] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory or inhibitory manner. Exemplary cytoplasmic signaling sequences are disclosed by the present Inventors in PCT International Application No. PCT/US2020/045250, which is incorporated by reference.

[0256] Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. In certain receptors of the disclosure, the cytoplasmic signaling domain contains 1, 2, 3, 4, or 5 ITAMs.

[0257] In certain receptors of the disclosure, the cytoplasmic domain comprises a CD3.zeta. activation domain. The CD3.zeta. activation domain may comprise a single ITAM, two ITAMs, or three ITAMs.

[0258] Further examples of ITAM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from TCR.zeta., FcR.gamma., FcR.beta., CD3.gamma., CD3.delta., CD3.epsilon., CD3.zeta., CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the instant invention comprises a cytoplasmic signaling sequence derived from CD3.zeta..

[0259] In certain receptors of the disclosure, the cytoplasmic domain of the CAR may comprise the CD3.zeta. signaling domain by itself or combined with any other desired cytoplasmic domain(s). For example, the cytoplasmic domain of the CAR can comprise a CD3.zeta. chain portion and a co-stimulatory domain.

[0260] For example, the CD3z activation domain may comprise a single ITAM and comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 11 and/or encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 12.

[0261] The co-stimulatory domain refers to a portion of a CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligands, that is required for an efficient response of lymphocytes to an antigen. In receptors of the disclosure, the costimulatory domain is selected from the group consisting of IL2R.beta., Fc Receptor gamma (FcR.gamma.), Fc Receptor beta (FcR.beta.), CD3g molecule gamma (CD3.gamma.), CD3.delta., CD3.epsilon., CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (OX40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen-1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional fragments thereof.

[0262] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides an example of a suitable linker.

[0263] The intracellular domains of CARs of the instant disclosure may include at least one co-stimulatory domain. The co-stimulatory domain may be isolated or derived from CD28.

[0264] An exemplary CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 13 and/or encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 14.

[0265] The intracellular domain of the CARs of the instant disclosure may include an interleukin-2 receptor beta-chain (IL-2Rbeta or IL-2R-beta) cytoplasmic domain. The IL-2Rbeta domain may be truncated. The IL-2Rbeta cytoplasmic domain may comprise one or more STAT5-recruitment motifs, which may be outside the IL-2Rbeta cytoplasmic domain.

[0266] An exemplary IL-2Rbeta intracellular domain may comprise an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 15 and/or encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 16.

[0267] Exemplary STAT5-recruitment motifs are provided by Passerini et al., (2008) STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25+ effector T cells, International Immunology, Vol. 20, No. 3, pp. 421-431, and by Kagoya et al., (2018) A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects. Nature Medicine doi:10.1038/nm.4478, which are each incorporated herein by reference.

[0268] An exemplary STAT-recruitment motif used herein may consist of SEQ ID NO: 17.

[0269] In certain blocking receptors of the disclosure, the inhibitory signal is transmitted through the intracellular domain of the receptor. Thus, the blocking receptor may comprise an inhibitory intracellular domain.

[0270] The inhibitory intracellular domain may comprise an immunoreceptor tyrosine-based inhibitory motif (ITIM). The inhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1. CTLA-4 and PD-1 are immune inhibitory receptors expressed on the surface of T cells, and play a pivotal role in attenuating or terminating T cell responses.

[0271] "ITIM" refers to a conserved sequence of amino acids with a consensus sequence provided in SEQ ID NO: 274. Exemplary ITIMs include, those having sequences of SEQ ID NOS: 67, 68, 69, and 70. In some embodiments, the intracellular domain comprises a sequence at least 95% identical to SEQ ID NOS: 71, 72, 73, 74, 75, or 76.

[0272] Inhibitory domains can also be isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.

[0273] The inhibitory domain may comprise an intracellular domain, a transmembrane or a combination thereof. Alternatively, the inhibitory domain comprises an intracellular domain, a transmembrane domain, a hinge region or a combination thereof. The inhibitory domain may contain an immunoreceptor tyrosine-based inhibitory motif (ITIM). The inhibitory domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1.

[0274] Inhibitory domains can be isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1. The inhibitory domain may be isolated or derived from a human protein, for example a human TRAIL receptor, CTLA-4, or PD-1 protein. In some embodiments, the TRAIL receptor comprises TR10A, TR10B or TR10D.

[0275] Endogenous TRAIL is expressed as a 281-amino acid type II trans-membrane protein, which is anchored to the plasma membrane and presented on the cell surface. TRAIL is expressed by natural killer cells, which, following the establishment of cell-cell contacts, can induce TRAIL-dependent apoptosis in target cells. Physiologically, the TRAIL-signaling system was shown to be essential for immune surveillance, for shaping the immune system through regulating T-helper cell 1 versus T-helper cell 2 as well as "helpless" CD8+ T-cell numbers, and for the suppression of spontaneous tumor formation.

[0276] The inhibitory domain may comprise an intracellular domain isolated or derived from a CD200 receptor. The cell surface glycoprotein CD200 receptor 1 (Uniprot ref: Q8TD46) represents another example of an inhibitory intracellular domain of the present invention. This inhibitory receptor for the CD200/OX2 cell surface glycoprotein limits inflammation by inhibiting the expression of proinflammatory molecules including TNF-alpha, interferons, and inducible nitric oxide synthase (iNOS) in response to selected stimuli.

[0277] The inhibitory domain may be isolated or derived from killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin like receptor B1 (LIR1), programmed cell death 1 (PD1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin like receptor K1 (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N and C terminal SH2 domains), or ZAP70 KI_K369A (kinase inactive ZAP70).

[0278] The inhibitory domain may be isolated or derived from a human protein. The blocking receptor may comprise a cytoplasmic domain and transmembrane domain isolated or derived from the same protein. For example, an ITIM containing protein. The blocking receptor may comprise a cytoplasmic domain, a transmembrane domain, and an extracellular domain or a portion thereof isolated or derived isolated or derived from the same protein. The blocking receptor may comprise a hinge region isolated or derived from isolated or derived from the same protein as the intracellular domain and/or transmembrane domain.

[0279] In certain immune cells of the disclosure, one or more of the activating and blocking receptors comprise a T Cell Receptor (TCR).

[0280] A "TCR", sometimes also called a "TCR complex" or "TCR/CD3 complex" refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and one or more of the invariant CD3 chains (zeta, gamma, delta and epsilon), sometimes referred to as subunits.

[0281] The TCR alpha and beta chains can be disulfide-linked to function as a heterodimer to bind to peptide-MHC complexes. Once the TCR alpha/beta heterodimer engages peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with downstream proteins, thereby transducing a primary stimulatory signal. In an exemplary TCR complex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3 epsilon and CD3 delta form a heterodimer, CD3 epsilon and CD3 gamma for a heterodimer, and two CD3 zeta form a homodimer.

[0282] The LBD of the activating or blocking receptors may be fused to an extracellular domain of a TCR subunit. The TCR subunit can be TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. Both the first and second ligand binding domains may be fused to the same TCR subunit in different TCR receptors. Alternatively, the first and second ligand binding domains are fused to different TCR subunits in different TCR receptors.

[0283] The LBD of the activating receptor and blocking receptor may each independently comprise an scFv domain or a V.beta.-only domain.

[0284] TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma and CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta or CD3 epsilon, or fragments or derivatives thereof, can be fused to one or more domains capable of providing a stimulatory signal of the disclosure, thereby enhancing TCR function and activity. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta or CD3 epsilon, or fragments or derivative thereof, can be fused to an inhibitory intracellular domain of the disclosure.

[0285] The receptors of the present disclosure may comprise TCRs comprising a TCR variable domain. The TCR variable domain specifically binds to an antigen in the absence of a second TCR variable domain (a V.beta.-only domain).

[0286] The TCRs may comprise additional elements besides the TCR variable domain, including additional amino acid sequences, additional protein domains (covalently associated, non-covalently associated or covalently and non-covalently associated with the TCR variable domain), fusion or non-covalent association of the TCR variable domain with other types of macromolecules (for example polynucleotides, polysaccharides, lipids, or a combination thereof), fusion or non-covalent association of the TCR variable domain with one or more small molecules, compounds, or ligands, or a combination thereof. Any additional element, as described, may be combined provided that the TCR variable domain is configured to specifically bind the epitope in the absence of a second TCR variable domain.

[0287] TCRs comprising a V.beta.-only domain as described herein may comprise a single TCR chain (e.g. .alpha., .beta., .gamma., or .delta. chain), or may comprise a single TCR variable domain (e.g. of .alpha., .beta., .gamma., or .delta. chain). If a TCR is a single TCR chain, then the TCR chain comprises a transmembrane domain, a constant (or C domain) and a variable (or V domain), but does not comprise a second TCR variable domain. The TCRs may comprise or consist of a TCR .alpha. chain, a TCR .beta. chain, a TCR .gamma. chain or a TCR .delta. chain. The TCRs may be a membrane bound proteins. The TCRs may alternatively be membrane associated proteins.

[0288] The TCRs may use a surrogate .alpha. chain that lacks a V.alpha. segment, which forms activation competent TCRs complexed with the six CD3 subunits. The TCRs may function independently of a surrogate .alpha. chain that lacks a V.alpha. segment. For example, one or more TCRs may be fused to transmembrane (e.g., CD3 and CD28) and intracellular domain proteins (e.g., CD3, CD28, and/or 4-1BB) that are capable of activating T cells in response to antigen.

[0289] TCRs may comprise one or more single TCR chains fused to the V.beta.-only domain described herein. For example, the TCRs may comprise, or consist essentially of single .alpha. TCR chain, a single .beta. TCR chain, a single .gamma. TCR chain, or a single .delta. TCR chain fused to one or more V.beta.-only domains.

[0290] The TCRs may engage antigens using complementarity determining regions (CDRs). Each TCR may contain three complement determining regions (CDR1, CDR2, and CDR3).

[0291] The first and/or second ligand binding V.beta.-only domain may be a human TCR variable domain. Alternatively, the first and/or second V.beta.-only domain may be a non-human TCR variable domain. The first and/or second V.beta.-only domain may be a mammalian TCR variable domain. The first and/or second V.beta.-only domain may be a vertebrate TCR variable domain.

[0292] Where V.beta.-only domain is incorporated into a fusion protein, for example a fusion protein comprising a TCR subunit, and optionally, an additional stimulatory intracellular domain, the fusion protein may comprise a V.beta.-only domain and any other protein domain or domains.

[0293] TCR receptors comprising transmembrane domains isolated or derived from any source are envisaged as within the scope of the fusion proteins of the disclosure.

[0294] The TCR transmembrane domain may be one that is associated with one of the other domains of the fusion protein, or isolated or derived from the same protein as one of the other domains of the fusion protein. The transmembrane domain and the second intracellular domain may be from the same protein, for example a TCR complex subunit such as TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma. The extracellular domain (svd-TCR), the transmembrane domain and the second intracellular domain may be from the same protein, for example a TCR complex subunit such as TCR alpha, TCR beta, CD3 delta, CD3 epsilon or CD3 gamma.

[0295] The TCR extracellular domain (comprising one or more ligand binding domains, such as V.beta.-only domain and scFv domains), the transmembrane domain and the intracellular domain(s) may be from different proteins. For example, the engineered svd-TCR may comprise a CD28 transmembrane domain with a CD28, 4-1BB and CD3.zeta. intracellular domain.

[0296] The TCR transmembrane domain may be derived from a natural or recombinant source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0297] The transmembrane domain may be capable of signaling to the intracellular domain(s) whenever the TCR complex is bound to a target. A transmembrane domain of particular use in this receptors of the disclosure may include at least the transmembrane region(s) of the alpha, beta, or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0298] The transmembrane domain can be attached to the extracellular region of the fusion protein, e.g., the antigen binding domain of the TCR alpha or beta chain, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge. The hinge may be isolated or derived from CD8.alpha. or CD28.

[0299] For example, an exemplary hinge isolated or derived from CD8a hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 1 and/or encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 2.

[0300] An exemplary CD28 hinge may comprise an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 3 and/or is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 4.

[0301] The transmembrane domain may comprise a TCR alpha transmembrane domain, a TCR beta transmembrane domain, or a CD3 zeta transmembrane domain, such as those disclosed by the present Inventors in PCT International Application No. PCT/US2020/045250, which is incorporated by reference.

[0302] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region).

[0303] The transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex or to minimize interactions with other receptors. This can help, for example, to ensure that the receptors remain at a sufficient distance apart on the surface of the immune cell to prevent blocking receptor inversion.

[0304] When present, the transmembrane domain may be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an artificial transmembrane domain. The transmembrane domain may be a membrane anchor domain.

[0305] Without limitation, a natural or artificial transmembrane domain may comprise a hydrophobic a helix of about 20 amino acids, often with positive charges flanking the transmembrane segment.

[0306] The transmembrane domain may have one transmembrane segment or more than one transmembrane segment. Prediction of transmembrane domains/segments may be made using publicly available prediction tools, e.g., TMHMM (Krogh et al., Journal of Molecular Biology 2001, 305(3):567-580) and TMpred (Hoppe-Seyler, Hofmann & Stoffel Biol. Chem. 1993; 347: 166), which are incorporated by reference. Non-limiting examples of membrane anchor systems include platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (added post-translationally to a signal sequence) and the like.

[0307] In certain aspects, transmembrane domain comprises a TCR alpha transmembrane domain. In some embodiments, the TCR alpha transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 26 and/or is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 27.

[0308] In some embodiments, the transmembrane domain comprises a TCR beta transmembrane domain. In some embodiments, the TCR beta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 28 or 35 and/or is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 20 or 36.

[0309] In some embodiments, the transmembrane comprises a CD3 zeta transmembrane domain. In some embodiments, the CD3 zeta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 29.

[0310] In some embodiments, the CD3 zeta transmembrane domain comprises, or consists essentially of, SEQ ID NO: 29. The disclosure provides fusion proteins comprising an intracellular domain. An "intracellular domain," refers to an intracellular portion of a protein. The TCR intracellular domain may comprise one or more domains capable of providing a stimulatory signal to a transmembrane domain. The intracellular domain may comprise a first intracellular domain capable of providing a stimulatory signal and a second intracellular domain capable of providing a stimulatory signal. The intracellular domain may comprise a first, second and third intracellular domain capable of providing a stimulatory signal.

[0311] The intracellular domains capable of providing a stimulatory signal may be selected from the group consisting of a CD28 molecule (CD28) domain, a LCK proto-oncogene, Src family tyrosine kinase (Lck) domain, a TNF receptor superfamily member 9 (4-1BB) domain, a TNF receptor superfamily member 18 (GITR) domain, a CD4 molecule (CD4) domain, a CD8a molecule (CD8a) domain, a FYN proto-oncogene, Src family tyrosine kinase (Fyn) domain, a zeta chain of T cell receptor associated protein kinase 70 (ZAP70) domain, a linker for activation of T cells (LAT) domain, lymphocyte cytosolic protein 2 (SLP76) domain, (TCR) alpha, TCR beta, CD3 delta, CD3 gamma and CD3 epsilon intracellular domains.

[0312] The TCR intracellular domain may comprise at least one intracellular signaling domain. An intracellular signaling domain generates a signal that promotes a function a cell, for example an immune effector function of a TCR containing cell, e.g., a TCR-expressing T cell. In certain methods and cells of the disclosure, the intracellular domain of the fusion proteins includes at least one intracellular signaling domain. For example, the intracellular domains of CD3 gamma, delta or epsilon comprise signaling domains.

[0313] The extracellular domain, transmembrane domain and intracellular domain may be isolated or derived from the same protein, for example T cell receptor (TCR) alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon.

[0314] Examples of intracellular domains for use in fusion proteins of the disclosure include the cytoplasmic sequences of the TCR alpha, TCR beta, CD3 zeta, and 4-1BB, and the intracellular signaling co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0315] The intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the proteins responsible for primary stimulation, or antigen dependent stimulation.

[0316] In some embodiments, the stimulatory domain comprises a CD28 intracellular domain. In some embodiments, the CD28 intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 37 and/or is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 30.

[0317] In some embodiments, the stimulatory domain comprises a 4-IBB intracellular domain. In some embodiments, the 4-IBB intracellular domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 39 and/or is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 40.

[0318] An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the fusion protein has been introduced. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

[0319] Thus, "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While in some cases the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire intracellular signaling domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0320] The intracellular domain may comprise the entirety or a portion of a CD3 delta intracellular domain, a CD3 epsilon intracellular domain, a CD3 gamma intracellular domain, or a CD3 zeta intracellular domain, such as those disclosed by the present inventors in PCT International Application No. PCT/US2020/045250, which is incorporated by reference.

[0321] The intracellular domain may comprise a TCR alpha intracellular domain or a TCR beta intracellular domain, such as those disclosed by the present inventors in PCT International Application No. PCT/US2020/045250, incorporated by reference.

[0322] The intracellular signaling domain may comprise at least one stimulatory intracellular domain. The intracellular signaling domain may comprise a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and one additional stimulatory intracellular domain, for example a co-stimulatory domain. The intracellular signaling domain may comprise a primary intracellular signaling domain, such as a CD3 delta, CD3 gamma and CD3 epsilon intracellular domain, and two additional stimulatory intracellular domains.

[0323] An exemplary CD3 delta intracellular domain may comprise, for example, an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 30 and/or is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 31.

[0324] An exemplary CD3 epsilon intracellular domain may comprise, for example, an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 32 and/or is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 19.

[0325] An exemplary CD3 gamma intracellular domain may comprise, for example, an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 22 and/or is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 9.

[0326] Exemplary co-stimulatory intracellular signaling domains include those derived from proteins responsible for co-stimulatory signals, or antigen independent stimulation.

[0327] The term "co-stimulatory molecule" refers to the cognate binding partner on a T-cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T-cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors. Co-stimulatory molecules and their ligands are required for an efficient immune response. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA, a Toll ligand receptor, as well as DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18) 4-1BB (CD137, TNF receptor superfamily member 9), and CD28 molecule (CD28).

[0328] A "co-stimulatory domain", sometimes referred to as "a co-stimulatory intracellular signaling domain" can be the intracellular portion of a co-stimulatory protein. A co-stimulatory domain can be a domain of a co-stimulatory protein that transduces the co-stimulatory signal. A co-stimulatory protein can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that specifically binds with CD83, CD4, and the like. The co-stimulatory domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

[0329] The stimulatory domain may comprise a co-stimulatory domain. The co-stimulatory domain may comprise a CD28 or 4-1BB co-stimulatory domain. CD28 and 4-1BB are well characterized co-stimulatory molecules required for full T cell activation and known to enhance T cell effector function. For example, CD28 and 4-1BB have been utilized in chimeric antigen receptors (CARs) to boost cytokine release, cytolytic function, and persistence over the first-generation CAR containing only the CD3 zeta signaling domain. Likewise, inclusion of co-stimulatory domains, for example CD28 and 4-1BB domains, in engineered TCR can increase T cell effector function and specifically allow co-stimulation in the absence of co-stimulatory ligand, which is typically down-regulated on the surface of tumor cells.

[0330] The stimulatory domain may comprise or be derived from a CD28 intracellular domain or a 4-1BB intracellular domain, such as those disclosed by the present inventors in PCT International Application No. PCT/US2020/045250, which is incorporated herein by reference.

[0331] The disclosure provides inhibitory intracellular domains which can be fused to the transmembrane or intracellular domain of any of the TCR subunits to generate a blocking TCR.

[0332] The inhibitory intracellular domain may comprise an immunoreceptor tyrosine-based inhibitory motif (ITIM). The inhibitory intracellular domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1.

[0333] Inhibitory domains can be isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1.

[0334] The inhibitory domain may comprise an intracellular domain, a transmembrane domain or a combination thereof. The inhibitory domain may comprise an intracellular domain, a transmembrane domain, a hinge region or a combination thereof. The inhibitory domain may comprise an immunoreceptor tyrosine-based inhibitory motif (ITIM). The inhibitory domain comprising an ITIM can be isolated or derived from an immune checkpoint inhibitor such as CTLA-4 and PD-1.

[0335] Inhibitory domains can be isolated from human tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor and CD200 receptor 1. The inhibitory domain can be isolated or derived from a human protein, for example a human TRAIL receptor, CTLA-4, or PD-1 protein. In some embodiments, the TRAIL receptor comprises TR10A, TR10B or TR10D.

[0336] Endogenous TRAIL is expressed as a 281-amino acid type II trans-membrane protein, which is anchored to the plasma membrane and presented on the cell surface. TRAIL is expressed by natural killer cells, which, following the establishment of cell-cell contacts, can induce TRAIL-dependent apoptosis in target cells. Physiologically, the TRAIL-signaling system was shown to be essential for immune surveillance, for shaping the immune system through regulating T-helper cell 1 versus T-helper cell 2 as well as "helpless" CD8+ T-cell numbers, and for the suppression of spontaneous tumor formation.

[0337] The inhibitory domain may comprise an intracellular domain isolated or derived from a CD200 receptor. The cell surface glycoprotein CD200 receptor 1 (Uniprot ref: Q8TD46) represents another example of an inhibitory intracellular domain of the present invention. This inhibitory receptor for the CD200/OX2 cell surface glycoprotein limits inflammation by inhibiting the expression of proinflammatory molecules including TNF-alpha, interferons, and inducible nitric oxide synthase (iNOS) in response to selected stimuli.

[0338] The inhibitory domain may be isolated or derived from killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3), leukocyte immunoglobulin like receptor B1 (LIR1), programmed cell death 1 (PD1), Fc gamma receptor IIB (FcgRIIB), killer cell lectin like receptor K1 (NKG2D), CTLA-4, a domain containing a synthetic consensus ITIM, a ZAP70 SH2 domain (e.g., one or both of the N and C terminal SH2 domains), or ZAP70 KI_K369A (kinase inactive ZAP70).

[0339] The inhibitory domain can be isolated or derived from a human protein.

[0340] The blocking receptor may comprise a cytoplasmic domain and transmembrane domain isolated or derived from the same protein, for example an ITIM containing protein. The blocking receptor may comprise a cytoplasmic domain, a transmembrane domain, and an extracellular domain or a portion thereof isolated or derived isolated or derived from the same protein, for example an ITIM containing protein. The blocking receptor may comprise a hinge region isolated or derived from isolated or derived from the same protein as the intracellular domain and/or transmembrane domain, for example an ITIM containing protein.

[0341] The blocking receptor may be a TCR comprising an inhibitory domain (an inhibitory TCR). The inhibitory TCR may comprise an inhibitory intracellular domain and/or an inhibitory transmembrane domain. The inhibitory intracellular domain can be fused to the intracellular domain of any one or more subunits of the TCR complex, including TCR alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon, or a portion of any thereof. The inhibitory intracellular domain can be fused to the transmembrane domain of TCR alpha, TCR beta, CD3 delta, CD3 gamma or CD3 epsilon.

[0342] The blocking receptor may comprise a hinge, transmembrane domain, and/or an intracellular domain derived from leukocyte immunoglobulin like receptor B1 (LILRBI). The blocking receptor may comprise the intracellular domain of the protein phosphoprotein membrane anchor with glycosphingolipid microdomains 1 (PAG1) or a functional variant thereof, and optionally hinge, a transmembrane domain, and/or one or more further intracellular domains. The transmembrane domain may be the transmembrane domain of PAG1. The hinge, transmembrane domain, and/or a further intracellular domain may be from leukocyte immunoglobulin like receptor B1 (LILRB1), PAG1 or a combination thereof. Examples of such blocking receptors have been disclosed by the Inventors of the present disclosure in U.S. Provisional Application Nos. 63/018,881 and 62/946,888 and PCT/US2021/030149, which are herein incorporated by reference in its entirety.

[0343] In some embodiments of the receptors having one or more domains isolated or derived from LILRB1, the one or more domains of LILRB1 comprise an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 65, 77, 78, 79, 80, 81, 82, 83, 84, or 85 and/or is encoded by a polynucleotide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is identical to a sequence or subsequence of SEQ ID NO: 66.

[0344] In various embodiments, an blocking receptor is provided, comprising a polypeptide, wherein the polypeptide comprises one or more of: an LILRB1 hinge domain or functional fragment or variant thereof; an LILRB1 transmembrane domain or a functional variant thereof; and an LILRB1 intracellular domain or an intracellular domain comprising at least one, or at least two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), wherein each ITIM is independently selected from and/or includes a sequence of SEQ ID NOS: 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.

[0345] Assays

[0346] Provided herein are assays that can be used to measure the activity of the engineered receptors and immune cells disclosed herein.

[0347] Receptor activity may be assayed using a cell line engineered to express a reporter of receptor activity such as a luciferase reporter. Exemplary cell lines include Jurkat T cells, although any suitable cell line known in the art may be used. For example, Jurkat cells expressing a luciferase reporter under the control of an NFAT promoter can be used as effector cells. Expression of luciferase by this cell line reflects TCR-mediated signaling.

[0348] Nuclear factor of activated T cells (NFAT) is a family of transcription factors shown to be important in immune response. The NFAT transcription factor family consists of five members NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. NFAT plays a role in regulating inflammation. As used herein, an NFAT promoter is a promoter that is regulated (i.e., activated or repressed) when NFAT is expressed in a cell. NFAT target promoters are described in Badran, B. M. et al., (2002) J. Biological Chemistry, Vol. 277: 47136-47148, incorporated herein by reference, and contain NFAT consensus sequences such as GGAAA.

[0349] The reporter cells can be transfected with each of the various fusion protein constructs, combinations of fusion protein constructs or controls described herein. Expression of the fusion proteins in reporter cells can be confirmed by using fluorescently labeled MHC tetramers, for example Alexa Fluor 647-labeled NY-ESO-1-MHC tetramer, to detect expression of the fusion protein.

[0350] To assay the activity of engineered receptors, target cells can be loaded with activating or blocking ligands prior to exposure to the cells comprising the reporter and the engineered receptor(s). For example, target cells can be loaded with ligands at least 12, 14, 16, 18, 20, 22 or 24 hours prior to exposure to immune cells. Exemplary target cells include A375 cells, although any suitable cells known in the art may be used. In some cases, target cells can be loaded with serially diluted concentrations of a ligand, such as NY-ESO-1 peptide. The immune cells can then be cocultured with target cells for a suitable period of time, for example 6 hours. Luciferase is then measured by luminescence reading after co-culture. Luciferase luminescence can be normalized to maximum and minimum intensity to allow comparison of activating peptide concentrations for each engineered receptor construct.

[0351] Provided herein are methods of determining the relative EC.sub.50 of engineered receptors of the disclosure. As used herein, "EC.sub.50" refers to the concentration of an inhibitor or agent to cause half the maximal response (or binding). Binding of the ligand, or probe to the engineered receptor can be measured by staining with labeled peptide or labeled peptide-MHC complex, for example MHC:NY-ESO-1 pMHC complex conjugated with fluorophore. EC.sub.50 can be obtained by nonlinear regression curve fitting of reporter signal with peptide titration. Probe binding and EC.sub.50 can be normalized to the levels of benchmark TCR without a fusion protein, e.g. NY-ESO-1 (clone 1G4).

[0352] Methods of assessing the effects of receptor activation on gene expression are known in the art, and include the use of reporter genes, whose expression can be quantified. Reporter genes can be used for identifying potentially transfected or transduced cells and for evaluating the functionality of regulatory sequences.

[0353] In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. See, e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82, which is incorporated herein by reference.

[0354] Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription. In exemplary embodiments, an NFAT promoter operably linked to a reporter gene is used to evaluate the expression of the receptors of the disclosure on NFAT signaling.

[0355] Exemplary assays have been disclosed by the present Inventors in PCT International Application Nos. PCT/US2019/037038, PCT/US2020/045250, PCT/US2020/045228, PCT/US2020/045373, and PCT/CA2016/051421, and U.S. Provisional Application Nos. 62/946,888, 62/934,419, 63/076,123, 63/068,244, 63/068,249, 63/068,245, 63/068,246, 63/065,324, and 63/037,975, which are each incorporated herein by reference.

[0356] Immune Cells

[0357] An immune cell can be a cell involved in the innate or adaptive (acquired) immune systems. Exemplary innate immune cells include phagocytic cells such as neutrophils, monocytes and macrophages, Natural Killer (NK) cells, polymophonuclear leukocytes such as neutrophils eosinophils and basophils and mononuclear cells such as monocytes, macrophages and mast cells. Immune cells with roles in acquired immunity include lymphocytes such as T-cells and B-cells. An engineered immune cell of the present disclosure can be derived from an innate immune cell and/or can be a modified innate immune cell.

[0358] A T cell is a type of lymphocyte that originates from a bone marrow precursor that develops in the thymus gland. There are several distinct types of T-cells which develop upon migration to the thymus, which include, helper CD4+ T-cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T-cells and stem memory T-cells. Different types of T cells can be distinguished by the ordinarily skilled artisan based on their expression of markers. Methods of distinguishing between T cell types will be readily apparent to the ordinarily skilled artisan.

[0359] The present disclosure also comprises methods of producing and modifying the engineered immune cells disclosed herein. The engineered immune cells of the present disclosure can be derived from any naturally occurring immune cell.

[0360] Methods of producing the disclosed immune cells may comprise introducing polynucleotide encoding the activating and blocking receptors into cells, optionally using vectors. The resulting cells express the polynucleotide encoding the receptors.

[0361] Methods transforming populations of immune cells, such as T cells, with vectors will be readily apparent to the person of ordinary skill in the art. For example, CD3+ T cells can be isolated from PBMCs using a CD3+ T cell negative isolation kit (Miltenyi), according to manufacturer's instructions. T cells can be cultured at a density of 1.times.10{circumflex over ( )}6 cells/mL in X-Vivo 15 media supplemented with 5% human AB serum and 1% Pen/strep in the presence of CD3/28 Dynabeads (1:1 cell to bead ratio) and 300 Units/mL of IL-2 (Miltenyi). After 2 days, T cells can be transduced with viral vectors, such as lentiviral vectors using methods known in the art. In some embodiments, the viral vector is transduced at a multiplicity of infection (MOI) of 5. Cells can then be cultured in IL-2 or other cytokines such as combinations of IL-7/15/21 for an additional 5 days prior to enrichment.

[0362] Methods of isolating and culturing other populations of immune cells, such as B cells, or other populations of T cells, will be readily apparent to the person of ordinary skill in the art. Although this method outlines a potential approach it should be noted that these methodologies are rapidly evolving. For example, high levels of viral transduction of peripheral blood mononuclear cells can be achieved after 5 days of growth to generate a >99% CD3+ highly transduced cell population.

[0363] In some embodiments, the first and second receptors are encoded by a single vector. Methods of encoding multiple polypeptides using a single vector will be known to persons of ordinary skill in the art, and include, inter alia, encoding multiple polypeptides under control of different promoters, or, if a single promoter is used to control transcription of multiple polypeptides, use of sequences encoding internal ribosome entry sites (IRES) and/or self-cleaving peptides. Exemplary self-cleaving peptides include T2A, P2A, E2A and F2A self-cleaving peptides. In some embodiments, the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 271). In some embodiments, the P2A self-cleaving peptide comprises a sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 192). In some embodiments, the E2A self-cleaving peptide comprises a sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 272). In some embodiments, the F2A selfcleaving peptide comprises a sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 273).

[0364] Methods of activating and culturing populations immune cells comprising the receptors, polynucleotides, or vectors of the disclosure will be readily apparent to the person of ordinary skill in the art.

[0365] Whether prior to or after genetic modification, the immune cells of the present disclosure can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, 10,040,846; and U.S. Pat. Appl. Pub. No. 2006/0121005, each of which are incorporated herein by reference.

[0366] Immune cells of the instant disclosure can be expanded and activated in vitro. Generally, the immune cells of the instant disclosure are expanded in vitro by contact with a surface having an attached agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the immune cells. Immune cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody. For co-stimulation of an accessory molecule on the surface of the immune cells, a ligand that binds the accessory molecule can be used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate to stimulate proliferation of the T cells. In order to stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art, such as in Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; and Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999, each of which is incorporated herein by reference.

[0367] The primary stimulatory signal and the co-stimulatory signal for an immune cell of the disclosure may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. The agent providing the co-stimulatory signal may be bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. Both agents can be in solution. The agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. U.S. Patent Application Publication Nos. 2004/0101519 and 2006/0034810, which are incorporated herein by reference, disclose artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding immune cells of the present invention.

[0368] The two agents may be immobilized on beads, either on the same bead, i.e., "cis," or to separate beads, i.e., "trans." By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts.

[0369] In certain methods of the disclosure, a 1:1 ratio of each antibody bound to the beads for CD4+ immune cell expansion and growth is used. The ratio of CD3:CD28 antibody bound to the beads may range from 100:1 to 1:100, and all integer values there between. In one aspect of the present disclosure, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain methods of the disclosure, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1.

[0370] Ratios of particles to cells from 1:500 to 500:1, and any integer values in between, may be used to stimulate immune cells, such as T cells, or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads may only bind a few cells, while larger beads can bind many.

[0371] In certain methods of the disclosure, the ratio of cells to particles ranges from 1:100 to 100:1, and any integer values in-between, can be used to stimulate the immune cells. In certain methods of the disclosure, the ratio comprises 1:9 to 9:1 and any integer values in between. In certain methods, a ratio of 1:1 cells to beads may be used. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, the ratios used will vary depending on particle size and on cell size and type.

[0372] In further methods of the present disclosure, the immune cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. Alternatively, prior to culture, the agent-coated beads and cells are not separated, but are cultured together. The beads and cells may initially be concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0373] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached to contact the immune cells. The cells (for example, CD4+ T cells) and beads (for example, DYNABEADS CD3/CD28 T paramagnetic beads at a ratio of 1:1) are combined in a buffer. Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. In certain methods, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, a concentration of about 2 billion cells/ml can be used. Alternatively, greater than 100 million cells/ml can be used. A concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml can be used. A concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml can be used. Concentrations of 125 or 150 million cells/ml can be used. In certain methods, cells are cultured at a density of 1.times.10.sup.6 cells/mL.

[0374] The mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. The beads and immune cells may be cultured together for 2-3 days. Conditions appropriate for immune cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-.gamma., IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF.beta., and TNF-.alpha. or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, .alpha.-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of immune cells. The media may comprise XVIVO-15 media supplemented with 5% human AB serum, 1% penicillin/streptomycin (pen/strep) and 300 Units/ml of IL-2 (Miltenyi).

[0375] The engineered immune cells can be maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37.degree. C.) and atmosphere (e.g., air plus 5% CO2).

[0376] Immune cells comprising receptors of the present disclosure may be autologous. Prior to expansion and genetic modification, a source of immune cells can obtained from a subject, such as a human patient. Immune cells, such as T cells, can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0377] In certain methods of the disclosure, any number of immune cell lines available in the art, may be used. Immune cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll.TM. separation.

[0378] Cells from the circulating blood of an individual can be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. The cells can be washed with phosphate buffered saline (PBS). The wash solution may lack calcium and magnesium or may lack many, if not all, divalent cations. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0379] Immune cells, such as T cells, can be isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL.TM. gradient or by counterflow centrifugal elutriation. Specific subpopulations of immune cells, such as T cells, B cells, or CD4+ T cells can be further isolated by positive or negative selection techniques. For example, T cells can be isolated by incubation with anti-CD4-conjugated beads, for a time period sufficient for positive selection of the desired T cells.

[0380] Enrichment of an immune cell population, such as a T cell population, by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immune-adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0381] For isolation of a desired population of immune cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads.

[0382] The cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10.degree. C. or at room temperature.

[0383] T cells for stimulation, or PBMCs from which immune cells such as T cells are isolated, can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80.degree. C. at a rate of 1.degree. per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20.degree. C. or in liquid nitrogen.

[0384] Exemplary immune cells and methods for producing the same include those that have been disclosed by the present Inventors in PCT International Application Nos. PCT/US2019/037038, PCT/US2020/045250, PCT/US2020/045228, PCT/US2020/045373, and PCT/CA2016/051421, and U.S. Provisional Application Nos. 62/946,888, 62/934,419, 63/076,123, 63/068,244, 63/068,249, 63/068,245, 63/068,246, 63/065,324, and 63/037,975, which are each incorporated herein by reference.

[0385] Target Ligands

[0386] The disclosure provides receptors comprising extracellular ligand binding domains. The ligand may be an antigen and the ligand binding domain may be an antigen binding domain.

[0387] Any suitable ligand binding domain is envisaged as within the scope of the receptors described herein.

[0388] The ligand binding domain of the activating or blocking receptors may comprise an antigen binding domain comprises an antibody fragment, a VP only domain, a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb).

[0389] The receptors may each comprise two polypeptides each having a part of a ligand-binding domain (e.g. cognates of a heterodimeric LDB, such as a TCR.alpha./.beta.- or Fab-based LBD). The disclosure further provides receptors having two polypeptides, each having a part of a ligand-binding domain (e.g. cognates of a heterodimeric LDB, such as a TCR.alpha./.beta.- or Fab-based LBD) and one part of the ligand binding domain is fused to a hinge or transmembrane domain, while the other part of the ligand binding domain has no intracellular domain. Further variations include receptors where each polypeptide has a hinge domain, and where each polypeptide has a hinge and transmembrane domain. Some receptors may not have a hinge domain.

[0390] The ligand binding domain of the receptors may comprise a Fab fragment of an antibody.

[0391] Receptors of the present disclosure may comprise a first polypeptide that comprises an antigen-binding fragment of the heavy chain of an antibody and an intracellular domain, and a second polypeptide of the receptor comprises an antigen-binding fragment of the light chain of the antibody. Alternatively, the first polypeptide may comprise an antigen-binding fragment of the light chain of the antibody and the intracellular domain, and the second polypeptide comprises an antigen-binding fragment of the heavy chain of the antibody.

[0392] The blocking and/or activating receptors may comprise an extracellular fragment of a T cell receptor (TCR).

[0393] Any macromolecule, including virtually all proteins or peptides, can serve as an antigen for the receptors described herein. Antigens can be derived from recombinant or genomic DNA. Any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response, encodes an antigen. An antigen need not be encoded solely by a full-length nucleotide sequence of a gene. An antigen need not be encoded by a gene. An antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.

[0394] In the engineered receptors of the present disclosure, the antigen-binding domain may specifically bind to a target selected from etiolate receptor, av.beta..beta. integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, ErbB2 (HER2), ErbB3 (Her3), ErbB4 (Her4), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11R, IL13R-a2, KDR, .kappa. light chain, .lamda., light chain, LeY, LI CAM, MAGE-A1, mesothelin, MHC presented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notch1, Notch2/3, NYESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2, and ROR1.

[0395] The antigen-binding domain may specifically bind peptide MHC (pMHC) as the antigen. Exemplary pMHC antigens include, but are not limited to, MAGE-A3 pMHC (e.g., FLWGPRALV and MPKVAELVHFL peptides), HPV E6 pMHC (e.g., TIHDIILECV peptide), HPV E7 pMHC (e.g., YMLDLQPET peptide) and NY-ESO-1 pMHC (e.g., LLEFYLAMPFA or SLLMWITQV peptides).

[0396] The antigen-binding domain may specifically bind to a target selected from CD33, CD38, a human leukocyte antigen (HLA), an organ specific antigen, a blood-brain barrier specific antigen, an Epithelial-mesenchymal transition (EMT) antigen, E-cadherin, cytokeratin, Opioid-binding protein/cell adhesion molecule (OPCML), HYLA2, Deleted in Colorectal Carcinoma (DCC), Scaffold/Matrix attachment region-binding protein 1 (SMAR1), cell surface carbohydrate and mucin type 0-glycan.

[0397] The antigen-binding domain of the blocking receptor may specifically bind to an antigen from a gene with high, homogeneous surface expression across tissues. High, homogeneous surface expression across tissues allows the blocking receptor to deliver a large, even inhibitory signal. The antigen may be encoded by a gene that is absent or polymorphic in in many tumors.

[0398] Methods of distinguishing the differential expression of blocking ligands (e.g., antigens) between target and non-target cells can be used in methods and systems of the invention. For example, the presence or absence of blocking ligands in nontarget and target cells can be assayed by immunohistochemistry with an antibody that binds to the inhibitor ligand, followed by microscopy or FACS, RNA expression profiling of target cells and non-target cells, or DNA sequencing of non-target and target cells to determine if the genomic locus of the blocking ligand comprises mutations in either the target or non-target cells.

[0399] Homozygous deletions in primary tumors are rare and small, and therefore unlikely to yield blocking ligand candidates. For example, in an analysis of 2218 primary tumors across 21 human cancer types, the top 4 candidates were CDKN2A, RB1, PTEN and N3PB2. However, CDKN2A (P16) was deleted in only 5% homozygous deletion across all cancers. Homozygous HLA-A deletions were found in less than 0.2% of cancers in Cheng et al., Nature Comm. 8:1221 (2017), incorporated herein by reference. In contrast, deletion of a single copy of gene in cancer cells due to loss of hemizygosity occurs far more frequently.

[0400] Thus, the blocking ligand may comprise an allele of a gene that is lost in target cells due to loss of heterozygosity, and the target cells may comprise cancer cells. Cancer cells undergo frequent genome rearrangements, including duplication and deletions. These deletions can lead to the deletion of one copy of one or more genes in the cancer cells.

[0401] Loss of heterozygosity (LOH) refers to a genetic change, whereby one of the two alleles in the genome of a cell or cells is deleted, leaving a single mono-allelic (hemizygous) locus.

[0402] The blocking ligand may comprise an HLA class I allele. The major histocompatibility complex (MHC) class I is a gene complex that encodes proteins that display antigens to cells of the immune system, triggering immune response. The Human Leukocyte Antigens (HLAs) corresponding to MHC class I are HLA-A, HLA-B and HLA-C.

[0403] The blocking ligand may comprise an HLA class I allele. The blocking ligand may comprise an allele of HLA class I that is lost in a target cell through LOH. HLA-A is a group of human leukocyte antigens (HLA) of the major histocompatibility complex (MHC) that are encoded by the HLA-A locus. HLA-A is one of three major types of human MHC class I cell surface receptors. The receptor is a heterodimer comprising a heavy .alpha. chain and smaller .beta. chain. The .alpha. chain is encoded by a variant of HLA-A, while the .beta. chain (.beta.2-microglobulin) is an invariant. There are several thousand variant HLA-A alleles, all of which fall within the scope of the instant disclosure.

[0404] The blocking ligand may comprise an HLA-B allele. The HLA-B gene has many possible variations (alleles). Hundreds of versions (alleles) of the HLA-B gene are known, each of which is given a particular number (such as HLAB27).

[0405] The blocking ligand may comprise an HLA-C allele. HLA-C belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). Over one hundred HLA-C alleles have been described.

[0406] The HLA class I allele may have broad or ubiquitous RNA expression. The HLA class I allele may have a known, or generally high minor allele frequency. The HLA class I allele may not require a peptide-MHC antigen, for example when the HLA class I allele is recognized by a pan-HLA ligand binding domain.

[0407] The blocking ligand may comprise an HLA-A allele. The HLA-A allele may comprise HLA-A*02. Various single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*02 are suitable for use in the present disclosure. Such scFvs include, for example and without limitation the following mouse and humanized scFv antibodies that bind HLA-A*02 in a peptide independent manner.

[0408] The blocking ligand (e.g., an antigen) may comprise a minor histocompatibility antigen (MiHA). The inhibitor ligand may comprise an allele of a MiHA that is lost in a target cell through LOH.

[0409] MiHAs are peptides derived from proteins that contain nonsynonymous differences between alleles and are displayed by common HLA alleles. The nonsynonymous differences can arise from SNPs, deletions, frameshift mutations or insertions in the coding sequence of the gene encoding the MiHA. Exemplary MiHAs can be about 9-12 amino acids in length and can bind to WIC class I and/or WIC class II proteins. Binding of the TCR to the MHC complex displaying the MiHA can activate T cells. The genetic and immunological properties of MiHAs will be known to the person of ordinary skill in the art. Candidate MiHAs are known peptides presented by known HLA class I alleles, are known to elicit T cell responses in the clinic (for example, in graft versus host disease, or transplant rejection), and allow for patient selection by simple SNP genotyping.

[0410] The MiHA may have broad or ubiquitous RNA expression. The MiHA may have high minor allele frequency. The MiHA may comprise a peptide derived from a Y chromosome gene.

[0411] The blocking ligand may comprise a Y chromosome gene, i.e. peptide encoded by a gene on the Y chromosome. The blocking ligand may comprise a peptide encoded by a Y chromosome gene that is lost in target cells through loss of Y chromosome (LoY). For example, about a third of the characterized MiHAs come from the Y chromosome. The Y chromosome contains over 200 protein coding genes, all of which are envisaged as within the scope of the instant disclosure.

[0412] As used herein, "loss of Y", or "LoY" refers a genetic change that occurs at high frequency in tumors whereby part or all of the Y chromosome is deleted, leading to a loss of Y chromosome encoded gene(s).

[0413] Loss of Y chromosome is known to occur in certain cancers. For example, there is a reported 40% somatic loss of Y chromosome in renal clear cell cancers (Arseneault et al., Sci. Rep. 7: 44876 (2017)). Similarly, clonal loss of the Y chromosome was reported in 5 out of 31 in male breast cancer subjects in Wong et al., Oncotarget 6(42):44927-40 (2015), incorporated herein by reference. Loss of the Y chromosome in tumors from male patients has been described as a "consistent feature" of head and neck cancer patients, as in el-Naggar et al., Am J Clin Pathol 105(1):102-8 (1996), incorporated herein by reference. Further, Y chromosome loss was associated with X chromosome disomy in four of seven male patients with gastric cancer in Saal et al., Virchows Arch B Cell Pathol (1993), incorporated herein by reference. Thus, Y chromosome genes can be lost in a variety of cancers, and can be used as blocking ligands with the engineered receptors of the instant disclosure targeting cancer cells.

[0414] The activating ligand may be a transferrin receptor (TFRC). Human transferrin receptor is described in NCBI record No. AAA61153.1, the contents of which are incorporated herein by reference.

[0415] The activating ligand may be a tumor specific antigen (TSA). The tumor specific antigen may be mesothelin (MSLN), CEACAM5 or EGFR. The TSA may be MSLN, CEA, EGFR, DLL4, CA125, GD2, ROR1 or HER2/NEU. The activating ligand may be a pan-HLA ligand, and the activating receptor ligand binding domain is a pan-HLA binding domain, i.e. a binding domain that binds to and recognizes an antigenic determinant shared among HLA I products, such as the HLA A, B and C loci. The activating ligand may also be another class I gene product; e.g., antigens encoded by HLA-E or F. Various single variable domains known in the art are suitable for use in embodiments. Such scFvs include, for example and without limitation the following mouse and humanized pan-HLA scFv antibodies. An exemplary pan-HLA ligand is W6/32, which recognizes a conformational epitope, reacting with HLA class I alpha3 and alpha2 domains. Further exemplary antibodies with broad HLA binding are known in the art and include HC-10 and TFL-006. Exemplary activating ligands and activating receptor ligand binding domains have been disclosed by the Inventors of the present disclosure in U.S. Provisional Application No. 63/018,881, which is herein incorporated by reference in its entirety.

[0416] Exemplary ligands and ligand binding domains of activating and blocking receptors include those that have been disclosed by the present Inventors in PCT International Application Nos. PCT/US2019/037038, PCT/US2020/045250, PCT/US2020/045228, PCT/US2020/045373, and PCT/CA2016/051421, and U.S. Provisional Application Nos. 62/946,888, 62/934,419, 63/076,123, 63/068,244, 63/068,249, 63/068,245, 63/068,246, 63/065,324, and 63/037,975, which are each incorporated herein by reference.

[0417] For example, in some embodiments of the immune cells of the disclosure, a first/activating ligand is EGFR or a peptide antigen thereof, and the first/activating ligand binding domain comprises a sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, or SEQ ID NO: 391, or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the first ligand binding domain comprises CDRs selected from SEQ ID NOs: 131-166.

[0418] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR binding domain. In some embodiments, the EGFR ligand binding domain comprises an ScFv domain. In some embodiments, the EGFR ligand binding domain comprises a sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118 or SEQ ID NO: 391. In some embodiments, the EGFR ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118 or SEQ ID NO: 391. In some embodiments, the EGFR ligand binding domain is encoded by a sequence comprising SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117 or SEQ ID NO: 119. In some embodiments, the EGFR ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117 or SEQ ID NO: 119.

[0419] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain comprises an EGFR ligand binding domain. In some embodiments, the EGFR binding domain comprises a VH and/or a VL domain selected from the group disclosed in Table 2 or a sequence having at least 90% identity thereto. In some embodiments, the EGFR ligand binding domain comprises a VH domain selected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128 and SEQ ID NO: 130. In some embodiments, the EGFR ligand binding domain comprises a VH selected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128 and SEQ ID NO: 130 or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the EGFR ligand binding domain comprises a VL domain selected from the group consisting of SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129 and SEQ ID NO: 131. In some embodiments, the EGFR ligand binding domain comprises a VH selected from the group consisting of SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129 and SEQ ID NO: 131 or a sequence having at least 90%, at least 95% or at least 99% identity thereto.

[0420] In some embodiments, the activator ligand is EGFR or a peptide antigen thereof, and the activator ligand binding domain is an EGFR ligand binding domain. In some embodiments, the EGFR binding domain comprises complementarity determining region (CDRs) selected from the group of CDRs disclosed in Table 3. In some embodiments, the EGFR ligand binding domain comprises CDRs having at least 95% sequence identity to CDRs disclosed in Table 3. In some embodiments, the EGFR ligand binding domain comprises CDRs selected from SEQ ID NOs: 131-166. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 1 (CDR HI) selected from the group consisting of SEQ ID NOs: 132-137. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 2 (CDR H2) selected from the group consisting of SEQ ID NOs: 138-143. In some embodiments, the EGFR ligand binding domain comprises a heavy chain CDR 3 (CDR H3) selected from the group consisting of SEQ ID NOs: 144-149. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 1 (CDR LI) selected from the group consisting of SEQ ID NOs: 150-155. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 2 (CDR L2) selected from the group consisting of SEQ ID NOs: 156-160. In some embodiments, the EGFR ligand binding domain comprises a light chain CDR 3 (CDR L3) selected from the group consisting of SEQ ID NOs: 161-166. In some embodiments, the EGFR ligand binding domain comprises a CDR HI selected from SEQ ID NOs: 132-137, a CDR H2 selected from SEQ ID NOs: 138-143, a CDR H3 selected from SEQ ID NOs: 144-149, a CDR LI selected from SEQ ID NOs: 150-155, a CDR L2 selected from SEQ ID NOs: 156-160, and a CDR L3 selected from SEQ ID NOs: 156-160.

[0177] Table 3. EGFR antigen binding domain CDRs.

[0421] In some embodiments of the immune cells of the disclosure, the first/activating ligand is MSLN or a peptide antigen thereof. In some embodiments, the first/activating ligand binding domain comprises a sequence of SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90 or SEQ P) NO: 92, or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the MSLN ligand binding domain is encoded by a sequence comprising SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 or SEQ ID NO: 93. In some embodiments, the MSLN ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 or SEQ ID NO: 93.

[0422] In some embodiments of the immune cells of the disclosure, the first/activating ligand is CEA or a peptide antigen thereof. In some embodiments, the first/activating ligand binding domain comprises SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 282, SEQ ID NO: 284, or SEQ ID NO: 286, or a sequence having at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the first/activating ligand binding domain comprises CDRs selected from SEQ ID NOs: 294-302. In some embodiments, the CEA ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287.

[0423] In some embodiments, the activator ligand is CEA or a peptide antigen thereof, and the activator ligand binding domain comprises a CEA binding domain. In some embodiments, the CEA ligand binding domain comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDF AYYVEAMD Y (SEQ ID NO: 296) or WDFAHYFQTMDY (SEQ ID NO: 297), a CDR-L1 of KASQNVGTNV A (SEQ ID NO: 298) or KASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRYS (SEQ ID NO: 300) or SASYRKR (SEQ ID NO: 301), and a CDR-L3 of HQ YYTYPLFT (SEQ ID NO: 302) or sequences having at least 85% or at least 95% identity thereto. In some embodiments, a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDF AYYVEAMD Y (SEQ ID NO: 296) or WDFAHYFQTMDY (SEQ ID NO: 297), a CDR-L1 of KASQNVGTNV A (SEQ ID NO: 298) or KASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRYS (SEQ ID NO: 300) or SASYRKR (SEQ ID NO: 301) and a CDR-L3 of HQ YYTYPLFT (SEQ ID NO: 302).

[0424] In some embodiments, a CEA binding domain comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296), a CDR-L1 of KASQNVGTNV A (SEQ ID NO: 298), a CDR-L2 of SASYRYS (SEQ ID NO: 300) and a CDR-L3 of HQ YYTYPLFT (SEQ ID NO: 302). In some embodiments, a CEA ScFv comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEATYVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAYYVEAMDY (SEQ ID NO: 296), a CDR-L1 of KASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRKR, and a CDR-L3 of HQ YYTYPLFT (SEQ ID NO: 302). In some embodiments, a CEA binding domain comprises a CDR-H1 of EFGMN (SEQ ID NO: 294), a CDR-H2 of WINTKTGEAT YVEEFKG (SEQ ID NO: 295), a CDR-H3 of WDFAHYFQTMD Y (SEQ ID NO: 297), a CDR-L1 of KASAAVGTYVA (SEQ ID NO: 299), a CDR-L2 of SASYRKR, and a CDR-L3 of HQ YYTYPLFT (SEQ ID NO: 302).

[0425] In some embodiments, the activator ligand is CEA or a peptide antigen thereof, and the activator receptor is a CEA CAR In some embodiments, the CEA CAR comprises sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 288, SEQ ID NO: 290 or SEQ ID NO: 292. In some embodiments, the CEA CAR comprises or consists essentially of SEQ ID NO: 288, SEQ ID NO: 290 or SEQ ID NO: 292. In some embodiments, the CEA CAR is encoded by a sequence comprising or consisting essentially of SEQ ID NO: 289, SEQ ID NO: 291 or SEQ ID NO: 293. In some embodiments, the CEA CAR is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to SEQ ID NO: 289, SEQ ID NO: 291 or SEQ ID NO: 293.

[0426] In some embodiments of the immune cells of the disclosure, the first/activating ligand is CD19 or a peptide antigen thereof, and the first ligand binding domain comprises SEQ ID NO: 275 or SEQ ID NO: 277, or a sequence having at least 90%, at least 95% or at least 99% identity thereto.

[0427] In some embodiments of the immune cells of the disclosure, the first/activating ligand is a pan-HLA ligand. In some embodiments, the first ligand binding domain comprises a sequence of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177, or a sequence having at least 90%, at least 95% or at least 99% identity thereto.

[0428] In some embodiments of the immune cells of the disclosure, the second/blocking ligand comprises HA-1. In some embodiments, and wherein the second/blocking ligand binding domain comprises a TCR alpha variable domain comprising SEQ ID NO: 199 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto, and a TCR beta variable domain comprising SEQ ID NO: 200 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the second/blocking ligand binding domain comprises a TCR alpha variable domain comprising SEQ ID NO: 199, and a TCR beta variable domain comprising SEQ ID NO: 200.

[0429] In some embodiments of the immune cells of the disclosure, the second/blocking ligand comprises an HLA-A*02 allele. In some embodiments, the second/blocking ligand binding domain comprises any one of SEQ ID NOs: 53-64 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the second/blocking ligand binding domain comprises CDRs selected from SEQ ID NOs: 41-52.

[0430] In some embodiments of the inhibitory/blocking receptors of the disclosure, the extracellular ligand binding domain has a higher affinity for an HA-1 (H) peptide of VLHDDLLEA (SEQ ID NO: 191) than for an HA-1(R) peptide of VLRDDLLEA (SEQ ID NO: 266). In some embodiments, the inhibitory/blocking receptor is activated by the HA-1(H) peptide of VLHDDLLEA (SEQ ID NO: 191) and is not activated, or activated to a lesser extent, by the HA-1(R) peptide of VLRDDLLEA (SEQ ID NO: 266). In some embodiments, the extracellular ligand binding domain comprises a TCR alpha variable domain comprising SEQ ID NO: 199 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto, and a TCR beta variable domain comprising SEQ ID NO: 200 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the extracellular ligand binding domain comprises a TCR alpha variable domain comprising SEQ ID NO: 199 and a TCR beta variable domain comprising SEQ ID NO: 200.

[0431] In some embodiments, the activator ligand is pan-HLA ligand, and the activator ligand binding domain comprises a pan-HLA ligand binding domain. In some embodiments, the pan-HLA ligand binding domain comprises an ScFv domain. In some embodiments, the pan-HLA ligand binding domain comprises a sequence of SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177. In some embodiments, the pan-HLA ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, or SEQ ID NO: 177. In some embodiments, the pan-HLA ligand binding domain is encoded by a sequence comprising SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, or SEQ ID NO: 178. In some embodiments, the pan-HLA ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, or SEQ ID NO: 178.

[0181] In some embodiments, the activator ligand is CD19 molecule (CD19) or a peptide antigen thereof, and the activator ligand binding domain comprises a CD 19 ligand binding domain. In some embodiments, the CD 19 ligand binding domain comprises an ScFv domain. In some embodiments, the CD 19 ligand binding domain comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 275 or SEQ ID NO: 277. In some embodiments, the CD-19 ligand binding domain comprises a sequence of SEQ ID NO: 275 or SEQ ID NO: 277. In some embodiments, the CD19 ligand binding domain is encoded by a sequence comprising SEQ ID NO: 276, or SEQ ID NO: 278. In some embodiments, the CD19 ligand binding domain is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 276 or SEQ ID NO: 278.

[0432] In some embodiments, activator ligand is CD19 molecule (CD19) or a peptide antigen thereof, and the activator receptor is a CAR In some embodiments, the CD 19 CAR comprises a sequence at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 279 or SEQ ID NO: 281. In some embodiments, the CD 19 CAR comprises or consists essentially of SEQ ID NO: 279 or SEQ ID NO: 281. In some embodiments, the CD19 CAR is encoded by a sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a sequence of SEQ ID NO: 280 or SEQ P) NO: 390. In some embodiments, the CD19 CAR is encoded by a sequence comprising or consisting essentially of SEQ ID NO: 280 or SEQ ID NO: 390.

[0433] In some embodiments, the one or more ligand comprises an HLA-A allele. In some embodiments the HLA-A allele comprises HLA-A*02. Various single variable domains known in the art or disclosed herein that bind to and recognize HLA-A*02 are suitable for use in embodiments. Such scFvs include, for example and without limitation, the following mouse and humanized scFv antibodies that bind HLA-A*02 in a peptide-independent way, which include binding domains having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to any one of SEQ ID NOS: 179-190.

[0434] In some embodiments, the scFv comprises the complementarity determined regions (CDRs) of any one of SEQ ID NOS: 41-52. In some embodiments, the scFv comprises a sequence at least 95% identical to any one of SEQ P) NOS: 41-52. In some embodiments, the scFv comprises a sequence identical to any one of SEQ ID NOS: 41-52. In some embodiments, the heavy chain of the antibody comprises the heavy chain CDRs of any one of SEQ ID NOS: 53-64, and wherein the light chain of the antibody comprises the light chain CDRs of any one of SEQ ID NOS: 53-64. In some embodiments, the heavy chain of the antibody comprises a sequence at least 95% identical to the heavy chain portion of any one of SEQ ID NOS: 53-64, and wherein the light chain of the antibody comprises a sequence at least 95% identical to the light chain portion of any one of SEQ ID NOS: 53-64.

[0209] In some embodiments, the heavy chain of the antibody comprises a sequence identical to the heavy chain portion of any one of SEQ ID NOS: 53-64, and wherein the light chain of the antibody comprises a sequence identical to the light chain portion of any one of SEQ ID NOS: 53-64.

[0435] In some embodiments, a ligand as used herein comprises a minor histocompatibility antigen (MiHA). In some embodiments, the blocking ligand comprises an allele of a MiHA that is lost in a target cell through LOH. Exemplary, but non-limiting, examples of MiHAs that are envisaged as within the scope of the instant invention include those having the sequence of any one of SEQ ID NOS: 273, 303-325, 327-356, 358-389, 34, and 23-25.

[0436] Exemplary ligand binding domains that selectively bind to HA-1 variant H peptide (VLHDDLLEA (SEQ ID NO: 191)) include the sequences of SEQ ID NO: 194, 201, 202, 196, and 198. TCR alpha and TCR beta sequences in SEQ ID NO: 193 are separated by a P2A self-cleaving polypeptide of sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 192) with an N terminal GSG linker.

[0437] In some embodiments, the TCR alpha and TCR beta variable domains separated by a self-cleaving polypeptide sequence comprise SEQ ID NO: 193, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR alpha and TCR beta variable domains are encoded by a sequence of SEQ ID NO: 194, or a sequence having at least 80% identity, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR alpha variable domain comprises SEQ ID NO: 199 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the TCR beta variable domain comprises SEQ ID NO: 200 or a sequence having at least 90%, at least 95%, or at least 99% identity thereto.

[0438] In some embodiments, the first or second ligand binding domain comprises a sequence of any one of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222 Or SEQ ID NO: 224, or a sequence having at least 90%, at least 95% or at least 99% identity thereto.

[0439] It will be appreciated by the person of ordinary skill that first, activator ligand binding domains for the first receptor may be isolated or derived from any source known in the art, including, but not limited to, art recognized T cell receptors, chimeric antigen receptors and antibody binding domains.

[0440] Methods of Treatment

[0441] The present disclosure provides methods of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the engineered immune cells of the present disclosure.

[0442] The engineered immune cells of the present disclosure may be used to treat a subject that has cancer. The cancer may comprise a liquid tumor or a solid tumor. Exemplary liquid tumors include leukemias and lymphomas. Further cancers that are liquid tumors can be those that occur, for example, in blood, bone marrow, and lymph nodes, and can include, for example, leukemia, myeloid leukemia, lymphocytic leukemia, lymphoma, Hodgkin's lymphoma, melanoma, and multiple myeloma. Leukemias include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and hairy cell leukemia. Exemplary solid tumors include sarcomas and carcinomas. Cancers can arise in virtually an organ in the body, including blood, bone marrow, lung, breast, colon, bone, central nervous system, pancreas, prostate and ovary. Further cancers that are solid tumors include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer, squamous cell skin cancer, renal cancer, head and neck cancers, throat cancer, squamous carcinomas that form on the moist mucosal linings of the nose, mouth, throat, bladder cancer, osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In some embodiments, the condition treated by the methods described herein is metastasis of melanoma cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroid cancer cells, stomach cancer cells, lung cancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin cancer cells, renal cancer cells, head or neck cancer cells, throat cancer cells, squamous carcinoma cells, bladder cancer cells, osteosarcoma cells, cervical cancer cells, endometrial cancer cells, esophageal cancer cells, liver cancer cells, or kidney cancer cells.

[0443] Treating cancer with the engineered immune cells of the present disclosure can result in a reduction in size of a tumor. A reduction in size of a tumor may also be referred to as "tumor regression". Preferably, after treatment, tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.

[0444] Treating cancer with the engineered immune cells of the present disclosure can result in a reduction in tumor volume. Preferably, after treatment, tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Tumor volume may be measured by any reproducible means of measurement.

[0445] Treating cancer using the engineered immune cells of the present disclosure may result in a decrease in number of tumors. Preferably, after treatment, tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. Number of tumors may be measured by any reproducible means of measurement. The number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2.times., 3.times., 4.times., 5.times., 10.times., or 50.times..

[0446] Treating cancer with the engineered immune cells of the present disclosure can result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2.times., 3.times., 4.times., 5.times., 10.times., or 50.times..

[0447] Treating cancer with the engineered immune cells of the present disclosure can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0448] Treating cancer with the engineered immune cells can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0449] Treating cancer with the engineered immune cells can result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0450] Treating cancer with the engineered immune cells can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.

[0451] Treating cancer with the engineered immune cells can result in a decrease in tumor growth rate. Preferably, after treatment, tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.

[0452] Treating cancer with the engineered immune cells can result in a decrease in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped.

[0453] Treating or preventing a cell proliferative disorder with the engineered immune cells can result in a reduction in the rate of cellular proliferation. Preferably, after treatment, the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of cellular proliferation may be measured by any reproducible means of measurement. The rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

[0454] Treating or preventing a cell proliferative disorder with the engineered immune cells can result in a reduction in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The proportion of proliferating cells may be measured by any reproducible means of measurement. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample. The proportion of proliferating cells can be equivalent to the mitotic index.

[0455] Treating or preventing a cell proliferative disorder with the engineered immune cells can result in a decrease in size of an area or zone of cellular proliferation. Preferably, after treatment, size of an area or zone of cellular proliferation is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement. The size of an area or zone of cellular proliferation may be measured as a diameter or width of an area or zone of cellular proliferation.

[0456] Treating or preventing a cell proliferative disorder with the engineered immune cells can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement. An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope. An abnormal cellular morphology can take the form of nuclear pleiomorphism.

[0457] Exemplary methods of treatment and conditions to be treated using the cells of the present invention including those that have been disclosed by the present Inventors in PCT International Application Nos. PCT/US2019/037038, PCT/US2020/045250, PCT/US2020/045228, PCT/US2020/045373, and PCT/CA2016/051421, and U.S. Provisional Application Nos. 62/946,888, 62/934,419, 63/076,123, 63/068,244, 63/068,249, 63/068,245, 63/068,246, 63/065,324, and 63/037,975, which are each incorporated herein by reference.

EXAMPLES

Example 1

[0458] FIG. 4 provides experimental results showing that, for the engineered immune cells of the present disclosure expressing activating and blocking receptors, surface levels of the activating receptor decrease when the immune cells are in the presence of non-target cells expressing both the activating and blocking ligand.

[0459] FIGS. 5-7 provides experimental results showing that this reduced surface expression of the activating receptors corresponds with the ability of the immune cells to kill other cells. Thus, advantageously, when the immune cells are in limited or no contact with target cells, their ability to kill is diminished, thereby reducing non-target effects.

Example 2

[0460] Another surprising facet of the immune cells of the present invention is that the reduced surface expression of the activating receptors only occurs when the immune cells contact non-target cells expressing both the activating and blocking ligands. This is shown in FIG. 7.

Example 3

[0461] FIGS. 8-16 provide an experimental protocol and results that indicate the reduced expression of activating receptors is reversible upon contact with target cells.

Example 4

[0462] FIGS. 17-19 provide experimental results showing that, unlike the activating receptor, the blocking receptor does not undergo reduced surface expression in an appreciable amount in the presence of non-target cells.

Example 5

[0463] FIG. 22 shows experimental results indicating that the blocking receptor provides a blocking signal that dominates and inhibits the activating signal from the activating receptor.

[0464] Jurkat cells were transfected with either an activating receptor (MP1-CAR) for a MAGE-A3 activating ligand or the activating receptor and a blocking receptor (ESO-Tmod) for a NY-ESO-1 blocking ligand.

[0465] Panel A shows the NFAT-luciferase signal of Jurkat cells transfected with either the activator alone or in combination with the blocker, after 6 hours of co-culture with activator and blocker peptide-loaded T2 cells. The T2 cells were loaded with titrated amounts of activator MAGE-A3 peptide and a fixed amount of blocker NY-ESO-1 peptide concentration. This reveals the activation dose-response of the transfected cells.

[0466] Panel B shows the NFAT-luciferase signal of Jurkat cells transfected with either the activator alone or in combination with the blocker, after 6 hours of co-culture with activator and blocker peptide-loaded T2 cells. The T2 cells were loaded with titrated amounts of blocker NY-ESO-1 peptide and a fixed amount of activator MAGE-A3 peptide concentration above the Emax concentration (.about.0.1 .mu.M). This reveals the inhibition dose-response of the transfected cells.

[0467] Panel C shows the NFAT-luciferase signal of Jurkat cells transfected with either the activator alone or in combination with the blocker, after 6 hours of co-culture with activator and blocker peptide-loaded T2 cells. The x-value blocker NY-ESO-1 peptide concentrations from panel B were normalized to the constant activator MAGE peptide concentrations used for each curve and plotted on the x-axis. The ratio of blocker peptide to activator peptide required for 50% blocking (IC50) are indicated for each curve. The B:A peptide ratio required is less than 1 indicating that, for this pair of activator CAR and blocker, similar (or fewer) blocker pMHC antigens may be sufficient on target cells to block activator pMHC antigens.

[0468] Since this ratio is less than 1, it can be inferred that the blocking signal dominates and inhibits the activating signal. Thus, a single blocking receptor can provide a blocking signal of sufficient strength to inhibit the activating signal of one or more activating receptors. As such, the quantity of activating and blocking ligands expressed by a non-target cell can form part of the basis for determining the appropriate relative amounts of activating and blocking receptors that should be expressed by an immune cell of the disclosure.

Example 6

[0469] FIG. 23 provides experimental results showing that the blocking receptors are ligand-dependent. For both CAR and TCR activating receptors, blocking receptors had minimal ligand-independent blocking activity. This impact is shown by the minimal effect on the EC.sub.50 of the activating receptors by the blocking receptor in the presence/absence of the blocking ligand.

Example 7

[0470] FIG. 25 provides experimental results showing the relative impact hinge length and flexibility has on the strength of a blocking receptor as a function of the EC.sub.50 of the activating receptor.

Example 8

[0471] FIG. 26 shows the large relative impact of the LBD on the activating receptor's structure activity relationship when compared with the effects provided by different hinges, transmembrane and intracellular domains. In this study, 45 separate activating receptors were created using various combinations of ligand binding domains, hinges, and intracellular domains. For each receptor one of five ligand binding domains that bind to the same activating ligand were selected. Despite all binding to the same target ligand, the identity of the ligand binding domain caused differences in the EC.sub.50 of the activating receptors that spanned orders of magnitude. The ligand binding domain was shown to have greater than 10.times. the impact on the receptors' EC.sub.50 compared to the hinge, transmembrane and intracellular domains.

Example 9

[0472] FIGS. 33-34 show the impact receptor cross-talk can have on the ability of the blocking receptor to inhibit the activation signal. Engineered immune cells were created with one of five different activating receptors. Though the activating receptors differed between the cells, each targeted the same activating ligand, epidermal growth factor receptor (EGFR), using a different LBD. As shown by five graph panels FIGS. 33-34, each of the different activating receptors provided the immune cells with equivalent abilities to kill target cells. Then, immune cells were created that had one of the five activating receptors and the same blocking receptor. Addition of the blocker caused some of the immune cells, like CT486-containing cells, to decrease their ability to kill target cells.

INCORPORATION BY REFERENCE

[0473] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

EQUIVALENTS

[0474] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Sequence CWU 1

1

391145PRTArtificial SequenceCD8alpha hinge 1Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala1 5 10 15Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 452135DNAArtificial SequenceCD8alpha hinge 2accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120gacttcgcct gtgat 135341PRTArtificial SequenceCD28 hinge 3Cys Thr Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys1 5 10 15Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser 20 25 30Pro Leu Phe Pro Gly Pro Ser Lys Pro 35 404123DNAArtificial SequenceCD28 hinge 4tgtaccattg aagttatgta tcctcctcct tacctagaca atgagaagag caatggaacc 60attatccatg tgaaagggaa acacctttgt ccaagtcccc tatttcccgg accttctaag 120ccc 123527PRTArtificial SequenceCD28 transmembrane domain 5Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu1 5 10 15Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25681DNAArtificial SequenceCD28 transmembrane domain 6ttctgggtgc tggtcgttgt gggcggcgtg ctggcctgct acagcctgct ggtgacagtg 60gccttcatca tcttttgggt g 81725PRTArtificial SequenceIL-2R beta transmembrane domain 7Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly1 5 10 15Phe Ile Ile Leu Val Tyr Leu Leu Ile 20 25875DNAArtificial SequenceIL-2R beta transmembrane domain 8attccgtggc tcggccacct cctcgtgggc ctcagcgggg cttttggctt catcatctta 60gtgtacttgc tgatc 759112PRTArtificial SequenceCD3 zeta activation domain 9Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 11010336DNAArtificial SequenceCD3 zeta activation domain 10agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gcgtagaggc 120cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240cggaggggca aggggcacga tggcctttac cagggactca gtacagccac caaggacacc 300tacgacgccc ttcacatgca ggccctgccc cctcgc 3361143PRTArtificial SequenceCD3 zeta activation domain 11Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu His Met Gln Ala Leu Pro Pro Arg 35 4012129DNAArtificial SequenceCD3 zeta activation domain 12agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60tataacgagc tcaatctagg acgaagagag gagtacgatg ttttgcacat gcaggccctg 120ccccctcgc 1291341PRTArtificial SequenceCD28 co-stimulatory domain 13Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr1 5 10 15Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 4014123DNAArtificial SequenceCD28 co-stimulatory domain 14aggagcaagc ggagcagact gctgcacagc gactacatga acatgacccc ccggaggcct 60ggccccaccc ggaagcacta ccagccctac gcccctccca gggatttcgc cgcctaccgg 120agc 1231594PRTArtificial SequenceIL-2-Rbeta intracellular domain 15Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn1 5 10 15Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly 20 25 30Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe 35 40 45Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu 50 55 60Arg Asp Lys Val Thr Gln Leu Leu Pro Leu Asn Thr Asp Ala Tyr Leu65 70 75 80Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 85 9016282DNAArtificial SequenceIL-2-Rbeta intracellular domain 16aactgcagga acaccgggcc atggctgaag aaggtcctga agtgtaacac cccagacccc 60tcgaagttct tttcccagct gagctcagag catggaggcg acgtccagaa gtggctctct 120tcgcccttcc cctcatcgtc cttcagccct ggcggcctgg cacctgagat ctcgccacta 180gaagtgctgg agagggacaa ggtgacgcag ctgctccccc tgaacactga tgcctacttg 240tctctccaag aactccaggg tcaggaccca actcacttgg tg 282174PRTArtificial SequenceSTAT5 recruitment motif 17Tyr Leu Ser Leu118760PRTHomo sapiens 18Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu1 5 10 15Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala 35 40 45Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly 50 55 60Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly65 70 75 80Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro 100 105 110Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys 115 120 125Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Ser Thr Ile 130 135 140Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln145 150 155 160Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe 165 170 175Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val 180 185 190Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg 195 200 205Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys225 230 235 240Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile 245 250 255Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu 260 265 270Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe 275 280 285Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly 290 295 300Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln305 310 315 320Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345 350Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser 355 360 365Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile 370 375 380Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp385 390 395 400His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala 405 410 415Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met 420 425 430Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr465 470 475 480Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val 485 490 495Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn 500 505 510Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp 515 520 525Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe 530 535 540Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp545 550 555 560Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585 590Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn 595 600 605Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp 610 615 620Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln625 630 635 640Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu 645 650 655Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys 660 665 670Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro 675 680 685Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690 695 700Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys705 710 715 720Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala 725 730 735Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp 740 745 750Val Trp Asp Ile Asp Asn Glu Phe 755 76019300DNAArtificial SequenceCD3 epsilon intracellular domain 19aagaatagaa aggccaaggc caagcctgtg acacgaggag cgggtgctgg cggcaggcaa 60aggggacaaa acaaggagag gccaccacct gttcccaacc cagactatga gcccatccgg 120aaaggccagc gggacctgta ttctggcctg aatcagcgca gaatcggcgg aagcaggagc 180aagcggagca gactgctgca cagcgactac atgaacatga ccccccggag gcctggcccc 240acccggaagc actaccagcc ctacgcccct cccagggatt tcgccgccta ccggagctag 3002069DNAArtificial SEquenceTCR beta transmembrane domain 20accatcctct atgagatctt gctagggaag gccaccttgt atgccgtgct ggtcagtgcc 60ctcgtgctg 69214PRTArtificial SequenceITAM motifX(2)..(3)X can be any amino acid 21Tyr Xaa Xaa Leu122270DNAArtificial SequenceCD3 gamma intracellular domain 22ggacaggatg gagttcgcca gtcgagagct tcagacaagc agactctgtt gcccaatgac 60cagctctacc agcccctcaa ggatcgagaa gatgaccagt acagccacct tcaaggaaac 120cagttgagga ggaatggcgg aagcaggagc aagcggagca gactgctgca cagcgactac 180atgaacatga ccccccggag gcctggcccc acccggaagc actaccagcc ctacgcccct 240cccagggatt tcgccgccta ccggagctag 2702310PRTArtificial SequenceUTY peptideX(1)..(1)X is R or GX(6)..(6)X is E or AX(8)..(8)X is V or P 23Xaa Glu Ser Glu Glu Xaa Ser Xaa Ser Leu1 5 10249PRTArtificial SequenceRPSY peptideX(8)..(8)X is V or L 24Thr Ile Arg Tyr Pro Asp Pro Xaa Ile1 5258PRTArtificial SequenceUTY peptideX(5)..(5)X is H or RX(7)..(7)X is D or N 25Leu Pro His Asn Xaa Thr Xaa Leu1 52623PRTArtificial SequenceTCR alpha transmembrane domain 26Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu1 5 10 15Leu Met Thr Leu Arg Leu Trp 202769DNAArtificial SequenceTCR alpha transmembrane domain 27gtgattgggt tccgaatcct cctcctgaaa gtggccgggt ttaatctgct catgacgctg 60cggctgtgg 692823PRTArtificial SequenceTCR beta transmembrane domain 28Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val1 5 10 15Leu Val Ser Ala Leu Val Leu 202921PRTArtificial SequenceCD3 zeta transmembrane domain 29Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu1 5 10 15Thr Ala Leu Phe Leu 203089PRTArtificial SequenceCD3 delta intracellular domain 30Gly His Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu1 5 10 15Leu Arg Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala 20 25 30Gln Tyr Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys Gly Gly Ser 35 40 45Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 50 55 60Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro65 70 75 80Pro Arg Asp Phe Ala Ala Tyr Arg Ser 8531269DNAArtificial SequenceCD3 delta intracellular domain 31ggacatgaga ctggaaggct gtctggggct gccgacacac aagctctgtt gaggaatgac 60caggtctatc agcccctccg agatcgagat gatgctcagt acagccacct tggaggaaac 120tgggctcgga acaagggcgg aagcaggagc aagcggagca gactgctgca cagcgactac 180atgaacatga ccccccggag gcctggcccc acccggaagc actaccagcc ctacgcccct 240cccagggatt tcgccgccta ccggagcta 2693299PRTArtificial SequenceCD3 epsilon intracellular domain 32Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala1 5 10 15Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro 20 25 30Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Tyr Ser 35 40 45Gly Leu Asn Gln Arg Arg Ile Gly Gly Ser Arg Ser Lys Arg Ser Arg 50 55 60Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro65 70 75 80Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 85 90 95Tyr Arg Ser3389PRTArtificial SequenceCD3 gamma intracellular domain 33Gly Gln Asp Gly Val Arg Gln Ser Arg Ala Ser Asp Lys Gln Thr Leu1 5 10 15Leu Pro Asn Asp Gln Leu Tyr Gln Pro Leu Lys Asp Arg Glu Asp Asp 20 25 30Gln Tyr Ser His Leu Gln Gly Asn Gln Leu Arg Arg Asn Gly Gly Ser 35 40 45Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 50 55 60Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro65 70 75 80Pro Arg Asp Phe Ala Ala Tyr Arg Ser 853411PRTArtificial SequenceSMCY peptideX(3)..(3)X is S or AX(8)..(8)X is R or Q 34Ser Pro Xaa Val Asp Lys Ala Xaa Ala Glu Leu1 5 103510PRTArtificial SequenceTCR beta intracellular domain 35Met Ala Met Val Lys Arg Lys Asp Ser Arg1 5 103630DNAArtificial SequenceTCR beta intracellular domain 36atggccatgg tcaagagaaa ggattccaga 303741PRTArtificial SequenceCD28 intracellular domain 37Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr1 5 10 15Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 4038123DNAArtificial SequenceCD28 intracellular domain 38aggagcaagc ggagcagact gctgcacagc gactacatga acatgacccc ccggaggcct 60ggccccaccc ggaagcacta ccagccctac gcccctccca gggatttcgc cgcctaccgg 120agc 1233942PRTArtificial Sequence4-1BB intracellular domain 39Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35

4040126DNAArtificial Sequence4-1BB intracellular domain 40aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag gccagtacaa 60actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg 1264116PRTArtificial SequenceHLA-A*02 CDR-L1 41Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 154212PRTArtificial SequenceHLA-A*02 CDR-L2 42Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg1 5 10439PRTArtificial SequenceHLA-A*02 CDR-L3 43Phe Gln Gly Ser His Val Pro Arg Thr1 54412PRTArtificial SequenceHLA-A*02 CDR-H1 44Ala Ser Gly Tyr Thr Phe Thr Ser Tyr His Ile His1 5 104518PRTArtificial SequenceHLA-A*02 CDR-H2 45Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe Lys1 5 10 15Gly Lys469PRTArtificial SequenceHLA-A*02 CDR-H3 46Glu Glu Ile Thr Tyr Ala Met Asp Tyr1 54716PRTArtificial SequenceHLA-A*02 CDR-L1 47Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Asp1 5 10 154812PRTArtificial SequenceHLA-A*02 CDR-L2 48Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg1 5 10499PRTArtificial SequenceHLA-A*02 CDR-L3 49Met Gln Gly Ser His Val Pro Arg Thr1 55011PRTArtificial SequenceHLA-A*02 CDR-H1 50Ser Gly Tyr Thr Phe Thr Ser Tyr His Met His1 5 105117PRTArtificial SequenceHLA-A*02 CDR-H2 51Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe Lys1 5 10 15Gly529PRTArtificial SequenceHLA-A*02 CDR-H3 52Glu Gly Thr Tyr Tyr Ala Met Asp Tyr1 553247PRTArtificial SequenceHLA-A*02 ScFv 53Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Arg Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly 130 135 140Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser145 150 155 160Tyr His Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp 165 170 175Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys 180 185 190Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 195 200 205Tyr Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe 210 215 220Cys Ala Arg Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly225 230 235 240Thr Ser Val Thr Val Ser Ser 24554247PRTArtificial SequenceHLA-A*02 ScFv 54Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Glu Ile Val Leu Thr Gln Ser Pro Gly 130 135 140Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 210 215 220Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu Ile Lys 24555247PRTArtificial SequenceHLA-A*02 ScFv 55Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Ile Val Met Thr Gln Thr Pro Leu 130 135 140Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly 210 215 220Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu Ile Lys 24556247PRTArtificial SequenceHLA-A*02 ScFv 56Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220Thr Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu Ile Lys 24557247PRTArtificial SequenceHLA-A*02 ScFv 57Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 210 215 220Thr Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gln225 230 235 240Gly Thr Lys Val Glu Val Lys 24558247PRTArtificial SequenceHLA-A*02 ScFv 58Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Tyr Pro Gly Asn Val Asn Thr Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Glu Glu Ile Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Val Gln Met Thr Gln Ser Pro Ser 130 135 140Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Met Glu Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 210 215 220Thr Tyr Tyr Cys His Gln Gly Ser His Val Pro Arg Thr Phe Gly Gln225 230 235 240Gly Thr Lys Val Glu Val Lys 24559247PRTArtificial SequenceHLA-A*02 ScFv 59Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Phe Cys 85 90 95Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Val Leu Met Thr Gln Thr Pro Leu 130 135 140Ser Leu Pro Val Ser Leu Gly Asp Gln Val Ser Ile Ser Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 210 215 220Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly225 230 235 240Gly Thr Lys Leu Glu Ile Lys 24560247PRTArtificial SequenceHLA-A*02 ScFv 60Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Ile Gln Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Asn Leu Asp Ser Val Ser Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Lys Gly Ser 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 210 215 220Thr Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Pro225 230 235 240Gly Thr Lys Val Asp Ile Lys 24561247PRTArtificial SequenceHLA-A*02 ScFv 61Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Ile Gln Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195

200 205Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 210 215 220Thr Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gln225 230 235 240Gly Thr Lys Val Glu Val Lys 24562247PRTArtificial SequenceHLA-A*02 ScFv 62Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Glu Ile Val Leu Thr Gln Ser Pro Gly 130 135 140Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr 165 170 175Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 210 215 220Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu Ile Lys 24563247PRTArtificial SequenceHLA-A*02 ScFv 63Gln Val Thr Leu Lys Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Thr Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu 35 40 45Gly Arg Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Met Asp Thr Ser Phe65 70 75 80Met Glu Leu Thr Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Leu Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Glu Ile Val Leu Thr Gln Ser Pro Gly 130 135 140Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Ala Trp Tyr 165 170 175Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Ser Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 210 215 220Val Tyr Tyr Cys Gln Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu Ile Lys 24564247PRTArtificial SequenceHLA-A*02 ScFv 64Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30His Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Ile Val Met Thr Gln Thr Pro Leu 130 135 140Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser145 150 155 160Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Asp Trp Tyr 165 170 175Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly 210 215 220Val Tyr Tyr Cys Met Gln Gly Ser His Val Pro Arg Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu Ile Lys 24565670PRTHomo sapiens 65Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly1 5 10 15Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 40 45Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60Lys Thr Ala Leu Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys65 70 75 80Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 85 90 95Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp 100 105 110Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln 130 135 140Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys Glu Gly145 150 155 160Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly 165 170 175Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185 190Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 195 200 205Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu225 230 235 240Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260 265 270Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser 290 295 300Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly305 310 315 320Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325 330 335Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340 345 350Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg 355 360 365Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser385 390 395 400Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu 405 410 415Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly 420 425 430Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly 435 440 445Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly 450 455 460Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe465 470 475 480Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly 530 535 540Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val545 550 555 560Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser 565 570 575Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln 580 585 590Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala 595 600 605Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg 610 615 620Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val625 630 635 640Pro Ser Ile Tyr Ala Thr Leu Ala Ile His Pro Ser Gln Glu Gly Pro 645 650 655Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 660 665 670663229DNAHomo sapiens 66aaatgagttt taaaaaggct tgtccaggaa gcacatatgg gagctggtca ctctgcattt 60tgggccctcc tggaggtgtt tagaccttcc gagagagaaa ctgagacaca tgagagggaa 120gaaatgactc agtggtgaga ccctgtggag tcccacccac aaccagcaca ctgtgaccca 180ctgcacaaac ctctagccca cagctcactt cctcctttaa gaagagaaga gaaaagagga 240gaggagagga ggaacagaaa agaaaagaaa agaaaaagtg ggaaacaaat aatctaagaa 300tgaggagaaa gcaagaagag tgaccccctt gtgggcactc cattggtttt atggcgcctc 360tactttctgg agtttgtgta aaacaaaaat attatggtct ttgtgcacat ttacatcaag 420ctcagcctgg gcggcacagc cagatgcgag atgcgtctct gctgatctga gtctgcctgc 480agcatggacc tgggtcttcc ctgaagcatc tccagggctg gagggacgac tgccatgcac 540cgagggctca tccatccaca gagcagggca gtgggaggag acgccatgac ccccatcctc 600acggtcctga tctgtctcgg gctgagtctg ggcccccgga cccacgtgca ggcagggcac 660ctccccaagc ccaccctctg ggctgaacca ggctctgtga tcacccaggg gagtcctgtg 720accctcaggt gtcagggggg ccaggagacc caggagtacc gtctatatag agaaaagaaa 780acagcaccct ggattacacg gatcccacag gagcttgtga agaagggcca gttccccatc 840ccatccatca cctgggaaca cacagggcgg tatcgctgtt actatggtag cgacactgca 900ggccgctcag agagcagtga ccccctggag ctggtggtga caggagccta catcaaaccc 960accctctcag cccagcccag ccccgtggtg aactcaggag ggaatgtaac cctccagtgt 1020gactcacagg tggcatttga tggcttcatt ctgtgtaagg aaggagaaga tgaacaccca 1080caatgcctga actcccagcc ccatgcccgt gggtcgtccc gcgccatctt ctccgtgggc 1140cccgtgagcc cgagtcgcag gtggtggtac aggtgctatg cttatgactc gaactctccc 1200tatgagtggt ctctacccag tgatctcctg gagctcctgg tcctaggtgt ttctaagaag 1260ccatcactct cagtgcagcc aggtcctatc gtggcccctg aggagaccct gactctgcag 1320tgtggctctg atgctggcta caacagattt gttctgtata aggacgggga acgtgacttc 1380cttcagctcg ctggcgcaca gccccaggct gggctctccc aggccaactt caccctgggc 1440cctgtgagcc gctcctacgg gggccagtac agatgctacg gtgcacacaa cctctcctcc 1500gagtggtcgg cccccagcga ccccctggac atcctgatcg caggacagtt ctatgacaga 1560gtctccctct cggtgcagcc gggccccacg gtggcctcag gagagaacgt gaccctgctg 1620tgtcagtcac agggatggat gcaaactttc cttctgacca aggagggggc agctgatgac 1680ccatggcgtc taagatcaac gtaccaatct caaaaatacc aggctgaatt ccccatgggt 1740cctgtgacct cagcccatgc ggggacctac aggtgctacg gctcacagag ctccaaaccc 1800tacctgctga ctcaccccag tgaccccctg gagctcgtgg tctcaggacc gtctgggggc 1860cccagctccc cgacaacagg ccccacctcc acatctggcc ctgaggacca gcccctcacc 1920cccaccgggt cggatcccca gagtggtctg ggaaggcacc tgggggttgt gatcggcatc 1980ttggtggccg tcatcctact gctcctcctc ctcctcctcc tcttcctcat cctccgacat 2040cgacgtcagg gcaaacactg gacatcgacc cagagaaagg ctgatttcca acatcctgca 2100ggggctgtgg ggccagagcc cacagacaga ggcctgcagt ggaggtccag cccagctgcc 2160gatgcccagg aagaaaacct ctatgctgcc gtgaagcaca cacagcctga ggatggggtg 2220gagatggaca ctcggagccc acacgatgaa gacccccagg cagtgacgta tgccgaggtg 2280aaacactcca gacctaggag agaaatggcc tctcctcctt ccccactgtc tggggaattc 2340ctggacacaa aggacagaca ggcggaagag gacaggcaga tggacactga ggctgctgca 2400tctgaagccc cccaggatgt gacctacgcc cagctgcaca gcttgaccct cagacgggag 2460gcaactgagc ctcctccatc ccaggaaggg ccctctccag ctgtgcccag catctacgcc 2520actctggcca tccactagcc caggggggga cgcagacccc acactccatg gagtctggaa 2580tgcatgggag ctgccccccc agtggacacc attggacccc acccagcctg gatctacccc 2640aggagactct gggaactttt aggggtcact caattctgca gtataaataa ctaatgtctc 2700tacaattttg aaataaagca acagacttct caataatcaa tgaagtagct gagaaaacta 2760agtcagaaag tgcattaaac tgaatcacaa tgtaaatatt acacatcaag cgatgaaact 2820ggaaaactac aagccacgaa tgaatgaatt aggaaagaaa aaaagtagga aatgaatgat 2880cttggctttc ctataagaaa tttagggcag ggcacggtgg ctcacgcctg taattccagc 2940actttgggag gccgaggcgg gcagatcacg agttcaggag atcgagacca tcttggccaa 3000catggtgaaa ccctgtctct cctaaaaata caaaaattag ctggatgtgg tggcagtgcc 3060tgtaatccca gctatttggg aggctgaggc aggagaatcg cttgaaccag ggagtcagag 3120gtttcagtga gccaagatcg caccactgct ctccagcctg gcgacagagg gagactccat 3180ctcaaattaa aaaaaaaaaa aaaaaagaaa gaaaaaaaaa aaaaaaaaa 3229676PRTArtificial SequenceITIM 67Asn Leu Tyr Ala Ala Val1 5686PRTArtificial SequenceITIM 68Val Thr Tyr Ala Glu Val1 5696PRTArtificial SequenceITIM 69Val Thr Tyr Ala Gln Leu1 5706PRTArtificial SequenceITIM 70Ser Ile Tyr Ala Thr Leu1 57135PRTArtificial SequenceITIM 1-2 71Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu1 5 10 15Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr 20 25 30Ala Glu Val 357258PRTArtificial SequenceITIM 2-3 72Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala1 5 10 15Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg 20 25 30Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu 35 40 45Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu 50 557336PRTArtificial SequenceITIM 3-4 73Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr1 5 10 15Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile 20 25 30Tyr Ala Thr Leu 357487PRTArtificial SequenceITIM 1-3 74Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu1 5 10 15Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr 20 25 30Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro 35 40 45Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu 50 55 60Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln65 70 75 80Asp Val Thr Tyr Ala Gln Leu 857588PRTArtificial SequenceITIM 2-4 75Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala1 5 10 15Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg 20 25 30Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu 35 40 45Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg 50 55 60Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala65 70 75 80Val Pro Ser Ile Tyr Ala Thr Leu 8576117PRTArtificial SequenceITIM 1-4 76Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu1 5 10 15Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr 20 25 30Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro 35 40 45Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu 50 55 60Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln65 70 75 80Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala 85 90 95Thr Glu Pro Pro Pro Ser

Gln Glu Gly Pro Ser Pro Ala Val Pro Ser 100 105 110Ile Tyr Ala Thr Leu 1157720PRTArtificial SequenceD3D4 domain 77Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp1 5 10 15Pro Leu Glu Leu 207843PRTArtificial Sequenceshort hinge 78Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly Pro1 5 10 15Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly Ser 20 25 30Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 35 407986PRTArtificial Sequencehinge- transmembrane 79Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp1 5 10 15Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro 20 25 30Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr 35 40 45Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val 50 55 60Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu65 70 75 80Leu Leu Phe Leu Ile Leu 8580190PRTArtificial Sequencehinge-transmembrane-intracellular domain 80Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp 20 25 30Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val 35 40 45Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala 50 55 60Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln65 70 75 80Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp 85 90 95Pro Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg 100 105 110Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr 115 120 125Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala 130 135 140Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu145 150 155 160Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro 165 170 175Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 180 185 19081167PRTArtificial SequenceLILRB1 intracellular domain 81Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg Lys Ala1 5 10 15Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr Asp Arg 20 25 30Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu Asn 35 40 45Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu Met 50 55 60Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala65 70 75 80Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro Ser 85 90 95Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu 100 105 110Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp 115 120 125Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr 130 135 140Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile145 150 155 160Tyr Ala Thr Leu Ala Ile His 16582253PRTArtificial SequenceLILRB1 hinge-transmemebrane-intracellular domain 82Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp1 5 10 15Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro 20 25 30Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr 35 40 45Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val 50 55 60Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu65 70 75 80Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr 85 90 95Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly 100 105 110Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala 115 120 125Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro 130 135 140Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro145 150 155 160Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu 165 170 175Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys 180 185 190Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala 195 200 205Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr 210 215 220Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser225 230 235 240Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 245 25083233PRTArtificial SequenceLILRB1 hinge-TM-intracellular domain 83Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly Pro1 5 10 15Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly Ser 20 25 30Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly Ile 35 40 45Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe Leu 50 55 60Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg65 70 75 80Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr 85 90 95Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu 100 105 110Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val 115 120 125Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr 130 135 140Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro145 150 155 160Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala 165 170 175Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro 180 185 190Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu 195 200 205Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro 210 215 220Ser Ile Tyr Ala Thr Leu Ala Ile His225 2308463PRTArtificial SequenceLILRB1 hinge domain 84Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp1 5 10 15Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro 20 25 30Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr 35 40 45Pro Thr Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly 50 55 608523PRTArtificial SequenceLILRB1 transmembrane domain 85Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Leu Phe Leu Ile Leu 2086237PRTArtificial SequenceMSLN binding domain 86Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Trp Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Asp Ile Val Met Thr Gln Ser Ser Ser Leu Ser Ala 130 135 140Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile145 150 155 160Arg Tyr Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 165 170 175Leu Leu Ile Tyr Thr Ala Ser Ile Leu Gln Asn Gly Val Pro Ser Arg 180 185 190Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 195 200 205Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Thr Tyr Thr 210 215 220Thr Pro Asp Phe Gly Pro Gly Thr Lys Val Glu Ile Lys225 230 23587711DNAArtificial SequenceMSLN binding domain 87caggtgcagc tggtgcagtc tggggctgag gtggagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat 180gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gtctggctgg 300gactttgact actggggcca gggaaccctg gtcaccgtgt cctcaggcgg aggtggaagc 360ggagggggag gatctggcgg cggaggaagc ggaggcgaca tcgtgatgac ccagtcttcc 420tccctgtctg catctgtcgg agacagagtc accatcactt gccgggccag tcagagcatt 480aggtactatt taagttggta tcagcagaaa ccaggaaaag cccctaagct cctgatctat 540actgcatcca ttttacaaaa tggggtccca tcaaggttca gtggcagtgg atctgggaca 600gatttcactc tcaccatcag cagcctgcaa cctgaggatt ttgcaactta ttactgcctc 660cagacttaca ctactccgga ctttggccca gggaccaagg tggaaatcaa a 71188246PRTArtificial SequenceMSLN binding domain 88Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Ala Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Gly Gly Ser Arg Ala Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Thr Ala Ser Cys Gly Gly Asp Cys Tyr Tyr Leu Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr 130 135 140Gln Ser Pro Pro Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile145 150 155 160Thr Cys Arg Ala Ser Glu Asn Val Asn Ile Trp Leu Ala Trp Tyr Gln 165 170 175Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ser Ser Ser 180 185 190Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala 195 200 205Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr 210 215 220Tyr Tyr Cys Gln Gln Tyr Gln Ser Tyr Pro Leu Thr Phe Gly Gly Gly225 230 235 240Thr Lys Val Glu Ile Lys 24589738DNAArtificial SequenceMSLN binding domain 89caggtgcagc tggtgcagtc tggggctgag gtgagggcac ctggggcctc agtgaagatt 60tcctgcaagg cttctggatt caccttcaga ggctactata tccactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaatc atcaacccta gtggtggtag cagagcctac 180gcacagaagt tccagggcag ggtcaccatg accagggaca cttccacgag cacagtctac 240atggagctga gcagcctgag atctgacgac acggccatgt attactgtgc gagaaccgca 300agttgtggtg gtgactgcta ctaccttgac tactggggcc agggaaccct ggtcaccgtg 360tcctcaggcg gaggtggaag cggaggggga ggatctggcg gcggaggaag cggaggcgac 420atccagatga cccagtctcc tcccaccctg tctgcatctg taggagacag agtcaccatc 480acttgccggg ccagtgagaa tgttaatatc tggttggcct ggtatcagca gaaaccaggg 540aaagccccta agctcctgat ctataagtca tccagtttag caagtggggt cccatcaagg 600ttcagtggca gtggatctgg ggcagaattc actctcacca tcagcagcct gcagcctgat 660gattttgcaa cttattactg ccaacagtat caaagttacc ccctcacttt cggcggaggg 720accaaggtgg aaatcaaa 73890242PRTArtificial SequenceMSLN binding domain 90Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe 50 55 60Arg Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro 130 135 140Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser145 150 155 160Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly 165 170 175Lys Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly 180 185 190Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu 195 200 205Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 210 215 220Gln Trp Ser Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu225 230 235 240Ile Lys91726DNAArtificial SequenceMSLN binding domain 91caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg cttctggata ctcattcacc ggctacacca tgaactgggt gaggcaggcc 120cctggacaaa gacttgagtg gatgggactt atcacccctt acaatggtgc ttctagctac 180aaccagaagt tcaggggcag ggtcacaatc actagagaca cgtcagccag cacagcctac 240atggagctct ccagcctgag atctgaagac actgcagtct attactgtgc aagggggggt 300tacgacggga ggggttttga ctactggggc cagggaacca cggtcaccgt gtcctcaggc 360ggaggtggaa gcggaggggg aggatctggc ggcggaggaa gcggaggcga catccagatg 420acccagtctc cttcaagctt gtctgcatct gtaggagaca gggtcaccat cacttgcagt 480gccagctcaa gtgtaagtta catgcactgg tatcagcaga aaccaggcaa ggcccctaag 540agattgatct atgacacatc caaattagca agtggggtcc caagtcgctt cagtggcagt 600ggatctggga ccgaattcac tctcaccatc agcagcttgc agcctgagga ttttgcaact 660tattactgcc agcagtggag tggttaccct ctcacgttcg gtcaggggac aaagttggaa 720atcaaa 72692242PRTArtificial SequenceMSLN binding domain 92Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Glu Leu Thr Gln Ser Pro 130 135 140Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser145 150 155 160Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly

165 170 175Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly 180 185 190Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu 195 200 205Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln 210 215 220Gln Trp Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu225 230 235 240Ile Lys93726DNAArtificial SequenceMSLN binding domain 93caggtgcagc tgcagcagtc tgggcctgag ctggagaagc ctggggcctc agtgaagatt 60tcctgcaagg cttctggata ctcattcacc ggctacacca tgaactgggt gaagcagagc 120catggaaaaa gccttgagtg gattggactt atcacccctt acaatggtgc ttctagctac 180aaccagaagt tcaggggcaa ggccacatta actgtagaca agtcatccag cacagcctac 240atggacctcc tcagcctgac atctgaagac tctgcagtct atttctgtgc aagggggggt 300tacgacggga ggggttttga ctactggggc cagggaacca cggtcaccgt gtcctcaggc 360ggaggtggaa gcggaggggg aggatctggc ggcggaggaa gcggaggcga catcgagctc 420acccagtctc ctgcaatcat gtctgcatct ccaggagaga aggtcaccat gacttgcagt 480gccagctcaa gtgtaagtta catgcactgg tatcagcaga aatcaggcac ctcccctaag 540agatggatct atgacacatc caaattggca agtggggtcc caggtcgctt cagtggcagt 600ggatctggga actcttactc tctcaccatc agcagcgtgg aggctgagga tgatgcaact 660tattactgcc agcagtggag tggttaccct ctcacgttcg gtgctgggac aaagttggaa 720atcaaa 72694243PRTArtificial SequenceCEA binding domain 94Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Thr1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30Tyr Met His Trp Leu Arg Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Asn Val Leu Thr Gln Ser 130 135 140Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys145 150 155 160Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro 165 170 175Gly Thr Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser 180 185 190Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 195 200 205Leu Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 210 215 220Gln Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu225 230 235 240Glu Leu Lys95729DNAArtificial SequenceCEA binding domain 95caggtccagc tgcagcagtc tggggcagag cttgtgaggt cagggacctc agtcaagttg 60tcctgcacag cttctggctt caacattaaa gactcctata tgcactggtt gaggcagggg 120cctgaacagg gcctggagtg gattggatgg attgatcctg agaatggtga tactgaatat 180gccccgaagt tccagggcaa ggccactttt actacagaca catcctccaa cacagcctac 240ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtaa tgaagggaca 300ccgacagggc catactattt tgactactgg ggtcaaggaa ccacagtcac cgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgagaacgtt 420ctcacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac cataacctgc 480agtgccagct caagtgtaag ttacatgcac tggttccagc agaagccagg cacttctccc 540aaactctgga tttatagcac atccaacctg gcttctggag tccctgctcg cttcagtggc 600agtggatctg ggacctctta ctctctcaca atcagccgaa tggaggctga agatgctgcc 660acttattact gccagcaaag gagtagttac ccgctcacgt tcggtgctgg gaccaagctg 720gagctgaaa 72996243PRTArtificial SequenceCEA binding domain 96Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Ile Val Leu Thr Gln Ser 130 135 140Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys145 150 155 160Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro 165 170 175Gly Leu Ala Pro Arg Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser 180 185 190Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220Gln Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu225 230 235 240Glu Ile Lys97729DNAArtificial SequenceCEA binding domain 97caggtccagc tggtgcagtc tggggcagag gtgaagaaac caggggcctc agtcaaggtg 60tcctgcaaag cttctggctt caacattaaa gactcctata tgcactgggt gaggcaggcg 120cctggacagg gcctggagtg gatgggatgg attgatcctg agaatggtga tactgaatat 180gccccgaagt tccagggcag ggtcactatg actacagaca catccacctc cacagcctac 240atggagctca ggagcctgag atctgacgac actgccgtct attactgtaa tgaagggaca 300ccgacagggc catactattt tgactactgg ggtcaaggaa ccacagtcac cgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgagatcgtt 420ctcacccagt ctccagcaac cttgtctctg tctccagggg agagggccac cctaagctgc 480agtgccagct caagtgtaag ttacatgcac tggtaccagc agaagccagg ccttgctccc 540agactcctga tttatagcac atccaacctg gcttctggaa tccctgatcg cttcagtggc 600agtggatctg ggaccgattt cactctcaca atcagccgac tggagcctga agatttcgcc 660gtttattact gccagcaaag gagtagttac ccgctcacgt tcggtcaggg gaccaagctg 720gagatcaaa 72998243PRTArtificial SequenceCEA binding domain 98Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asp 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Asn Glu Phe Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Ser Ser Glu Leu Thr Gln Asp Pro Ala Val 130 135 140Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser145 150 155 160Leu Arg Ser Ser Tyr Ala Ser Trp Tyr Arg Gln Arg Pro Gly Gln Ala 165 170 175Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro 180 185 190Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile 195 200 205Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Trp Asn Ser Ser 210 215 220Tyr Ala Trp Leu Pro Tyr Val Val Phe Gly Gly Gly Thr Lys Leu Thr225 230 235 240Val Leu Gly99729DNAArtificial SequenceCEA binding domain 99gaggtgcagc tggcggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc agcgatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aaagtctaat 300gagtttcttt ttgactactg gggccaaggt accctggtca ccgtgtcgag tggcggaggt 360ggaagcggag ggggaggatc tggcggcgga ggaagcggag gctcttctga gctgactcag 420gaccctgctg tgtctgtggc cttgggacag acagtcagga tcacatgcca aggagacagc 480ctcagaagct cttatgcaag ctggtaccgg cagaggccag gacaggcccc tgtacttgtc 540atctatggta aaaacaaccg gccctcaggg atcccagacc gattctctgg ctccagctca 600ggaaacacag cttccttgac catcactggg gctcaggcgg aagatgaggc tgactattac 660tggaactcca gctacgcttg gctgccctac gtggtattcg gcggagggac caagctgacc 720gtcctaggt 729100243PRTArtificial SequenceCEA binding domain 100Gln Val Gln Leu Glu Gln Ser Gly Ala Gly Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser 20 25 30Tyr Met His Trp Leu Arg Gln Gly Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ala Asn Thr Ala Tyr65 70 75 80Leu Gly Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Asn Val Leu Thr Gln Ser 130 135 140Pro Ser Ser Met Ser Val Ser Val Gly Asp Arg Val Asn Ile Ala Cys145 150 155 160Ser Ala Ser Ser Ser Val Pro Tyr Met His Trp Leu Gln Gln Lys Pro 165 170 175Gly Lys Ser Pro Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Ala Ser 180 185 190Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser 195 200 205Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Ala Ala Thr Tyr Tyr Cys 210 215 220Gln Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu225 230 235 240Glu Ile Lys101729DNAArtificial SequenceCEA binding domain 101caggtccagc tggagcagtc tggggcaggg gttgtgaagc caggggcctc agtcaagttg 60tcctgcaaag cttctggctt caacattaaa gactcctata tgcactggtt gaggcagggg 120cctggacagc gcctggagtg gattggatgg attgatcctg agaatggtga tactgaatat 180gccccgaagt tccagggcaa ggccactttt actacagaca catccgccaa cacagcctac 240ctggggctca gcagcctgag acctgaggac actgccgtct attactgtaa tgaagggaca 300ccgacagggc catactattt tgactactgg ggtcaaggaa ccctagtcac cgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgagaacgtt 420ctcacccagt ctccaagctc tatgtctgta tctgtcgggg acagggtcaa catcgcctgc 480agtgccagct caagtgtacc ttacatgcac tggctccagc agaagccagg caaatctccc 540aaactcctga tttatctcac atccaacctg gcttctggag tccctagccg cttcagtggc 600agtggatctg ggaccgatta ctctctcaca atcagctcag tgcagcctga agatgctgcc 660acttattact gccagcaaag gagtagttac ccgctcacgt tcggtggtgg gaccaagctg 720gagatcaaa 729102249PRTArtificial SequenceEGFR ScFv 102Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile 130 135 140Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg145 150 155 160Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Val 165 170 175Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp 180 185 190Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Glu 195 200 205Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 210 215 220Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe225 230 235 240Gly Gly Gly Thr Lys Val Glu Ile Lys 245103747DNAArtificial SequenceEGFR ScFv 103caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt acctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatgggatg atggaagtta taaatactat 180ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatggt 300attactatgg ttcggggagt tatgaaggac tactttgact actggggcca gggaaccctg 360gtcaccgtct cctcaggcgg aggtggaagc ggagggggag gatctggcgg cggaggaagc 420ggaggcgcca tccagttgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 480gtcaccatca cttgccgggc aagtcaggac attagcagtg ctttagtctg gtatcagcag 540aaaccaggga aagctcctaa gctcctgatc tatgatgcct ccagtttgga aagtggggtc 600ccatcaaggt tcagcggcag tgaatctggg acagatttca ctctcaccat cagcagcctg 660cagcctgaag attttgcaac ttattactgt caacagttta atagttaccc gctcactttc 720ggcggaggga ccaaggtgga gatcaaa 747104249PRTArtificial SequenceEGFR ScFv 104Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gln Val Gln Leu 115 120 125Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu 130 135 140Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Gly Met His Trp145 150 155 160Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp 165 170 175Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe 180 185 190Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 195 200 205Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gly 210 215 220Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe Asp Tyr Trp Gly225 230 235 240Gln Gly Thr Leu Val Thr Val Ser Ser 245105747DNAArtificial SequenceEGFR ScFv 105gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattagc agtgctttag tctggtatca gcagaaacca 120gggaaagctc ctaagctcct gatctatgat gcctccagtt tggaaagtgg ggtcccatca 180aggttcagcg gcagtgaatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag tttaatagtt acccgctcac tttcggcgga 300gggaccaagg tggagatcaa aggcggaggt ggaagcggag ggggaggatc tggcggcgga 360ggaagcggag gccaggtgca gctggtggag tctgggggag gcgtggtcca gcctgggagg 420tccctgagac tctcctgtgc agcgtctgga ttcaccttca gtacctatgg catgcactgg 480gtccgccagg ctccaggcaa

ggggctggag tgggtggcag ttatatggga tgatggaagt 540tataaatact atggagactc cgtgaagggc cgattcacca tctccagaga caattccaag 600aacacgctgt atctgcaaat gaacagcctg agagccgagg acacggctgt gtattactgt 660gcgagagatg gtattactat ggttcgggga gttatgaagg actactttga ctactggggc 720cagggaaccc tggtcaccgt ctcctca 747106247PRTArtificial SequenceEGFR ScFv 106Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Pro Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp Met 35 40 45Gly Met Ile Tyr Thr Asp Ile Gly Lys Pro Thr Tyr Ala Glu Glu Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Val Arg Asp Arg Tyr Asp Ser Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Asp Val Val Met Thr Gln Thr Pro Leu 130 135 140Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser145 150 155 160Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr 165 170 175Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser 180 185 190Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 210 215 220Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gly225 230 235 240Gly Thr Lys Leu Glu Ile Lys 245107741DNAArtificial SequenceEGFR ScFv 107cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60tcctgcaagg cctctgggta taccttcaca gaatatccaa tacactgggt gaagcaggct 120ccaggaaagg gtttcaagtg gatgggcatg atatacaccg acattggaaa gccaacatat 180gctgaagagt tcaagggacg gtttgccttc tctttggaga cctctgccag cactgcctat 240ttgcagatca acaacctcaa gaatgaggac acggctacat atttctgtgt aagagatcga 300tatgattccc tctttgacta ctggggccaa ggcaccactc tcacagtctc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgatgt tgtgatgacc 420caaactccac tctccctgcc tgtcagtctt ggagatcaag cctccatctc ttgcagatct 480agtcagagcc ttgtacacag taatggaaac acctatttac attggtacct gcagaagcca 540ggccagtctc caaagctcct gatctacaaa gtttccaacc gattttctgg ggtcccagac 600aggttcagtg gcagtggatc agggacagat ttcacactca agatcagcag agtggaggct 660gaggatctgg gagtttattt ctgctctcaa agtacacatg ttccgtggac gttcggtgga 720ggcaccaagc tggaaatcaa a 741108247PRTArtificial SequenceEGFR ScFv 108Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly 130 135 140Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu145 150 155 160Tyr Pro Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp 165 170 175Met Gly Met Ile Tyr Thr Asp Ile Gly Lys Pro Thr Tyr Ala Glu Glu 180 185 190Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala 195 200 205Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe 210 215 220Cys Val Arg Asp Arg Tyr Asp Ser Leu Phe Asp Tyr Trp Gly Gln Gly225 230 235 240Thr Thr Leu Thr Val Ser Ser 245109741DNAArtificial SequenceEGFR ScFv 109gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300tggacgttcg gtggaggcac caagctggaa atcaaaggcg gaggtggaag cggaggggga 360ggatctggcg gcggaggaag cggaggccag atccagttgg tgcagtctgg acctgagctg 420aagaagcctg gagagacagt caagatctcc tgcaaggcct ctgggtatac cttcacagaa 480tatccaatac actgggtgaa gcaggctcca ggaaagggtt tcaagtggat gggcatgata 540tacaccgaca ttggaaagcc aacatatgct gaagagttca agggacggtt tgccttctct 600ttggagacct ctgccagcac tgcctatttg cagatcaaca acctcaagaa tgaggacacg 660gctacatatt tctgtgtaag agatcgatat gattccctct ttgactactg gggccaaggc 720accactctca cagtctcctc a 741110249PRTArtificial SequenceEGFR ScFv 110Glu Met Gln Leu Val Glu Ser Gly Gly Gly Phe Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser His Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro Lys Gln Arg Leu Glu Trp Val 35 40 45Ala Tyr Ile Ala Ser Gly Gly Asp Ile Thr Tyr Tyr Ala Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys 85 90 95Ser Arg Ser Ser Tyr Gly Asn Asn Gly Asp Ala Leu Asp Phe Trp Gly 100 105 110Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Val Val Met Thr Gln 130 135 140Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser145 150 155 160Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu 165 170 175His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 180 185 190Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu 210 215 220Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Leu Thr Phe225 230 235 240Gly Ser Gly Thr Lys Leu Glu Ile Lys 245111747DNAArtificial SequenceEGFR ScFv 111gaaatgcagc tggtggagtc tgggggaggc ttcgtgaagc ctggagggtc cctgaaactc 60tcatgtgcag cctctggatt cgctttcagt cactatgaca tgtcttgggt tcgccagact 120ccgaagcaga ggctggagtg ggtcgcatac attgctagtg gtggtgatat cacctactat 180gcagacactg tgaagggccg attcaccatc tccagagaca atgcccagaa caccctgtac 240ctgcaaatga gcagtctgaa gtctgaggac acagccatgt tttactgttc acgatcctcc 300tatggtaaca acggagatgc cctggacttc tggggtcaag gtacctcagt caccgtctcc 360tcaggcggag gtggaagcgg agggggagga tctggcggcg gaggaagcgg aggcgatgtt 420gtgatgaccc aaactccact ctccctgcct gtcagtcttg gagatcaagc ctccatctct 480tgcagatcta gtcagagcct tgttcacagt aatggaaaca cctatttaca ttggtacctg 540cagaagccag gccagtctcc aaagctcctg atctacaaag tttccaaccg attttctggg 600gtcccagaca ggttcagtgg cagtggatca gggacagatt tcacactcaa gatcagcaga 660gtggaggctg aggatctggg agtttatttc tgctctcaaa gtacacatgt tctcacgttc 720ggctcgggga caaagttgga aataaaa 747112249PRTArtificial SequenceEGFR ScFv 112Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Glu Met Gln Leu Val Glu Ser Gly Gly Gly Phe Val Lys Pro Gly Gly 130 135 140Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser His Tyr145 150 155 160Asp Met Ser Trp Val Arg Gln Thr Pro Lys Gln Arg Leu Glu Trp Val 165 170 175Ala Tyr Ile Ala Ser Gly Gly Asp Ile Thr Tyr Tyr Ala Asp Thr Val 180 185 190Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr 195 200 205Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys 210 215 220Ser Arg Ser Ser Tyr Gly Asn Asn Gly Asp Ala Leu Asp Phe Trp Gly225 230 235 240Gln Gly Thr Ser Val Thr Val Ser Ser 245113747DNAArtificial SequenceEGFR ScFv 113gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagccttgtt cacagtaatg gaaacaccta tttacattgg 120tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttctc 300acgttcggct cggggacaaa gttggaaata aaaggcggag gtggaagcgg agggggagga 360tctggcggcg gaggaagcgg aggcgaaatg cagctggtgg agtctggggg aggcttcgtg 420aagcctggag ggtccctgaa actctcatgt gcagcctctg gattcgcttt cagtcactat 480gacatgtctt gggttcgcca gactccgaag cagaggctgg agtgggtcgc atacattgct 540agtggtggtg atatcaccta ctatgcagac actgtgaagg gccgattcac catctccaga 600gacaatgccc agaacaccct gtacctgcaa atgagcagtc tgaagtctga ggacacagcc 660atgttttact gttcacgatc ctcctatggt aacaacggag atgccctgga cttctggggt 720caaggtacct cagtcaccgt ctcctca 747114243PRTArtificial SequenceEGFR ScFv 114Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Leu Leu Thr Gln Ser Pro 130 135 140Val Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg145 150 155 160Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr 165 170 175Asn Gly Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser 180 185 190Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205Leu Ser Ile Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys 210 215 220Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu225 230 235 240Glu Leu Lys115729DNAArtificial SequenceEGFR ScFv 115caggtgcagc tgaagcagtc cggccccggc ctggtgcagc cctcccagtc cctgtccatc 60acctgcaccg tgtccggctt ctccctgacc aactacggcg tgcactgggt gcggcagtcc 120cccggcaagg gcctggagtg gctgggcgtg atctggtccg gcggcaacac cgactacaac 180acccccttca cctcccggct gtccatcaac aaggacaact ccaagtccca ggtgttcttc 240aagatgaact ccctgcagtc caacgacacc gccatctact actgcgcccg ggccctgacc 300tactacgact acgagttcgc ctactggggc cagggcaccc tggtgaccgt gtccgccggc 360ggaggtggaa gcggaggggg aggatctggc ggcggaggaa gcggaggcga catcctgctg 420acccagtccc ccgtgatcct gtccgtgtcc cccggcgagc gggtgtcctt ctcctgccgg 480gcctcccagt ccatcggcac caacatccac tggtaccagc agcggaccaa cggctccccc 540cggctgctga tcaagtacgc ctccgagtcc atctccggca tcccctcccg gttctccggc 600tccggctccg gcaccgactt caccctgtcc atcaactccg tggagtccga ggacatcgcc 660gactactact gccagcagaa caacaactgg cccaccacct tcggcgccgg caccaagctg 720gagctgaag 729116244PRTArtificial SequenceEGFR ScFv 116Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Phe Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Met Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Arg Asp Tyr Asp Tyr Asp Gly Arg Tyr Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln 130 135 140Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr145 150 155 160Cys Ser Ala Ser Ser Ser Val Thr Tyr Met Tyr Trp Tyr Gln Gln Lys 165 170 175Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala 180 185 190Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 195 200 205Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr 210 215 220Cys Gln Gln Trp Ser Ser His Ile Phe Thr Phe Gly Gln Gly Thr Lys225 230 235 240Val Glu Ile Lys117731DNAArtificial SequenceEGFR ScFv 117aggtgcagct ggtgcagtcc ggcgccgagg tgaagaagcc cggcgcctcc gtgaaggtgt 60cctgcaaggc ctccggctac accttcacct cccactggat gcactgggtg cggcaggccc 120ccggccaggg cctggagtgg atcggcgagt tcaacccctc caacggccgg accaactaca 180acgagaagtt caagtccaag gccaccatga ccgtggacac ctccaccaac accgcctaca 240tggagctgtc ctccctgcgg tccgaggaca ccgccgtgta ctactgcgcc tcccgggact 300acgactacga cggccggtac ttcgactact ggggccaggg caccctggtg accgtgtcct 360ccggcggagg tggaagcgga gggggaggat ctggcggcgg aggaagcgga ggcgacatcc 420agatgaccca gtccccctcc tccctgtccg cctccgtggg cgaccgggtg accatcacct 480gctccgcctc ctcctccgtg acctacatgt actggtacca gcagaagccc ggcaaggccc 540ccaagctgct gatctacgac acctccaacc tggcctccgg cgtgccctcc cggttctccg 600gctccggctc cggcaccgac tacaccttca ccatctcctc cctgcagccc gaggacatcg 660ccacctacta ctgccagcag tggtcctccc acatcttcac cttcggccag ggcaccaagg 720tggagatcaa g 731118243PRTArtificial SequenceEGFR ScFv 118Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25 30Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr 85 90 95Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser

Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro 130 135 140Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln145 150 155 160Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 165 170 175Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr 180 185 190Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys 210 215 220Gln His Phe Asp His Leu Pro Leu Ala Phe Gly Gly Gly Thr Lys Val225 230 235 240Glu Ile Lys119729DNAArtificial SequenceEGFR ScFv 119caggtgcagc tgcaggagtc cggccccggc ctggtgaagc cctccgagac cctgtccctg 60acctgcaccg tgtccggcgg ctccgtgtcc tccggcgact actactggac ctggatccgg 120cagtcccccg gcaagggcct ggagtggatc ggccacatct actactccgg caacaccaac 180tacaacccct ccctgaagtc ccggctgacc atctccatcg acacctccaa gacccagttc 240tccctgaagc tgtcctccgt gaccgccgcc gacaccgcca tctactactg cgtgcgggac 300cgggtgaccg gcgccttcga catctggggc cagggcacca tggtgaccgt gtcctccggc 360ggaggtggaa gcggaggggg aggatctggc ggcggaggaa gcggaggcga catccagatg 420acccagtccc cctcctccct gtccgcctcc gtgggcgacc gggtgaccat cacctgccag 480gcctcccagg acatctccaa ctacctgaac tggtaccagc agaagcccgg caaggccccc 540aagctgctga tctacgacgc ctccaacctg gagaccggcg tgccctcccg gttctccggc 600tccggctccg gcaccgactt caccttcacc atctcctccc tgcagcccga ggacatcgcc 660acctacttct gccagcactt cgaccacctg cccctggcct tcggcggcgg caccaaggtg 720gagatcaag 729120125PRTArtificial SequenceEGFR VH 120Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125121107PRTArtificial SequenceEGFR VL 121Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105122118PRTArtificial SequenceEGFR VH 122Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Pro Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp Met 35 40 45Gly Met Ile Tyr Thr Asp Ile Gly Lys Pro Thr Tyr Ala Glu Glu Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Val Arg Asp Arg Tyr Asp Ser Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser 115123112PRTArtificial SequenceEGFR VL 123Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110124121PRTArtificial SequenceEGFR VH 124Glu Met Gln Leu Val Glu Ser Gly Gly Gly Phe Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser His Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro Lys Gln Arg Leu Glu Trp Val 35 40 45Ala Tyr Ile Ala Ser Gly Gly Asp Ile Thr Tyr Tyr Ala Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys 85 90 95Ser Arg Ser Ser Tyr Gly Asn Asn Gly Asp Ala Leu Asp Phe Trp Gly 100 105 110Gln Gly Thr Ser Val Thr Val Ser Ser 115 120125111PRTArtificial SequenceEGFR VL 125Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Val Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110126119PRTArtificial SequenceEGR VH 126Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala 115127107PRTArtificial SequenceEGFR VL 127Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser65 70 75 80Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105128121PRTArtificial SequenceEGFR VH 128Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Phe Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr Met Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Arg Asp Tyr Asp Tyr Asp Gly Arg Tyr Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120129106PRTArtificial SequenceEGFR VL 129Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met 20 25 30Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser His Ile Phe Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105130119PRTArtificial SequenceEGFR VH 130Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly 20 25 30Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr 85 90 95Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser 115131107PRTArtificial SequenceEGFR VL 131Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu 85 90 95Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 1051325PRTArtificial SequenceEGFR CDR-H1 132Thr Tyr Gly Met His1 51335PRTArtificial SequenceEGFR CDR-H1 133Glu Tyr Pro Ile His1 51345PRTArtificial SEquenceEGFR CDR-H1 134His Tyr Asp Met Ser1 51355PRTArtificial SequenceEGFR CDR-H1 135Asn Tyr Gly Val His1 51365PRTArtificial SequenceEGFR CDR-H1 136Ser His Trp Met His1 51377PRTArtificial SequenceEGFR CDR-H1 137Ser Gly Asp Tyr Tyr Trp Thr1 513817PRTArtificial SequenceEGFR CDR-H2 138Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val Lys1 5 10 15Gly13917PRTArtificial SequenceEGFR CDR-H2 139Met Ile Tyr Thr Asp Ile Gly Lys Pro Thr Tyr Ala Glu Glu Phe Lys1 5 10 15Gly14017PRTArtificial SequenceEGFR CDR-H2 140Tyr Ile Ala Ser Gly Gly Asp Ile Thr Tyr Tyr Ala Asp Thr Val Lys1 5 10 15Gly14116PRTArtificial SequenceEGFR CDR-H2 141Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser1 5 10 1514217PRTArtificial SequenceEGFR CDR-H2 142Glu Phe Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys1 5 10 15Ser14316PRTArtificial SequenceEGFR CDR-H2 143His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10 1514416PRTArtificial SequenceEGFR CDR-H3 144Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe Asp Tyr1 5 10 151459PRTArtificial SequenceEGFR CDR-H3 145Asp Arg Tyr Asp Ser Leu Phe Asp Tyr1 514612PRTArtificial SequenceEGFR CDR-H3 146Ser Ser Tyr Gly Asn Asn Gly Asp Ala Leu Asp Phe1 5 1014711PRTArtificial SequenceEGFR CDR-H3 147Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr1 5 1014812PRTArtificial SequenceEGFR CDR-H3 148Arg Asp Tyr Asp Tyr Asp Gly Arg Tyr Phe Asp Tyr1 5 101499PRTArtificial SequenceEGFR CDR-H3 149Asp Arg Val Thr Gly Ala Phe Asp Ile1 515011PRTArtificial SequenceEGFR CDR-L1 150Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Val1 5 1015116PRTArtificial SequenceEGFR CDR-L1 151Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His1 5 10 1515216PRTArtificial SequenceEGFRR CDR-L1 152Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His1 5 10 1515311PRTArtificial SequenceEGFR CDR-L1 153Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His1 5 1015410PRTArtificial SequenceEGFR CDR-L1 154Ser Ala Ser Ser Ser Val Thr Tyr Met Tyr1 5 1015511PRTArtificial SequenceEGFR CDR-L1 155Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5 101567PRTArtificial SequenceEGFR CDR-L2 156Asp Ala Ser Ser Leu Glu Ser1 51577PRTArtificial SequenceEGFR CDR-L2 157Lys Val Ser Asn Arg Phe Ser1 51587PRTArtificial SequenceEGFR CDR-L2 158Tyr Ala Ser Glu Ser Ile Ser1 51597PRTArtificial SequenceEGFR CDR-L2 159Asp Thr Ser Asn Leu Ala Ser1 51607PRTArtificial SequenceEGFR CDR-L2 160Asp Ala Ser Asn Leu Glu Thr1 51619PRTArtificial SequenceEGFR CDR-L3 161Gln Gln Phe Asn Ser Tyr Pro Leu Thr1 51629PRTArtificial SequenceEGFR CDR-L3 162Ser Gln Ser Thr His Val Pro Trp Thr1 51638PRTArtificial SequenceEGFR CDR-L3 163Ser Gln Ser Thr His Val Leu Thr1 51649PRTArtificial SequenceEGFR CDR-L3 164Gln Gln Asn Asn Asn Trp Pro Thr Thr1 51659PRTArtificial SequenceEGFR CDR-L3 165Gln Gln Trp Ser Ser His Ile Phe Thr1 51669PRTArtificial SequenceEGFR CDR-L3 166Gln His Phe Asp His Leu Pro Leu Ala1 5167246PRTArtificial Sequencepan-HLA ScFv 167Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Thr Phe Thr Thr Ser Thr Ser Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Ile Val Met Thr Gln Thr 130 135 140Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp Tyr Gln Gln Lys 165 170 175Pro Gly Gln Ser Pro Ile Cys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg 180 185 190Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp 195 200 205Phe Thr Phe Thr Ile Ser Thr Val Gln Ala Glu Asp Leu Ala Val Tyr 210 215 220Phe Cys Gln Gln Asp Tyr Ser Ser Pro Pro Trp

Thr Phe Gly Gly Gly225 230 235 240Thr Lys Leu Glu Ile Arg 245168738DNAArtificial Sequencepan-HLA ScFv 168caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccctg 60acctgcacag tctctggttt ctcattaact agttatggcg tacactgggt tcgccagcct 120ccaggaaagg gtctggagtg gctgggagtg atctggagtg gtggaagcac agactataat 180gctgctttca tatccagact gagcatcagg aaggacaact ccaagagcca agtcttcttt 240aaaatgaaca gtctgcaagc tgatgacaca gccatatact actgtgccag aacctttact 300acgtctacct cggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cagcatcgtg 420atgacccaga ctccaaaatt cctgcttgtg tctgcgggag acagagtcac catcacttgc 480aaggcgagtc agtctgtgag caacgacgta gcttggtatc agcagaaacc agggcaatct 540cctatctgtc tcctgatcta ctatgcatct aatcggtata caggggtccc tgataggttc 600accggaagtg gatatgggac agatttcact ttcaccatca gcaccgtgca ggctgaagat 660cttgcagtat atttctgtca acaggattat agtagtcctc cgtggacttt cggcggaggg 720accaagttgg agatcaga 738169245PRTArtificial Sequencepan-HLA ScFv 169Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Thr Phe Thr Thr Ser Thr Ser Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Val Met Thr Gln Ser 130 135 140Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys145 150 155 160Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp Tyr Gln Gln Lys 165 170 175Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr 180 185 190Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr 210 215 220Cys Gln Gln Asp Tyr Ser Ser Pro Pro Trp Thr Phe Gly Gly Gly Thr225 230 235 240Lys Val Glu Ile Lys 245170735DNAArtificial Sequencepan-HLA ScFv 170caggtgcagc tgcaggagtc cggacctggc ctagtgaagc cctcacagac cctgtccctg 60acctgcacag tctctggttt ctcattaact agctatggtg tacactggat tagacagcct 120ccaggaaagg gtctggagtg gattggagtg atctggagtg gtggaagcac agactataat 180gctgctttca tatccagagt gaccatcagc gtggacacct ccaagaacca attctccctt 240aaactgagca gtgtgacagc tgccgacaca gccgtatact actgtgccag aacctttact 300acgtctacct cggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctcttca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatcgtg 420atgacccaga gtccagattc cctggctgtg tctctgggag agagagccac catcaattgc 480aaggcgagtc agtctgtgag caacgacgta gcttggtatc agcagaaacc agggcaacct 540cctaaactcc tgatctacta tgcatctaat cggtatacag gggtccctga taggttcagc 600ggaagtggat ctgggacaga tttcactctc accatcagca gcctgcaggc tgaagatgtt 660gcagtatatt actgtcaaca ggattatagt agtcctccgt ggactttcgg cggagggacc 720aaggtggaga tcaaa 735171245PRTArtificial Sequencepan-HLA ScFv 171Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Thr Phe Thr Thr Ser Thr Ser Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp Tyr Gln Gln Lys 165 170 175Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr 180 185 190Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr 210 215 220Cys Gln Gln Asp Tyr Ser Ser Pro Pro Trp Thr Phe Gly Gly Gly Thr225 230 235 240Lys Val Glu Ile Lys 245172735DNAArtificial Sequencepan-HLA ScFv 172gaggtgcagc tgctggagtc cggaggtggc ctagtgcagc ccggagggag cctgcgcctg 60agctgcgcag cctctggttt ctcattaact agctatggtg tacactgggt tagacaggct 120ccaggaaagg gtctggagtg ggttagcgtg atctggagtg gtggaagcac agactataat 180gctgctttca tatccagatt taccatcagc cgggacaact ccaagaacac actctacctt 240caaatgaaca gtttgagagc tgaagacaca gccgtatact actgtgccag aacctttact 300acgtctacct cggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctcttca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatccag 420atgacccaga gtccaagctc cctgtctgcg tctgtgggag acagagtcac catcacttgc 480aaggcgagtc agtctgtgag caacgacgta gcttggtatc agcagaaacc agggaaagct 540cctaaactcc tgatctacta tgcatctaat cggtatacag gggtccctag taggttcagc 600ggaagtggat ctgggacaga tttcactttc accatcagca gcctgcagcc tgaagatatt 660gcaacatatt actgtcaaca ggattatagt agtcctccgt ggactttcgg cggagggacc 720aaggtggaga tcaaa 735173245PRTArtificial Sequencepan-HLA ScFv 173Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Thr Phe Thr Thr Ser Thr Ser Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Val Met Thr Gln Thr 130 135 140Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys145 150 155 160Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp Tyr Leu Gln Lys 165 170 175Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr 180 185 190Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 210 215 220Cys Gln Gln Asp Tyr Ser Ser Pro Pro Trp Thr Phe Gly Gly Gly Thr225 230 235 240Lys Val Glu Ile Lys 245174735DNAArtificial Sequencepan-HLA ScFv 174caggtgcagc tgcaggagtc cggacctggc ctagtgaagc cctcagaaac cctgtccctg 60acctgcacag tctctggttt ctcattaact agctatggtg tacactggat tagacagcct 120ccaggaaagg gtctggagtg gattggagtg atctggagtg gtggaagcac agactataat 180gctgctttca tatccagagt gaccatcagc agggacacct ccaagaacca attctccctt 240aaactgagca gtgtgacagc tgccgacaca gccgtatact actgtgccag aacctttact 300acgtctacct cggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctcttca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatcgtg 420atgacccaga ctccactttc cctgtctgtg actccgggac agccagccag catcagttgc 480aaggcgagtc agtctgtgag caacgacgta gcttggtatc tgcagaaacc agggcaatct 540cctcaactcc tgatctacta tgcatctaat cggtatacag gggtccctga taggttcagc 600ggaagtggat ctgggacaga tttcactttg aagatcagca gggtggaggc tgaagatgtt 660ggagtatatt actgtcaaca ggattatagt agtcctccgt ggactttcgg cggagggacc 720aaggtggaga tcaaa 735175245PRTArtificial Sequencepan-HLA ScFv 175Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys Thr Phe Thr Thr Ser Thr Ser Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Ile Val Leu Thr Gln Ser 130 135 140Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys145 150 155 160Arg Ala Ser Gln Ser Val Ser Asn Asp Leu Ala Trp Tyr Gln Gln Lys 165 170 175Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr 180 185 190Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr 210 215 220Cys Gln Gln Asp Tyr Ser Ser Pro Pro Trp Thr Phe Gly Gln Gly Thr225 230 235 240Lys Val Glu Ile Lys 245176735DNAArtificial Sequencepan-HLA ScFv 176caggtgcagc tggtggagtc cggaggtggc gtagtgcagc ccggaaggag cctgcgcctg 60agctgcgcag tctctggttt ctcattaact agctatggta tgcactgggt tagacaggct 120ccaggaaagg gtctggagtg ggttgcagtg atctggagtg gtggaagcac agactataat 180gctgctttca tatccagatt taccatcagc cgggacaact ccaagaacac actctacctt 240caaatgaaca gtttgagagc tgaagacaca gccgtatact actgtgccaa aacctttact 300acgtctacct cggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctcttca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgagatcgtg 420ctgacccaga gtccagctac cctgtctctg tctccgggag agagagccac cctcagttgc 480agggcgagtc agtctgtgag caacgaccta gcttggtatc agcagaaacc agggcaagct 540cctagactcc tgatctacta tgcatctaat cggtatacag gggtccctga taggttcagc 600ggaagtggat ctgggacaga tttcactctc accatcagca gcctggagcc tgaagatttt 660gcagtatatt actgtcaaca ggattatagt agtcctccgt ggactttcgg ccaagggacc 720aaggtggaga tcaaa 735177245PRTArtificial Sequencepan-HLA ScFv 177Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Thr Phe Thr Thr Ser Thr Ser Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Gln Ala Ser Gln Ser Val Ser Asn Asp Leu Asn Trp Tyr Gln Gln Lys 165 170 175Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr 180 185 190Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr 210 215 220Cys Gln Gln Asp Tyr Ser Ser Pro Pro Trp Thr Phe Gly Gly Gly Thr225 230 235 240Lys Val Glu Ile Lys 245178735DNAArtificial Sequencepan-HLA ScFv 178caggtgcagc tggtggagtc cggaggtggc gtagtgcagc ccggaaggag cctgcgcctg 60agctgcgcag tctctggttt ctcattaact agctatggta tgcactgggt tagacaggct 120ccaggaaagg gtctggagtg ggttgcagtg atctggagtg gtggaagcac agactataat 180gctgctttca tatccagatt taccatcagc cgggacaact ccaagaacac actctacctt 240caaatgaaca gtttgagagc tgaagacaca gccgtatact actgtgccag aacctttact 300acgtctacct cggcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctcttca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatccag 420atgacccaga gtccaagctc cctgtctgcg tctgtgggag acagagtcac catcacttgc 480caggcgagtc agtctgtgag caacgaccta aattggtatc agcagaaacc agggaaagct 540cctaaactcc tgatctacta tgcatctaat cggtatacag gggtccctga taggttcagc 600ggaagtggat ctgggacaga tttcactttc accatcagca gcctgcagcc tgaagatatt 660gcaacatatt actgtcaaca ggattatagt agtcctccgt ggactttcgg cggagggacc 720aaggtggaga tcaaa 735179741DNAArtificial SequenceHLA-A*02 antigen binding domain 179gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240agtagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300cggacgtccg gtggaggcac caagctggaa atcaaaggcg gaggtggaag cggaggggga 360ggatctggcg gcggaggaag cggaggccag gtccagctgc agcagtctgg acctgagctg 420gtgaagcctg gggcttcagt gaggatatcc tgcaaggctt ctggctacac cttcacaagt 480taccatatac attgggtgaa gcagaggcct ggacagggac ttgagtggat tggatggatt 540tatcctggaa atgttaatac tgagtacaat gagaagttca agggcaaggc cacactgact 600gcagacaaat cgtccagcac agcctacatg cacctcagca gcctgacctc tgaggactct 660gcggtctatt tctgtgccag agaggagatt acctatgcta tggactactg gggtcaagga 720acctcagtca ccgtgtcctc a 741180741DNAArtificial SequenceHLA-A*02 antigen binding domain 180caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg cttctggata caccttcact agctatcata tacattgggt gcgccaggcc 120cccggacaag ggcttgagtg gatgggatgg atctaccctg gcaatgttaa cacagaatat 180aatgagaagt tcaagggcaa agccaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagggaggaa 300attacctacg ctatggacta ctggggccag ggaaccacag tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgagat tgtattgacc 420cagagcccag gcaccctgag cctctctcca ggagagcggg ccaccctcag ttgtagatcc 480agtcagagta ttgtacacag taatgggaac acctatttgg aatggtatca gcagaaacca 540ggtcaagccc caagattgct catctacaaa gtctctaaca gatttagtgg tattccagac 600aggttcagcg gttccggaag tggtactgat ttcaccctca cgatctccag gctcgagcca 660gaagatttcg ccgtttatta ctgttttcaa ggttcacatg tgccgcgcac attcggtggg 720ggtactaaag tagaaatcaa a 741181741DNAArtificial SequenceHLA-A*02 antigen binding domain 181caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg cttctggata caccttcact agctatcata tacattgggt gcgccaggcc 120cccggacaag ggcttgagtg gatgggatgg atctaccctg gcaatgttaa cacagaatat 180aatgagaagt tcaagggcaa agccaccatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagggaggaa 300attacctacg ctatggacta ctggggccag ggaaccacag tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgacat tgtaatgacc 420cagaccccac tcagcctgcc cgtcactcca ggagagccgg ccagcatcag ttgtagatcc 480agtcagagta ttgtacacag taatgggaac acctatttgg aatggtatct gcagaaacca 540ggtcaatccc cacaattgct catctacaaa gtctctaaca gatttagtgg tgtaccagac 600aggttcagcg gttccggaag tggtactgat ttcaccctca agatctccag ggtcgaggca 660gaagatgtcg gcgtttatta ctgttttcaa ggttcacatg tgccgcgcac

attcggtggg 720ggtactaaag tagaaatcaa a 741182741DNAArtificial SequenceHLA-A*02 antigen binding domain 182gaggtgcagc tggtggagtc tgggggtggg ctggtgaagc ctgggggctc actgaggctt 60tcctgcgcgg cttctggata caccttcact agctatcata tacattgggt gcgccaggcc 120cccggaaaag ggcttgagtg ggtgggatgg atctaccctg gcaatgttaa cacagaatat 180aatgagaagt tcaagggcag attcaccatt agcagggacg attccaagaa cacactctac 240ctgcagatga acagcctgaa aactgaagac acggctgtgt attactgtgc gagggaggaa 300attacctacg ctatggacta ctggggccag ggaaccacag tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgacat tcaaatgacc 420cagagcccat ccagcctgag cgcatctgta ggtgaccggg tcaccatcac ttgtagatcc 480agtcagagta ttgtacacag taatgggaac acctatttgg aatggtatca gcagaaacca 540ggtaaagccc caaaattgct catctacaaa gtctctaaca gatttagtgg tgtaccaagc 600aggttcagcg gttccggaag tggtactgat ttcaccctca cgatctcctc tctccagcca 660gaagatttcg ccacttatta ctgttttcaa ggttcacatg tgccgcgcac attcggtggg 720ggtactaaag tagaaatcaa a 741183741DNAArtificial SequenceHLA-A*02 antigen binding domain 183caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg cttctggata caccttcact agctatcata tacattgggt gcgccaggcc 120cccggacaag ggcttgagtg gatcggatgg atctaccctg gcaatgttaa cacagaatat 180aatgagaagt tcaagggcaa agccaccatt accgcggacg aatccacgaa cacagcctac 240atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagggaggaa 300attacctacg ctatggacta ctggggccag ggaaccctgg tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgacat tcaaatgacc 420cagagcccat ccaccctgag cgcatctgta ggtgaccggg tcaccatcac ttgtagatcc 480agtcagagta ttgtacacag taatgggaac acctatttgg aatggtatca gcagaaacca 540ggtaaagccc caaaattgct catctacaaa gtctctaaca gatttagtgg tgtaccagcc 600aggttcagcg gttccggaag tggtactgaa ttcaccctca cgatctcctc tctccagcca 660gatgatttcg ccacttatta ctgttttcaa ggttcacatg tgccgcgcac attcggtcag 720ggtactaaag tagaagtcaa a 741184741DNAArtificial SequenceHLA-A*02 antigen binding domain 184caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc agtgaaggtt 60tcctgcaagg cttctggata caccttcact agctatcata tgcattgggt gcgccaggcc 120cccggacaag ggcttgagtg gatcggatac atctaccctg gcaatgttaa cacagaatat 180aatgagaagt tcaagggcaa agccaccctt accgcggaca aatccacgaa cacagcctac 240atggagctga gcagcctgag atctgaagac acggctgtgt atttctgtgc gagggaggaa 300attacctacg ctatggacta ctggggccag ggaaccctgg tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgacgt tcaaatgacc 420cagagcccat ccaccctgag cgcatctgta ggtgaccggg tcaccatcac ttgtagctcc 480agtcagagta ttgtacacag taatgggaac acctatatgg aatggtatca gcagaaacca 540ggtaaagccc caaaattgct catctacaaa gtctctaaca gatttagtgg tgtaccagac 600aggttcagcg gttccggaag tggtactgaa ttcaccctca cgatctcctc tctccagcca 660gatgatttcg ccacttatta ctgtcatcaa ggttcacatg tgccgcgcac attcggtcag 720ggtactaaag tagaagtcaa a 741185741DNAArtificial SequenceHLA-A*02 antigen binding domain 185caggtgcagc tgcagcagtc tgggcctgag ctggtgaagc ctggggcctc agtgaagatg 60tcctgcaagg cttctggata caccttcact agctatcata tccagtgggt gaagcagagg 120cctggacaag ggcttgagtg gatcggatgg atctaccctg gcgatggtag tacacagtat 180aatgagaagt tcaagggcaa aaccaccctt accgcggaca aatcctccag cacagcctac 240atgttgctga gcagcctgac ctctgaagac tctgctatct atttctgtgc gagggagggg 300acctactacg ctatggacta ctggggccag ggaacctcag tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgatgt tttgatgacc 420cagactccac tctccctgcc tgtctctctt ggagaccaag tctccatctc ttgtagatcc 480agtcagagta ttgtacacag taatgggaac acctatttag aatggtatct gcagaaacca 540ggtcagtctc caaagttgct catctacaaa gtctctaaca gatttagtgg tgtaccagac 600aggttcagcg gttccggaag tggtactgat ttcaccctca agatctcgag agtggaggct 660gaggatctgg gagtttatta ctgttttcaa ggttcacatg tgccgcgcac attcggtgga 720ggtactaaac tggaaatcaa a 741186741DNAArtificial SequenceHLA-A*02 antigen binding domain 186cagctgcagc tgcaggagtc tgggcccggg ctggtgaagc cttcggaaac gctgagcctc 60acctgcacgg tttctggata caccttcacc agctatcata tccagtggat ccgacagccc 120cctggaaaag ggcttgagtg gatcggatgg atctaccctg gcgatggttc aacacagtac 180aatgagaagt tcaagggcag agccacgatt agcgtggaca catccaagaa ccaattctcc 240ctgaacctgg acagcgtgag tgctgcggac acggccattt attactgtgc gagagaggga 300acttactacg ctatggacta ctggggcaaa gggagcacgg tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgacat ccagatgacc 420cagagcccaa gctccctgag tgcgtccgtg ggcgaccgcg tgaccatcac ttgcagatcc 480tctcagtcca tcgtgcactc caacggcaac acgtacctcg agtggtacca gcagaagccc 540gggaaggccc cgaaactgct catctacaag gtgagcaacc ggttctccgg cgtccccagc 600cgcttctcag ggtccggctc ggggacggat ttcaccttca cgattagcag cttgcagccc 660gaagacatcg ccacgtacta ctgctttcag ggaagtcacg tgccgcgtac cttcgggccg 720ggcacgaaag tggatattaa g 741187741DNAArtificial SequenceHLA-A*02 antigen binding domain 187gaggtgcagc tggtgcagtc tggggccgag ctgaagaagc ctgggtcctc ggtgaaggtg 60tcctgcaagg cttctggata caccttcacc agctatcata tccagtgggt aaaacaggcc 120cctggacaag ggcttgagtg gatcggatgg atctaccctg gcgatggttc aacacagtac 180aatgagaagt tcaagggcaa agccacgctt accgtggaca aatccacgaa cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtat attactgtgc gagagaggga 300acttactacg ctatggacta ctggggccaa gggaccctgg tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgacat ccagatgacc 420cagagcccat ccaccctgag tgcgtccgtg ggcgaccgcg tgaccatcac ttgcagatcc 480tctcagtcca tcgtgcactc caacggcaac acgtacctcg agtggtacca gcagaagccc 540gggaaggccc cgaaactgct catctacaag gtgagcaacc ggttctccgg cgtccccagc 600cgcttctcag ggtccggctc ggggacggat ttcaccctca cgattagcag cttgcagccc 660gatgacttcg ccacgtacta ctgctttcag ggaagtcacg tgccgcgtac cttcgggcag 720ggcacgaaag tggaagttaa g 741188741DNAArtificial SequenceHLA-A*02 antigen binding domain 188caggtgcagc tggtgcagtc tggggccgag gtgaagaagc ctgggtcctc ggtgaaggtg 60tcctgcaagg cttctggata caccttcacc agctatcata tccagtgggt acgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atctaccctg gcgatggttc aacacagtac 180aatgagaagt tcaagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtat attactgtgc gagagaggga 300acttactacg ctatggacta ctggggccaa gggaccacgg tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgagat cgtcctgacc 420cagagcccag ggaccctgag tttgtccccg ggcgagcgcg cgaccctcag ttgcagatcc 480tctcagtcca tcgtgcactc caacggcaac acgtacctcg agtggtacca gcagaagccc 540gggcaggccc cgcgactgct catctacaag gtgagcaacc ggttctccgg catccccgac 600cgcttctcag ggtccggctc ggggacggat ttcaccctca cgattagccg cttggagccc 660gaagacttcg ccgtgtacta ctgctttcag ggaagtcacg tgccgcgtac cttcgggggg 720ggcacgaaag tggaaattaa g 741189741DNAArtificial SequenceHLA-A*02 antigen binding domain 189caggtgaccc tgaagcagtc tggggccgag gtgaagaagc ctgggtcctc ggtgaaggtg 60tcctgcacgg cttctggata caccttcacc agctatcatg tcagctgggt acgacaggcc 120cctggacaag ggcttgagtg gttgggaagg atctaccctg gcgatggttc aacacagtac 180aatgagaagt tcaagggcaa agtcacgatt accgcggaca aatccatgga cacatccttc 240atggagctga ccagcctgac atctgaggac acggccgtat attactgtgc gagagaggga 300acttactacg ctatggacct ctggggccaa gggaccctgg tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgagat cgtcctgacc 420cagagcccag ggaccctgag tttgtccccg ggcgagcgcg cgaccctcag ttgcagatcc 480tctcagtcca tcgtgcactc caacggcaac acgtacctcg cgtggtacca gcagaagccc 540gggcaggccc cgcgactgct catctccaag gtgagcaacc ggttctccgg cgtccccgac 600cgcttctcag ggtccggctc ggggacggat ttcaccctca cgattagccg cttggagccc 660gaagacttcg ccgtgtacta ctgccaacag ggaagtcacg tgccgcgtac cttcgggggg 720ggcacgaaag tggaaattaa g 741190741DNAArtificial SequenceHLA-A*02 antigen binding domain 190caggtgcagc tggtgcagtc tggggccgag gtgaagaagc ctggggcctc ggtgaaggtg 60tcctgcaagg cttctggata caccttcacc agctatcata tgcactgggt acgacaggcc 120cctggacaaa ggcttgagtg gatgggatgg atctaccctg gcgatggttc aacacagtac 180aatgagaagt tcaagggcaa agtcacgatt acccgggaca catccgcgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtat attactgtgc gagagaggga 300acttactacg ctatggacta ctggggccaa gggaccctgg tcaccgtgtc ctcaggcgga 360ggtggaagcg gagggggagg atctggcggc ggaggaagcg gaggcgacat cgtcatgacc 420cagaccccac tgtccctgcc tgtgaccccg ggcgagcccg cgagcatcag ttgcagatcc 480tctcagtcca tcgtgcactc caacggcaac acgtacctcg actggtacct gcagaagccc 540gggcagtccc cgcaactgct catctacaag gtgagcaacc ggttctccgg cgtccccgac 600cgcttctcag ggtccggctc ggggacggat ttcaccctca agattagccg cgtggaggcc 660gaagacgtcg gcgtgtacta ctgcatgcag ggaagtcacg tgccgcgtac cttcgggggg 720ggcacgaaag tggaaattaa g 7411919PRTArtificial SequenceHA-1 variant H peptide 191Val Leu His Asp Asp Leu Leu Glu Ala1 519219PRTArtificial SequenceP2A self cleaving peptide 192Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn1 5 10 15Pro Gly Pro193608PRTArtificial SequenceHA-1H TCR alpha and beta 193Met Val Lys Ile Arg Gln Phe Leu Leu Ala Ile Leu Trp Leu Gln Leu1 5 10 15Ser Cys Val Ser Ala Ala Lys Asn Glu Val Glu Gln Ser Pro Gln Asn 20 25 30Leu Thr Ala Gln Glu Gly Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser 35 40 45Val Gly Ile Ser Ala Leu His Trp Leu Gln Gln His Pro Gly Gly Gly 50 55 60Ile Val Ser Leu Phe Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg65 70 75 80Leu Ile Ala Thr Ile Asn Ile Gln Glu Lys His Ser Ser Leu His Ile 85 90 95Thr Ala Ser His Pro Arg Asp Ser Ala Val Tyr Ile Cys Ala Val Arg 100 105 110Ser Val Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr 115 120 125Lys Leu Ser Val Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr 130 135 140Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr145 150 155 160Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val 165 170 175Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys 180 185 190Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala 195 200 205Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser 210 215 220Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr225 230 235 240Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile 245 250 255Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu 260 265 270Trp Ser Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala 275 280 285Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Thr Ser Leu Leu Cys 290 295 300Trp Met Ala Leu Cys Leu Leu Gly Ala Asp His Ala Asp Thr Gly Val305 310 315 320Ser Gln Asn Pro Arg His Lys Ile Thr Lys Arg Gly Gln Asn Val Thr 325 330 335Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu Tyr Trp Tyr Arg 340 345 350Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr Phe Gln Asn Glu 355 360 365Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg Phe Ser Ala Glu 370 375 380Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln Arg Thr Glu Gln385 390 395 400Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ile Asp Ser Phe Asn 405 410 415Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu 420 425 430Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala 435 440 445Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly 450 455 460Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu465 470 475 480Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro 485 490 495Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser 500 505 510Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln 515 520 525Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys 530 535 540Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys545 550 555 560Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile 565 570 575Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val 580 585 590Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly 595 600 6051941827DNAArtificial SequenceHA-1(H) TCR alpha and beta 194atggtgaaga tccggcaatt tttgttggct attttgtggc ttcagctaag ctgtgtaagt 60gccgccaaaa atgaagtgga gcagagtcct cagaacctga ctgcccagga aggagaattt 120atcacaatca actgcagtta ctcggtagga ataagtgcct tacactggct gcaacagcat 180ccaggaggag gcattgtttc cttgtttatg ctgagctcag ggaagaagaa gcatggaaga 240ttaattgcca caataaacat acaggaaaag cacagctccc tgcacatcac agcctcccat 300cccagagact ctgccgtcta catctgtgct gtcagaagcg tgtccggggc cggctcctac 360cagctcacct ttgggaaggg gaccaaatta tcagtcattc caaatatcca gaaccctgac 420cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagcggatc cggagccacc 840aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc catgggcacc 900agcctcctct gctggatggc cctgtgtctc ctgggggcag atcacgcaga tactggagtc 960tcccagaacc ccagacacaa gatcacaaag aggggacaga atgtaacttt caggtgtgat 1020ccaatttctg aacacaaccg cctttattgg taccgacaga ccctggggca gggcccagag 1080tttctgactt acttccagaa tgaagctcaa ctagaaaaat caaggctgct cagtgatcgg 1140ttctctgcag agaggcctaa gggatctttc tccaccttgg agatccagcg cacagagcag 1200ggggactcgg ccatgtatct ctgtgccagc agcatcgact ccttcaacga gcagttcttc 1260gggccgggca ccaggctcac ggtcctcgag gacctgaaaa acgtgttccc acccgaggtc 1320gctgtgtttg agccatcaga agcagagatc tcccacaccc aaaaggccac actggtgtgc 1380ctggccacag gcttctaccc cgaccacgtg gagctgagct ggtgggtgaa tgggaaggag 1440gtgcacagtg gggtctgcac agacccgcag cccctcaagg agcagcccgc cctcaatgac 1500tccagatact gcctgagcag ccgcctgagg gtgtcggcca ccttctggca gaacccccgc 1560aaccacttcc gctgtcaagt ccagttctac gggctctcgg agaatgacga gtggacccag 1620gatagggcca aacctgtcac ccagatcgtc agcgccgagg cctggggtag agcagactgt 1680ggcttcacct ccgagtctta ccagcaaggg gtcctgtctg ccaccatcct ctatgagatc 1740ttgctaggga aggccacctt gtatgccgtg ctggtcagtg ccctcgtgct gatggccatg 1800gtcaagagaa aggattccag aggctag 1827195440PRTArtificial SequenceFtcr alpha LIR1 HA-1H 195Met Val Lys Ile Arg Gln Phe Leu Leu Ala Ile Leu Trp Leu Gln Leu1 5 10 15Ser Cys Val Ser Ala Ala Lys Asn Glu Val Glu Gln Ser Pro Gln Asn 20 25 30Leu Thr Ala Gln Glu Gly Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser 35 40 45Val Gly Ile Ser Ala Leu His Trp Leu Gln Gln His Pro Gly Gly Gly 50 55 60Ile Val Ser Leu Phe Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg65 70 75 80Leu Ile Ala Thr Ile Asn Ile Gln Glu Lys His Ser Ser Leu His Ile 85 90 95Thr Ala Ser His Pro Arg Asp Ser Ala Val Tyr Ile Cys Ala Val Arg 100 105 110Ser Val Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr 115 120 125Lys Leu Ser Val Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr 130 135 140Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr145 150 155 160Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val 165 170 175Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys 180 185 190Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala 195 200 205Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser 210 215 220Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr225 230 235

240Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Val Ile Gly Ile Leu 245 250 255Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe Leu Ile 260 265 270Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg Lys 275 280 285Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr Asp 290 295 300Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu305 310 315 320Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly Val Glu 325 330 335Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr Tyr 340 345 350Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser Pro Pro 355 360 365Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu 370 375 380Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln385 390 395 400Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala 405 410 415Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser 420 425 430Ile Tyr Ala Thr Leu Ala Ile His 435 4401961279DNAArtificial SequenceFtcr alpha LIR1 HA-1H 196atggtgaaga tccggcaatt tttgttggct attttgtggc ttcagctaag ctgtgtaagt 60gccgccaaaa atgaagtgga gcagagtcct cagaacctga ctgcccagga aggagaattt 120atcacaatca actgcagtta ctcggtagga ataagtgcct tacactggct gcaacagcat 180ccaggaggag gcattgtttc cttgtttatg ctgagctcag ggaagaagaa gcatggaaga 240ttaattgcca caataaacat acaggaaaag cacagctccc tgcacatcac agcctcccat 300cccagagact ctgccgtcta catctgtgct gtcagaagcg tgtccggggc cggctcctac 360cagctcacct ttgggaaggg gaccaaatta tcagtcattc caaatatcca gaaccctgac 420cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720gatacgaacc taaactttca aaacctgtca gttgtgatcg gcatcttggt ggccgtcatc 780ctactgctcc tcctcctcct cctcctcttc ctcatcctcc gacatcgacg tcagggcaaa 840cactggacat cgacccagag aaaggctgat ttccaacatc ctgcaggggc tgtggggcca 900gagcccacag acagaggcct gcagtggagg tccagcccag ctgccgatgc ccaggaagaa 960aacctctatg ctgccgtgaa gcacacacag cctgaggatg gggtggagat ggacactcgg 1020agcccacacg atgaagaccc ccaggcagtg acgtatgccg aggtgaaaca ctccagacct 1080aggagagaaa tggcctctcc tccttcccca ctgtctgggg aattcctgga cacaaaggac 1140agacaggcgg aagaggacag gcagatggac actgaggctg ctgcatctga agccccccag 1200gatgtgacct acgcccagct gcacagcttg accctcagac gggaggcaac tgagcctcct 1260ccatcccagg aagggccct 1279197466PRTArtificial SequenceFtcr beta LIR1 HA-1H 197Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala1 5 10 15Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr 20 25 30Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His 35 40 45Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe 50 55 60Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu65 70 75 80Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu 85 90 95Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala 100 105 110Ser Ser Ile Asp Ser Phe Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg 115 120 125Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr145 150 155 160Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu225 230 235 240Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270Gly Val Leu Ser Val Val Ile Gly Ile Leu Val Ala Val Ile Leu Leu 275 280 285Leu Leu Leu Leu Leu Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln 290 295 300Gly Lys His Trp Thr Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro305 310 315 320Ala Gly Ala Val Gly Pro Glu Pro Thr Asp Arg Gly Leu Gln Trp Arg 325 330 335Ser Ser Pro Ala Ala Asp Ala Gln Glu Glu Asn Leu Tyr Ala Ala Val 340 345 350Lys His Thr Gln Pro Glu Asp Gly Val Glu Met Asp Thr Arg Ser Pro 355 360 365His Asp Glu Asp Pro Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser 370 375 380Arg Pro Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu385 390 395 400Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp 405 410 415Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln 420 425 430Leu His Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser 435 440 445Gln Glu Gly Pro Ser Pro Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala 450 455 460Ile His4651981401DNAArtificial SequenceFtcr beta LIR1 HA-1H 198atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60actggagtct cccagaaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300acagagcagg gggactcggc catgtatctc tgtgccagca gcatcgactc cttcaacgag 360cagttcttcg ggccgggcac caggctcacg gtcctcgagg acctgaaaaa cgtgttccca 420cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 540gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 600ctcaatgact ccagatactg cctgagcagc cgcctgaggg tgtcggccac cttctggcag 660aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 780gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgt tgtgatcggc 840atcttggtgg ccgtcatcct actgctcctc ctcctcctcc tcctcttcct catcctccga 900catcgacgtc agggcaaaca ctggacatcg acccagagaa aggctgattt ccaacatcct 960gcaggggctg tggggccaga gcccacagac agaggcctgc agtggaggtc cagcccagct 1020gccgatgccc aggaagaaaa cctctatgct gccgtgaagc acacacagcc tgaggatggg 1080gtggagatgg acactcggag cccacacgat gaagaccccc aggcagtgac gtatgccgag 1140gtgaaacact ccagacctag gagagaaatg gcctctcctc cttccccact gtctggggaa 1200ttcctggaca caaaggacag acaggcggaa gaggacaggc agatggacac tgaggctgct 1260gcatctgaag ccccccagga tgtgacctac gcccagctgc acagcttgac cctcagacgg 1320gaggcaactg agcctcctcc atcccaggaa gggccctctc cagctgtgcc cagcatctac 1380gccactctgg ccatccacta g 1401199250PRTArtificial SequenceHA-1H TCR alpha 199Met Val Lys Ile Arg Gln Phe Leu Leu Ala Ile Leu Trp Leu Gln Leu1 5 10 15Ser Cys Val Ser Ala Ala Lys Asn Glu Val Glu Gln Ser Pro Gln Asn 20 25 30Leu Thr Ala Gln Glu Gly Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser 35 40 45Val Gly Ile Ser Ala Leu His Trp Leu Gln Gln His Pro Gly Gly Gly 50 55 60Ile Val Ser Leu Phe Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg65 70 75 80Leu Ile Ala Thr Ile Asn Ile Gln Glu Lys His Ser Ser Leu His Ile 85 90 95Thr Ala Ser His Pro Arg Asp Ser Ala Val Tyr Ile Cys Ala Val Arg 100 105 110Ser Val Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr 115 120 125Lys Leu Ser Val Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr 130 135 140Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr145 150 155 160Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val 165 170 175Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys 180 185 190Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala 195 200 205Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser 210 215 220Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr225 230 235 240Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser 245 250200276PRTArtificial SequenceHA-1H TCR beta 200Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala1 5 10 15Asp His Ala Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr 20 25 30Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His 35 40 45Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe 50 55 60Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu65 70 75 80Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu 85 90 95Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala 100 105 110Ser Ser Ile Asp Ser Phe Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg 115 120 125Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr145 150 155 160Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu225 230 235 240Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270Gly Val Leu Ser 275201750DNAArtificial SequenceHA-1H TCR alpha 201atggtgaaga tccggcaatt tttgttggct attttgtggc ttcagctaag ctgtgtaagt 60gccgccaaaa atgaagtgga gcagagtcct cagaacctga ctgcccagga aggagaattt 120atcacaatca actgcagtta ctcggtagga ataagtgcct tacactggct gcaacagcat 180ccaggaggag gcattgtttc cttgtttatg ctgagctcag ggaagaagaa gcatggaaga 240ttaattgcca caataaacat acaggaaaag cacagctccc tgcacatcac agcctcccat 300cccagagact ctgccgtcta catctgtgct gtcagaagcg tgtccggggc cggctcctac 360cagctcacct ttgggaaggg gaccaaatta tcagtcattc caaatatcca gaaccctgac 420cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720gatacgaacc taaactttca aaacctgtca 750202828DNAArtificial SequenceHA-1H TCR beta 202atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60actggagtct cccagaaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300acagagcagg gggactcggc catgtatctc tgtgccagca gcatcgactc cttcaacgag 360cagttcttcg ggccgggcac caggctcacg gtcctcgagg acctgaaaaa cgtgttccca 420cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 540gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 600ctcaatgact ccagatactg cctgagcagc cgcctgaggg tgtcggccac cttctggcag 660aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 780gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtct 82820313PRTArtificial SequenceNY-ESO CDR 203Cys Ala Ser Ser Leu Gly Leu Gly Tyr Glu Gln Tyr Phe1 5 1020420PRTArtificial SequenceNY-ESO CDR 204Cys Ala Ser Ser Leu Gly Gly Pro Arg Gly Leu Ala Gly Leu Arg Gly1 5 10 15Asp Glu Gln Phe 2020512PRTArtificial SequenceNY-ESO CDR 205Cys Ala Ser Ser Leu Arg Arg Asp Asn Glu Gln Phe1 5 1020619PRTArtificial SequenceMAGE-A3 CDR 206Cys Ala Ser Ser Leu Glu Val Leu Leu Gly Ala Asp Phe Pro Asp Thr1 5 10 15Gln Tyr Phe20718PRTArtificial SequenceMAGE-A3 CDR 207Cys Ala Ser Ser Phe Pro Ala Gly His Gly Ala Asp Leu Asp Asn Glu1 5 10 15Gln Phe20814PRTArtificial SequenceMAGE-A3 CDR 208Cys Ala Ser Ser Glu Ile Thr Gly Arg Ile Gly Glu Gln Phe1 5 1020918PRTArtificial SequenceMAGE-A3 CDR 209Cys Ala Ser Ser Leu Gly Gly Asp Glu Leu Gly Ala Asp Gly Asn Glu1 5 10 15Gln Phe210241PRTArtificial SequenceNY-ESO-1 ScFv 210Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Gln Leu 115 120 125Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140Ser Cys Ala Ala Ser Gly Phe Thr Val Tyr Asp Tyr Met Ser Trp Val145 150 155 160Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser 165 170 175Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ser Tyr Tyr 210 215 220Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser225 230 235 240Ser211723DNAArtificial SequenceNY-ESO-1 ScFv 211gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacagta cccctctcac tttcggcggc 300ggaacaaagg tggagatcaa gggcggaggt ggaagcggag ggggaggatc tggcggcgga 360ggaagcggag gcgaagtgca gctggtggaa agcggcggag gcctggtgca gcctggcggc 420agcctgagac tgtcttgcgc cgccagcggc ttcaccgtgt acgactacat gagctgggtc 480cgccaggccc ctggcaaggg actggaatgg gtgtccgtga tctacagcgg cggcagcacc 540tactacgccg acagcgtgaa gggtcgattc accatcagcc gggacaacag caagaacacc 600ctgtacctgc agatgaacag cctgcgggcc gaggacaccg ccgtgtatta ctgtgcgagg 660tactcctact

actactacta catggacgtc tggggcaaag ggaccacggt caccgtgtcc 720tca 723212244PRTArtificial SequenceMAGE-A3 antigen binding domain 212Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Ser 20 25 30Asn Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Pro Phe Gly Asp Trp Trp Tyr Phe Asp Leu Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys 165 170 175Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln 180 185 190Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220Cys Gln Gln Ser Tyr Ser Phe Val Leu Thr Phe Gly Gly Gly Thr Lys225 230 235 240Val Glu Ile Lys213732DNAArtificial SequenceMAGE-A3 antigen binding domain 213caggtgcagc tgcaggaaag cggccctggc ctggtgaaac ccagcgacac cctgagcctg 60acctgcgccg tgtccggcta cagcatcagc agcagcaatt ggtggggctg gatcagacag 120ccccctggca agggcctgga atggatcggc tacatctact acagcggcag cacctactac 180aaccccagcc tgaagtccag agtgaccatg agcgtggaca ccagcaagaa ccagttcagc 240ctgaagctga gcagcgtgac cgccgtcgat accgctgtgt attactgtgc gagaataccc 300tttggggatt ggtggtactt cgatctctgg ggccgtggca ccctggtcac tgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatccag 420atgacccaga gccccagcag cctgagcgcc agcgtgggcg acagagtgac catcacctgt 480cgggccagcc agtcgatcag cagctacctg aactggtatc agcagaagcc cggcaaggcc 540cccaagctgc tgatctacgc cgccagctcc ctgcagagcg gcgtgccaag cagattcagc 600ggcagcggct ccggcaccga cttcaccctg accatcagca gcctgcagcc cgaggacttc 660gccacctact actgccagca gagttacagt ttcgttctca ctttcggcgg agggaccaag 720gtggagatca aa 732214248PRTArtificial SequenceMAGE-A3 antigen binding domain 214Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Asp Leu Tyr Ser Ser Ser Trp Tyr Cys Asp Ala Phe Asp Ile 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met 130 135 140Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr145 150 155 160Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 180 185 190Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220Thr Tyr Tyr Cys Gln Gln Ser Trp Ala Ser Thr Pro Leu Thr Phe Gly225 230 235 240Gly Gly Thr Lys Val Glu Ile Lys 245215744DNAArtificial SequenceMAGE-A3 antigen binding domain 215caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaaggtg 60tcctgcaagg tgtccggcta caccctgacc gagctgtcga tgcactgggt ccgccaggca 120cctggcaagg gactggaatg gatgggcggc tttgaccccg aggacggcga gacaatctac 180gcccagaaat tccagggcag agtgaccatg accgaggaca ccagcaccga caccgcctac 240atggaactga gcagcctgcg gagcgaggac accgctgtgt attactgtgc aacagatctg 300tatagcagca gctggtactg tgatgctttt gatatctggg gccaagggac aatggtcacc 360gtgtcctcag gcggaggtgg aagcggaggg ggaggatctg gcggcggagg aagcggaggc 420gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 480atcacctgtc gggccagcca gtcgatcagc agctacctga actggtatca gcagaagccc 540ggcaaggccc ccaagctgct gatctacgcc gccagctccc tgcagagcgg cgtgccaagc 600agattcagcg gcagcggctc cggcaccgac ttcaccctga ccatcagcag cctgcagccc 660gaggacttcg ccacctacta ctgccagcag agttgggcca gcacccctct cactttcggc 720ggagggacca aggtggagat caaa 744216245PRTArtificial SequenceHPV E6 antigen binding domain 216Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Asn Gly Val Val Lys Trp Tyr Phe Asp Leu Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys 165 170 175Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln 180 185 190Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220Cys Gln Gln Ser Tyr Ser Thr Pro Leu Phe Pro Phe Gly Gly Gly Thr225 230 235 240Lys Val Glu Ile Lys 245217735DNAArtificial SequenceHPV E6 antigen binding domain 217gaagtgcagc tggtggaaag cggcggaggc ctggtgcagc ctggcggcag cctgagactg 60tcttgcgccg ccagcggctt caccttcagc agctactgga tgcactgggt ccgccaggcc 120cctggcaagg gactggtctg ggtgtctcga atcaacagcg acggcagcag caccagctac 180gccgacagcg tgaagggccg gttcaccatc agccgggaca acgccaagaa caccctgtac 240ctgcagatga acagcctgcg ggccgaggac accgccgtgt attactgtgc cagggagaac 300ggcgtggtga agtggtactt cgacctgtgg ggccgtggca ccctggtcac tgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatccag 420atgacccaga gccccagcag cctgagcgcc agcgtgggcg acagagtgac catcacctgt 480cgggccagcc agtcgatcag cagctacctg aactggtatc agcagaagcc cggcaaggcc 540cccaagctgc tgatctacgc cgccagctcc ctgcagagcg gcgtgccaag cagattcagc 600ggcagcggct ccggcaccga cttcaccctg accatcagca gcctgcagcc cgaggacttc 660gccacctact actgccagca gagttacagt acccctctct ttcccttcgg cggagggacc 720aaggtggaga tcaaa 735218247PRTArtificial SequenceHPV E6 antigen binding domain 218Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Lys Asp Gly Arg Gly Ser Pro Phe Tyr Gly Gly Ala Phe Asp Ile 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met 130 135 140Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr145 150 155 160Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 180 185 190Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu Ile Lys 245219741DNAArtificial SequenceHPV E6 antigen binding domain 219gaagtgcagc tggtggaaag cggcggaggc ctggtgcagc ccggtcgaag cctgagactg 60agctgcgccg ccagcggctt cacctttgac gactacgcca tgcactgggt ccgccaggcc 120cctggcaagg gactggaatg ggtgtccggc atcagctgga acagcggcag catcggctac 180gccgacagcg tgaagggccg gttcaccatc agccgggaca acgccaagaa cagcctgtac 240ctgcagatga acagcctgcg ggccgaggac accgccttgt attactgtgc caaggacggc 300aggggctccc ccttctacgg cggcgccttc gacatctggg gccaagggac aatggtcacc 360gtgtcctcag gcggaggtgg aagcggaggg ggaggatctg gcggcggagg aagcggaggc 420gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 480atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 540gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 600aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 660gaagattttg caacttacta ctgtcaacag agttacagta cccctctcac tttcggcggc 720ggaacaaagg tggagatcaa g 741220251PRTArtificial SequenceHPV E7 antigen binding domain 220Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr Ser Tyr Asp Tyr Leu Leu Asn Pro Tyr Arg Trp Asn 100 105 110Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly145 150 155 160Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 165 170 175Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 180 185 190Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 195 200 205Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 210 215 220Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu225 230 235 240Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 245 250221753DNAArtificial SequenceHPV E7 antigen binding domain 221gaagtgcagc tggtggaaag cggcggaggc ctggtgaaac ctggcggcag cctgagactg 60agctgcgccg ccagcggctt caccttctcg aacgcctgga tgagctgggt ccgccaggcc 120cctggcaagg gactggaatg ggtcggacgg atcaagagca agaccgacgg cggcaccacc 180gactacgctg cccccgtgaa gggccggttc accatcagcc gggacgacag caagaacacc 240ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtatta ctgtaccacc 300tcctacgatt accttctcaa tccttatcgt tggaactggt tcgacccctg gggccaggga 360accctggtca ccgtgtcctc aggcggaggt ggaagcggag ggggaggatc tggcggcgga 420ggaagcggcg gagacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga 480gacagagtca ccatcacttg ccgggcaagt cagagcatta gcagctattt aaattggtat 540cagcagaaac cagggaaagc ccctaagctc ctgatctatg ctgcatccag tttgcaaagt 600ggggtcccat caaggttcag tggcagtgga tctgggacag atttcactct caccatcagc 660agtctgcaac ctgaagattt tgcaacttac tactgtcaac agagttacag tacccctctc 720actttcggcg gcggaacaaa ggtggagatc aag 753222246PRTArtificial SequenceMAGE-A3 antigen binding domain 222Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Met Asp Thr Phe Ser Met Val Thr Leu Phe Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr 130 135 140Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile145 150 155 160Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln 165 170 175Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser 180 185 190Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 210 215 220Tyr Tyr Cys Gln Gln Ser Tyr Ser Trp Pro Leu Thr Phe Gly Gly Gly225 230 235 240Thr Lys Val Glu Ile Lys 245223738DNAArtificial SequenceMAGE-A3 antigen binding domain 223caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ccggcagcag cgtgaaggtg 60tcctgcaagg ccagcggcgg caccttcagc agctacgcca tcagctgggt ccgccaggct 120cctggacagg gactggaatg gatgggcggc atcatcccca tcttcggcac cgccaactac 180gcccagaaat tccagggcag agtgaccatc accgccgacg agagcaccag caccgcctac 240atggaactga gcagccttcg aagcgaggac accgctgtgt attactgtgc cagggacatg 300gacaccttct ccatggtgac cctgttcgac tactggggcc agggcaccct ggtcaccgtg 360tcctcaggcg gaggtggaag cggaggggga ggatctggcg gcggaggaag cggaggcgac 420atccagatga cccagagccc cagcagcctg agcgccagcg tgggcgacag agtgaccatc 480acctgtcggg ccagccagtc gatcagcagc tacctgaact ggtatcagca gaagcccggc 540aaggccccca agctgctgat ctacgccgcc agctccctgc agagcggcgt gccaagcaga 600ttcagcggca gcggctccgg caccgacttc accctgacca tcagcagcct gcagcccgag 660gacttcgcca cctactactg ccagcagagt tacagttggc ctctcacttt cggcggaggg 720accaaggtgg agatcaaa 738224245PRTArtificial SequenceKRAS G12V antigen binding domain 224Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Cys Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser 50 55 60Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Tyr Asp Glu Leu Tyr Tyr Phe Asp

Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Arg Ala Ser Gln Ser Ile Trp Thr Ser Tyr Leu Asn Trp Tyr Gln Gln 165 170 175Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu 180 185 190Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 210 215 220Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr225 230 235 240Lys Val Glu Ile Lys 245225735DNAArtificial SequenceKRAS G12V antigen binding domain 225caggtcacac tgagagagtc cggccctgcc ctggtgaaac ccacccagac cctgaccctg 60acatgcacct tcagcggctt cagcctgagc accagcggga tgtgcgtgtc ctggattcga 120cagccccctg gcaaggccct ggaatggctg gccctgattg actgggacga cgacaagtac 180tacagcacca gcctgaaaac ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cgtattactg tgcacggagt 300tacgacgagc tctactactt tgactactgg ggccagggaa ccctggtcac cgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatccag 420atgacccaga gccccagctc cctctctgca tctgtgggcg acagagtgac catcacctgt 480cgggccagcc agtcgatctg gaccagctac ctgaactggt atcagcagaa gcccggcaag 540gcccccaagc tgctgatcta cgccgccagc tccctgcaga gcggcgtgcc aagcagattc 600agcggcagcg gctccggcac cgacttcacc ctgaccatca gcagcctgca gcccgaggac 660ttcgccacct actactgcca gcagagttac agtacccctc tcactttcgg cggagggacc 720aaggtggaga tcaaa 7352265PRTArtificial Sequencelinker 226Gly Gly Gly Gly Ser1 522710PRTArtificial Sequencelinker 227Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 1022815PRTArtificial Sequencelinker 228Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 1522915PRTArtificial Sequencelinker 229Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 1523020PRTArtificial Sequencelinker 230Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser 202314PRTArtificial Sequencelinker 231Gly Gly Gly Ser1232247PRTArtificial SequenceKRAS ScFv 232Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Phe Thr Arg Asp Tyr Tyr Tyr Tyr Tyr Tyr Met Asp Val 100 105 110Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met 130 135 140Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr145 150 155 160Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr 165 170 175Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 180 185 190Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly225 230 235 240Gly Thr Lys Val Glu Ile Lys 245233741DNAArtificial SequenceKRAS ScFv 233caggtgcagc tggtggaaag cggcggaggc ctggtgaaac ctggcggcag cctgagactg 60agctgcgccg ccagcggctt caccttcagc gactactaca tgagctggat cagacaggcc 120cctggcaagg gactggaatg ggtgtcctac atcagcagca gcggctcgac catctactac 180gccgacagcg tgaagggccg gttcaccatc agccgggaca acgccaagaa cagcctgtac 240ctgcagatga acagcctgcg ggccgaggac accgccgtgt attactgtgc cagggacttc 300accagggact actactacta ctactacatg gacgtgtggg gcaaagggac cacggtcacc 360gtgtcctcag gcggaggtgg aagcggaggg ggaggatctg gcggcggagg aagcggaggc 420gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 480atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 540gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 600aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 660gaagattttg caacttacta ctgtcaacag agttacagta cccctctcac tttcggcggc 720ggaacaaagg tggagatcaa g 741234244PRTArtificial SequenceKRAS ScFv 234Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Cys Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser 50 55 60Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Tyr Asp Glu Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys 165 170 175Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln 180 185 190Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys225 230 235 240Val Glu Ile Lys235732DNAArtificial SequenceKRAS ScFv 235caggtcacac tgagagagtc cggccctgcc ctggtgaaac ccacccagac cctgaccctg 60acatgcacct tcagcggctt cagcctgagc accagcggga tgtgcgtgtc ctggattcga 120cagccccctg gcaaggccct ggaatggctg gccctgattg actgggacga cgacaagtac 180tacagcacca gcctgaaaac ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cgtattactg tgcacggagt 300tacgacgagc tctactactt tgactactgg ggccagggaa ccctggtcac cgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatccag 420atgacccagt ctccatcctc cctgtctgca tctgtaggag acagagtcac catcacttgc 480cgggcaagtc agagcattag cagctattta aattggtatc agcagaaacc agggaaagcc 540cctaagctcc tgatctatgc tgcatccagt ttgcaaagtg gggtcccatc aaggttcagt 600ggcagtggat ctgggacaga tttcactctc accatcagca gtctgcaacc tgaagatttt 660gcaacttact actgtcaaca gagttacagt acccctctca ctttcggcgg cggaacaaag 720gtggagatca ag 732236245PRTArtificial SequenceKRAS ScFv 236Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Cys Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser 50 55 60Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Tyr Asp Glu Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Arg Ala Ser Gln Ser Ile Trp Thr Ser Tyr Leu Asn Trp Tyr Gln Gln 165 170 175Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu 180 185 190Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 210 215 220Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr225 230 235 240Lys Val Glu Ile Lys 245237735DNAArtificial SequenceKRAS ScFv 237caggtcacac tgagagagtc cggccctgcc ctggtgaaac ccacccagac cctgaccctg 60acatgcacct tcagcggctt cagcctgagc accagcggga tgtgcgtgtc ctggattcga 120cagccccctg gcaaggccct ggaatggctg gccctgattg actgggacga cgacaagtac 180tacagcacca gcctgaaaac ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cgtattactg tgcacggagt 300tacgacgagc tctactactt tgactactgg ggccagggaa ccctggtcac cgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatccag 420atgacccaga gccccagctc cctctctgca tctgtgggcg acagagtgac catcacctgt 480cgggccagcc agtcgatctg gaccagctac ctgaactggt atcagcagaa gcccggcaag 540gcccccaagc tgctgatcta cgccgccagc tccctgcaga gcggcgtgcc aagcagattc 600agcggcagcg gctccggcac cgacttcacc ctgaccatca gcagcctgca gcccgaggac 660ttcgccacct actactgcca gcagagttac agtacccctc tcactttcgg cggagggacc 720aaggtggaga tcaaa 735238244PRTArtificial SequenceKRAS ScFv 238Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Cys Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser 50 55 60Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Tyr Asp Glu Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser 130 135 140Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys145 150 155 160Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys 165 170 175Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln 180 185 190Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220Cys Gln Gln Ser Tyr Ser Thr Arg Leu Thr Phe Gly Gly Gly Thr Lys225 230 235 240Val Glu Ile Lys239732DNAArtificial SequenceKRAS ScFv 239caggtcacac tgagagagtc cggccctgcc ctggtgaaac ccacccagac cctgaccctg 60acatgcacct tcagcggctt cagcctgagc accagcggga tgtgcgtgtc ctggattcga 120cagccccctg gcaaggccct ggaatggctg gccctgattg actgggacga cgacaagtac 180tacagcacca gcctgaaaac ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cgtattactg tgcacggagt 300tacgacgagc tctactactt tgactactgg ggccagggaa ccctggtcac cgtgtcctca 360ggcggaggtg gaagcggagg gggaggatct ggcggcggag gaagcggagg cgacatccag 420atgacccaga gccccagctc cctctctgca tctgtgggcg acagagtgac catcacctgt 480cgggccagcc agtcgatcag cagctacctg aactggtatc agcagaagcc cggcaaggcc 540cccaagctgc tgatctacgc cgccagctcc ctgcagagcg gcgtgccaag cagattcagc 600ggcagcggct ccggcaccga cttcaccctg accatcagca gcctgcagcc cgaggacttc 660gccacctact actgccagca gagttacagt acccggctca ctttcggcgg agggaccaag 720gtggagatca aa 732240244PRTArtificial SequenceKRAS TCR alpha 240Met Gln Arg Asn Leu Gly Ala Val Leu Gly Ile Leu Trp Val Gln Ile1 5 10 15Cys Trp Val Arg Gly Asp Gln Val Glu Gln Ser Pro Ser Ala Leu Ser 20 25 30Leu His Glu Gly Thr Asp Ser Ala Leu Arg Cys Asn Phe Thr Thr Thr 35 40 45Met Arg Ser Val Gln Trp Phe Arg Gln Asn Ser Arg Gly Ser Leu Ile 50 55 60Ser Leu Phe Tyr Leu Ala Ser Gly Thr Lys Glu Asn Gly Arg Leu Lys65 70 75 80Ser Ala Phe Asp Ser Lys Glu Arg Arg Tyr Ser Thr Leu His Ile Arg 85 90 95Asp Ala Gln Leu Glu Asp Ser Gly Thr Tyr Phe Cys Ala Ala Asp Ser 100 105 110Ser Asn Thr Gly Tyr Gln Asn Phe Tyr Phe Gly Lys Gly Thr Ser Leu 115 120 125Thr Val Ile Pro Asn Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu 130 135 140Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe145 150 155 160Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile 165 170 175Thr Asp Lys Cys Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn 180 185 190Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile 195 200 205Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp 210 215 220Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe225 230 235 240Gln Asn Leu Ser241732DNAArtificial SequenceKRAS TCR alpha 241atgcagagga acctgggagc tgtgctgggg attctgtggg tgcagatttg ctgggtgaga 60ggggatcagg tggagcagag tccttcagcc ctgagcctcc acgagggaac cgattctgct 120ctgagatgca attttacgac caccatgagg agtgtgcagt ggttccgaca gaattccagg 180ggcagcctca tcagtttgtt ctacttggct tcaggaacaa aggagaatgg gaggctaaag 240tcagcatttg attctaagga gcggcgctac agcaccctgc acatcaggga tgcccagctg 300gaggactcag gcacttactt ctgtgctgct gactcttcga acacgggtta ccagaacttc 360tattttggga aaggaacaag tttgactgtc attccaaaca tccagaaccc agaacctgct 420gtgtaccagt taaaagatcc tcggtctcag gacagcaccc tctgcctgtt caccgacttt 480gactcccaaa tcaatgtgcc gaaaaccatg gaatctggaa cgttcatcac tgacaaatgt 540gtgctggaca tgaaagctat ggattccaag agcaatgggg ccattgcctg gagcaaccag 600acaagcttca cctgccaaga tatcttcaaa gagaccaacg ccacctaccc cagttcagac 660gttccctgtg atgccacgtt gaccgagaaa agctttgaaa cagatatgaa cctgaacttt 720caaaacctgt ct 732242283PRTArtificial SequenceKRAS TCR beta 242Met Ser Asn Thr Ala Phe Pro Asp Pro Ala Trp Asn Thr Thr Leu Leu1 5 10 15Ser Trp Val Ala Leu Phe Leu Leu Gly Thr Ser Ser Ala Asn Ser Gly 20 25 30Val Val Gln Ser Pro Arg Tyr Ile Ile Lys Gly Lys Gly Glu Arg Ser 35 40 45Ile Leu Lys Cys Ile Pro Ile Ser Gly His Leu Ser Val Ala Trp Tyr 50 55 60Gln Gln Thr Gln Gly Gln Glu Leu Lys Phe Phe Ile Gln His Tyr Asp65 70 75 80Lys Met Glu Arg Asp Lys Gly Asn Leu Pro Ser Arg Phe Ser Val Gln 85 90 95Gln Phe Asp Asp Tyr His Ser Glu Met Asn Met Ser Ala Leu Glu Leu 100 105 110Glu Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu Thr Asp Pro Leu 115 120 125Asp Ser Asp Tyr Thr Phe Gly Ser Gly Thr Arg Leu Leu Val Ile Glu 130 135 140Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro Ser145 150 155 160Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu Ala

165 170 175Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 180 185 190Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Ala Tyr Lys Glu 195 200 205Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr 210 215 220Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe His225 230 235 240Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro Val 245 250 255Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile 260 265 270Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser 275 280243849DNAArtificial SequenceKRAS TCR beta 243atgtctaaca ctgccttccc tgaccccgcc tggaacacca ccctgctatc ttgggttgct 60ctctttctcc tgggaacaag ttcagcaaat tctggggttg tccagtctcc aagatacata 120atcaaaggaa agggagaaag gtccattcta aaatgtattc ccatctctgg acatctctct 180gtggcctggt atcaacagac tcaggggcag gaactaaagt tcttcattca gcattatgat 240aaaatggaga gagataaagg aaacctgccc agcagattct cagtccaaca gtttgatgac 300tatcactctg agatgaacat gagtgccttg gagctagagg actctgccgt gtacttctgt 360gccagctctc tcacagatcc gctagactcc gactacacct tcggctcagg gaccaggctt 420ttggtaatag aggatctgag aaatgtgact ccacccaagg tctccttgtt tgagccatca 480aaagcagaga ttgcaaacaa acaaaaggct accctcgtgt gcttggccag gggcttcttc 540cctgaccacg tggagctgag ctggtgggtg aatggcaagg aggtccacag tggggtctgc 600acggaccctc aggcctacaa ggagagcaat tatagctact gcctgagcag ccgcctgagg 660gtctctgcta ccttctggca caatcctcgc aaccacttcc gctgccaagt gcagttccat 720gggctttcag aggaggacaa gtggccagag ggctcaccca aacctgtcac acagaacatc 780agtgcagagg cctggggccg agcagactgt gggattacct cagcatccta tcaacaaggg 840gtcttgtct 849244283PRTArtificial SequenceKRAS TCR beta 244Met Ser Asn Thr Ala Phe Pro Asp Pro Ala Trp Asn Thr Thr Leu Leu1 5 10 15Ser Trp Val Ala Leu Phe Leu Leu Gly Thr Ser Ser Ala Asn Ser Gly 20 25 30Val Val Gln Ser Pro Arg Tyr Ile Ile Lys Gly Lys Gly Glu Arg Ser 35 40 45Ile Leu Lys Cys Ile Pro Ile Ser Gly His Leu Ser Val Ala Trp Tyr 50 55 60Gln Gln Thr Gln Gly Gln Glu Leu Lys Phe Phe Ile Gln His Tyr Asp65 70 75 80Lys Met Glu Arg Asp Lys Gly Asn Leu Pro Ser Arg Phe Ser Val Gln 85 90 95Gln Phe Asp Asp Tyr His Ser Glu Met Asn Met Ser Ala Leu Glu Leu 100 105 110Glu Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu Thr Asp Pro Leu 115 120 125Asp Ser Asp Tyr Thr Phe Gly Ser Gly Thr Arg Leu Leu Val Ile Glu 130 135 140Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro Ser145 150 155 160Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu Ala 165 170 175Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 180 185 190Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Ala Tyr Lys Glu 195 200 205Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr 210 215 220Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe His225 230 235 240Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro Val 245 250 255Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile 260 265 270Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser 275 280245849DNAArtificial SequenceKRAS TCR beta 245atgtctaaca ctgccttccc tgaccccgcc tggaacacca ccctgctatc ttgggttgct 60ctctttctcc tgggaacaag ttcagcaaat tctggggttg tccagtctcc aagatacata 120atcaaaggaa agggagaaag gtccattcta aaatgtattc ccatctctgg acatctctct 180gtggcctggt atcaacagac tcaggggcag gaactaaagt tcttcattca gcattatgat 240aaaatggaga gagataaagg aaacctgccc agcagattct cagtccaaca gtttgatgac 300tatcactctg agatgaacat gagtgccttg gagctagagg actctgccgt gtacttctgt 360gccagctctc tcacagatcc gctagactcc gactacacct tcggctcagg gaccaggctt 420ttggtaatag aggatctgag aaatgtgact ccacccaagg tctccttgtt tgagccatca 480aaagcagaga ttgcaaacaa acaaaaggct accctcgtgt gcttggccag gggcttcttc 540cctgaccacg tggagctgag ctggtgggtg aatggcaagg aggtccacag tggggtctgc 600acggaccctc aggcctacaa ggagagcaat tatagctact gcctgagcag ccgcctgagg 660gtctctgcta ccttctggca caatcctcgc aaccacttcc gctgccaagt gcagttccat 720gggctttcag aggaggacaa gtggccagag ggctcaccca aacctgtcac acagaacatc 780agtgcagagg cctggggccg agcagactgt gggattacct cagcatccta tcaacaaggg 840gtcttgtct 849246248PRTArtificial SequenceKRAS TCRa 246Met Lys Thr Val Thr Gly Pro Leu Phe Leu Cys Phe Trp Leu Gln Leu1 5 10 15Asn Cys Val Ser Arg Gly Glu Gln Val Glu Gln Arg Pro Pro His Leu 20 25 30Ser Val Arg Glu Gly Asp Ser Ala Val Ile Thr Cys Thr Tyr Thr Asp 35 40 45Pro Asn Ser Tyr Tyr Phe Phe Trp Tyr Lys Gln Glu Pro Gly Ala Ser 50 55 60Leu Gln Leu Leu Met Lys Val Phe Ser Ser Thr Glu Ile Asn Glu Gly65 70 75 80Gln Gly Phe Thr Val Leu Leu Asn Lys Lys Asp Lys Arg Leu Ser Leu 85 90 95Asn Leu Thr Ala Ala His Pro Gly Asp Ser Ala Ala Tyr Phe Cys Ala 100 105 110Val Ser Gly Gly Thr Asn Ser Ala Gly Asn Lys Leu Thr Phe Gly Ile 115 120 125Gly Thr Arg Val Leu Val Arg Pro Asp Ile Gln Asn Pro Glu Pro Ala 130 135 140Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu145 150 155 160Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser 165 170 175Gly Thr Phe Ile Thr Asp Lys Cys Val Leu Asp Met Lys Ala Met Asp 180 185 190Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr 195 200 205Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 210 215 220Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met225 230 235 240Asn Leu Asn Phe Gln Asn Leu Ser 245247744DNAArtificial SequenceKRAS TCRa 247atgaagacgg tgactggacc tttgttcctg tgcttctggc tgcagctgaa ctgtgtgagc 60agaggcgagc aggtggagca gcgccctcct cacctgagtg tccgggaggg agacagtgcc 120gttatcacct gcacctacac agaccctaac agttattact tcttctggta caagcaagag 180ccgggggcaa gtcttcagtt gcttatgaag gttttctcaa gtacggaaat aaacgaagga 240caaggattca ctgtcctact gaacaagaaa gacaaacgac tctctctgaa cctcacagct 300gcccatcctg gggactcagc cgcgtacttc tgcgcagtca gtggagggac taacagtgca 360gggaacaagc taacttttgg aattggaacc agggtgctgg tcaggccaga catccagaac 420ccagaacctg ctgtgtacca gttaaaagat cctcggtctc aggacagcac cctctgcctg 480ttcaccgact ttgactccca aatcaatgtg ccgaaaacca tggaatctgg aacgttcatc 540actgacaaat gtgtgctgga catgaaagct atggattcca agagcaatgg ggccattgcc 600tggagcaacc agacaagctt cacctgccaa gatatcttca aagagaccaa cgccacctac 660cccagttcag acgttccctg tgatgccacg ttgaccgaga aaagctttga aacagatatg 720aacctgaact ttcaaaacct gtct 744248272PRTArtificial SequenceKRAS TCRb 248Met Gly Cys Arg Leu Leu Ser Cys Val Ala Phe Cys Leu Leu Gly Ile1 5 10 15Gly Pro Leu Glu Thr Ala Val Phe Gln Thr Pro Asn Tyr His Val Thr 20 25 30Gln Val Gly Asn Glu Val Ser Phe Asn Cys Lys Gln Thr Leu Gly His 35 40 45Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Leu Leu Lys Ile 50 55 60Met Phe Ser Tyr Asn Asn Lys Gln Leu Ile Val Asn Glu Thr Val Pro65 70 75 80Arg Arg Phe Ser Pro Gln Ser Ser Asp Lys Ala His Leu Asn Leu Arg 85 90 95Ile Lys Ser Val Glu Pro Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser 100 105 110Ser Arg Asp Trp Gly Pro Ala Glu Gln Phe Phe Gly Pro Gly Thr Arg 115 120 125Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser 130 135 140Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr145 150 155 160Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu 195 200 205Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys 210 215 220Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly225 230 235 240Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg 245 250 255Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser 260 265 270249816DNAArtificial SequenceKRAS TCRb 249atgggctgta ggctcctaag ctgtgtggcc ttctgcctct tgggaatagg ccctttggag 60acagctgttt tccagactcc aaactatcat gtcacacagg tgggaaatga agtgtctttc 120aattgtaagc aaactctggg ccacgatact atgtattggt acaagcaaga ctctaagaaa 180ttgctgaaga ttatgtttag ctacaataat aagcaactca ttgtaaacga aacagttcca 240aggcgcttct cacctcagtc ttcagataaa gctcatttga atcttcgaat caagtctgta 300gagccggagg actctgctgt gtatctctgt gccagcagtc gggactgggg gcctgctgag 360cagttcttcg gaccagggac acgactcacc gtcctagagg atctgagaaa tgtgactcca 420cccaaggtct ccttgtttga gccatcaaaa gcagagattg caaacaaaca aaaggctacc 480ctcgtgtgct tggccagggg cttcttccct gaccacgtgg agctgagctg gtgggtgaat 540ggcaaggagg tccacagtgg ggtctgcacg gaccctcagg cctacaagga gagcaattat 600agctactgcc tgagcagccg cctgagggtc tctgctacct tctggcacaa tcctcgaaac 660cacttccgct gccaagtgca gttccatggg ctttcagagg aggacaagtg gccagagggc 720tcacccaaac ctgtcacaca gaacatcagt gcagaggcct ggggccgagc agactgtgga 780atcacttcag catcctatca tcagggggtt ctgtct 816250272PRTArtificial SequenceKRAS TCRb 250Met Gly Cys Arg Leu Leu Ser Cys Val Ala Phe Cys Leu Leu Gly Ile1 5 10 15Gly Pro Leu Glu Thr Ala Val Phe Gln Thr Pro Asn Tyr His Val Thr 20 25 30Gln Val Gly Asn Glu Val Ser Phe Asn Cys Lys Gln Thr Leu Gly His 35 40 45Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Leu Leu Lys Ile 50 55 60Met Phe Ser Tyr Asn Asn Lys Gln Leu Ile Val Asn Glu Thr Val Pro65 70 75 80Arg Arg Phe Ser Pro Gln Ser Ser Asp Lys Ala His Leu Asn Leu Arg 85 90 95Ile Lys Ser Val Glu Pro Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser 100 105 110Ser Arg Asp Trp Gly Pro Ala Glu Gln Phe Phe Gly Pro Gly Thr Arg 115 120 125Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser 130 135 140Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr145 150 155 160Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu 195 200 205Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys 210 215 220Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly225 230 235 240Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg 245 250 255Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser 260 265 270251816DNAArtificial SequenceKRAS TCRb 251atgggctgta ggctcctaag ctgtgtggcc ttctgcctct tgggaatagg ccctttggag 60acagctgttt tccagactcc aaactatcat gtcacacagg tgggaaatga agtgtctttc 120aattgtaagc aaactctggg ccacgatact atgtattggt acaagcaaga ctctaagaaa 180ttgctgaaga ttatgtttag ctacaataat aagcaactca ttgtaaacga aacagttcca 240aggcgcttct cacctcagtc ttcagataaa gctcatttga atcttcgaat caagtctgta 300gagccggagg actctgctgt gtatctctgt gccagcagtc gggactgggg gcctgctgag 360cagttcttcg gaccagggac acgactcacc gtcctagagg atctgagaaa tgtgactcca 420cccaaggtct ccttgtttga gccatcaaaa gcagagattg caaacaaaca aaaggctacc 480ctcgtgtgct tggccagggg cttcttccct gaccacgtgg agctgagctg gtgggtgaat 540ggcaaggagg tccacagtgg ggtctgcacg gaccctcagg cctacaagga gagcaattat 600agctactgcc tgagcagccg cctgagggtc tctgctacct tctggcacaa tcctcgaaac 660cacttccgct gccaagtgca gttccatggg ctttcagagg aggacaagtg gccagagggc 720tcacccaaac ctgtcacaca gaacatcagt gcagaggcct ggggccgagc agactgtgga 780atcacttcag catcctatca tcagggggtt ctgtct 816252244PRTArtificial SequenceTCRa control 252Met Gln Arg Asn Leu Gly Ala Val Leu Gly Ile Leu Trp Val Gln Ile1 5 10 15Cys Trp Val Arg Gly Asp Gln Val Glu Gln Ser Pro Ser Ala Leu Ser 20 25 30Leu His Glu Gly Thr Asp Ser Ala Leu Arg Cys Asn Phe Thr Thr Thr 35 40 45Met Arg Ser Val Gln Trp Phe Arg Gln Asn Ser Arg Gly Ser Leu Ile 50 55 60Ser Leu Phe Tyr Leu Ala Ser Gly Thr Lys Glu Asn Gly Arg Leu Lys65 70 75 80Ser Ala Phe Asp Ser Lys Glu Arg Arg Tyr Ser Thr Leu His Ile Arg 85 90 95Asp Ala Gln Leu Glu Asp Ser Gly Thr Tyr Phe Cys Ala Ala Asp Ser 100 105 110Ser Asn Thr Gly Tyr Gln Asn Phe Tyr Phe Gly Lys Gly Thr Ser Leu 115 120 125Thr Val Ile Pro Asn Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu 130 135 140Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe145 150 155 160Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile 165 170 175Thr Asp Lys Cys Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn 180 185 190Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile 195 200 205Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp 210 215 220Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe225 230 235 240Gln Asn Leu Ser253732DNAArtificial SequenceTCRa control 253atgcagagga acctgggagc tgtgctgggg attctgtggg tgcagatttg ctgggtgaga 60ggggatcagg tggagcagag tccttcagcc ctgagcctcc acgagggaac cgattctgct 120ctgagatgca attttacgac caccatgagg agtgtgcagt ggttccgaca gaattccagg 180ggcagcctca tcagtttgtt ctacttggct tcaggaacaa aggagaatgg gaggctaaag 240tcagcatttg attctaagga gcggcgctac agcaccctgc acatcaggga tgcccagctg 300gaggactcag gcacttactt ctgtgctgct gactcttcga acacgggtta ccagaacttc 360tattttggga aaggaacaag tttgactgtc attccaaaca tccagaaccc agaacctgct 420gtgtaccagt taaaagatcc tcggtctcag gacagcaccc tctgcctgtt caccgacttt 480gactcccaaa tcaatgtgcc gaaaaccatg gaatctggaa cgttcatcac tgacaaatgt 540gtgctggaca tgaaagctat ggattccaag agcaatgggg ccattgcctg gagcaaccag 600acaagcttca cctgccaaga tatcttcaaa gagaccaacg ccacctaccc cagttcagac 660gttccctgtg atgccacgtt gaccgagaaa agctttgaaa cagatatgaa cctgaacttt 720caaaacctgt ct 732254248PRTArtificial SequenceTCRa control 254Met Lys Thr Val Thr Gly Pro Leu Phe Leu Cys Phe Trp Leu Gln Leu1 5 10 15Asn Cys Val Ser Arg Gly Glu Gln Val Glu Gln Arg Pro Pro His Leu 20 25 30Ser Val Arg Glu Gly Asp Ser Ala Val Ile Thr Cys Thr Tyr Thr Asp 35 40 45Pro Asn Ser Tyr Tyr Phe Phe Trp Tyr Lys Gln Glu Pro Gly Ala Ser 50 55 60Leu Gln Leu Leu Met Lys Val Phe Ser Ser Thr Glu Ile Asn Glu Gly65 70 75 80Gln Gly Phe Thr Val Leu Leu Asn Lys Lys Asp Lys Arg Leu Ser Leu 85 90 95Asn Leu Thr Ala Ala His Pro Gly Asp Ser Ala Ala Tyr Phe Cys Ala 100 105 110Val Ser Gly Gly Thr Asn Ser Ala Gly Asn Lys Leu Thr Phe Gly Ile 115 120 125Gly Thr Arg Val Leu Val Arg Pro Asp Ile Gln Asn Pro Glu Pro Ala 130 135

140Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu145 150 155 160Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser 165 170 175Gly Thr Phe Ile Thr Asp Lys Cys Val Leu Asp Met Lys Ala Met Asp 180 185 190Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr 195 200 205Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 210 215 220Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met225 230 235 240Asn Leu Asn Phe Gln Asn Leu Ser 245255744DNAArtificial SequenceTCRa control 255atgaagacgg tgactggacc tttgttcctg tgcttctggc tgcagctgaa ctgtgtgagc 60agaggcgagc aggtggagca gcgccctcct cacctgagtg tccgggaggg agacagtgcc 120gttatcacct gcacctacac agaccctaac agttattact tcttctggta caagcaagag 180ccgggggcaa gtcttcagtt gcttatgaag gttttctcaa gtacggaaat aaacgaagga 240caaggattca ctgtcctact gaacaagaaa gacaaacgac tctctctgaa cctcacagct 300gcccatcctg gggactcagc cgcgtacttc tgcgcagtca gtggagggac taacagtgca 360gggaacaagc taacttttgg aattggaacc agggtgctgg tcaggccaga catccagaac 420ccagaacctg ctgtgtacca gttaaaagat cctcggtctc aggacagcac cctctgcctg 480ttcaccgact ttgactccca aatcaatgtg ccgaaaacca tggaatctgg aacgttcatc 540actgacaaat gtgtgctgga catgaaagct atggattcca agagcaatgg ggccattgcc 600tggagcaacc agacaagctt cacctgccaa gatatcttca aagagaccaa cgccacctac 660cccagttcag acgttccctg tgatgccacg ttgaccgaga aaagctttga aacagatatg 720aacctgaact ttcaaaacct gtct 7442569PRTArtificial SequencemH-Y-2Db peptide 256Lys Cys Ser Arg Asn Arg Gln Tyr Leu1 5257614PRTArtificial SequenceH-Y TCR alpha and beta 257Met Phe Pro Val Thr Ile Leu Leu Leu Ser Ala Phe Phe Ser Leu Arg1 5 10 15Gly Asn Ser Ala Gln Ser Val Asp Gln Pro Asp Ala His Val Thr Leu 20 25 30Ser Glu Gly Ala Ser Leu Glu Leu Arg Cys Ser Tyr Ser Tyr Ser Ala 35 40 45Ala Pro Tyr Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln Ser Leu Gln 50 55 60Phe Leu Leu Lys Tyr Ile Thr Gly Asp Thr Val Val Lys Gly Thr Lys65 70 75 80Gly Phe Glu Ala Glu Phe Arg Lys Ser Asn Ser Ser Phe Asn Leu Lys 85 90 95Lys Ser Pro Ala His Trp Ser Asp Ser Ala Lys Tyr Phe Cys Ala Leu 100 105 110Glu Gly Gln Asp Gln Gly Gly Ser Ala Lys Leu Ile Phe Gly Glu Gly 115 120 125Thr Lys Leu Thr Val Ser Ser Pro Asp Ile Gln Asn Pro Glu Pro Ala 130 135 140Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu145 150 155 160Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser 165 170 175Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp 180 185 190Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr 195 200 205Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 210 215 220Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met225 230 235 240Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu 245 250 255Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 275 280 285Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Asn Thr 290 295 300Ala Phe Pro Asp Pro Ala Trp Asn Thr Thr Leu Leu Ser Trp Val Ala305 310 315 320Leu Phe Leu Leu Gly Thr Lys His Met Glu Ala Ala Val Thr Gln Ser 325 330 335Pro Arg Asn Lys Val Ala Val Thr Gly Gly Lys Val Thr Leu Ser Cys 340 345 350Asn Gln Thr Asn Asn His Asn Asn Met Tyr Trp Tyr Arg Gln Asp Thr 355 360 365Gly His Gly Leu Arg Leu Ile His Tyr Ser Tyr Gly Ala Gly Ser Thr 370 375 380Glu Lys Gly Asp Ile Pro Asp Gly Tyr Lys Ala Ser Arg Pro Ser Gln385 390 395 400Glu Asn Phe Ser Leu Ile Leu Glu Leu Ala Thr Pro Ser Gln Thr Ser 405 410 415Val Tyr Phe Cys Ala Ser Gly Asp Asn Ser Ala Glu Thr Leu Tyr Phe 420 425 430Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr 435 440 445Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn 450 455 460Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp465 470 475 480His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly 485 490 495Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys 500 505 510Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg 515 520 525Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp 530 535 540Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala545 550 555 560Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His 565 570 575Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys 580 585 590Ala Thr Leu Tyr Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met 595 600 605Val Lys Lys Lys Asn Ser 6102581845DNAArtificial SequenceH-Y TCR alpha and beta 258atgttccccg ttaccatatt gctcctgagt gctttcttca gtttgcgagg gaactctgcc 60caatctgtcg atcagccaga tgcacacgtt accctttccg agggtgcgtc acttgaactg 120aggtgttcct atagctactc agcagcccct tatctgtttt ggtacgtaca atacccaggg 180cagagtctcc aatttctcct taaatatata accggggaca ctgtagtgaa ggggactaaa 240ggttttgaag cagagtttag gaagagcaac agctccttca accttaagaa gtcacccgca 300cactggtcag actccgcgaa atacttctgt gctctggaag ggcaagacca gggtggaagt 360gccaaattga tatttggtga gggtactaaa ttgactgtta gctcaccaga catccagaac 420ccagaacctg ctgtgtacca gttaaaagat cctcggtctc aggacagcac cctctgcctg 480ttcaccgact ttgactccca aatcaatgtg ccgaaaacca tggaatctgg aacgttcatc 540actgacaaaa ctgtgctgga catgaaagct atggattcca agagcaatgg ggccattgcc 600tggagcaacc agacaagctt cacctgccaa gatatcttca aagagaccaa cgccacctac 660cccagttcag acgttccctg tgatgccacg ttgaccgaga aaagctttga aacagatatg 720aacctaaact ttcaaaacct gtcagttatg ggactccgaa tcctcctgct gaaagtagcg 780ggatttaacc tgctcatgac gctgaggctg tggtccagtc gggccaagcg gtccggatcc 840ggagccacca acttcagcct gctgaagcag gccggcgacg tggaggagaa ccccggcccc 900atgtctaata ctgcatttcc agatccggcc tggaatacta cactccttag ttgggttgcc 960ctcttcctcc ttggcacgaa gcacatggaa gccgccgtca cccagagtcc gagaaacaaa 1020gtagcggtca ccggcggtaa ggtaacattg agttgtaacc aaacgaacaa ccataacaat 1080atgtactggt atagacaaga cacaggccat ggcttgcgcc tgattcacta cagctatgga 1140gcggggagta cggagaaggg tgatattccc gatggctata aagcctctcg gcctagccag 1200gagaatttta gtttgatcct ggagctcgca acccccagtc agactagcgt ctacttttgt 1260gcctcaggcg acaatagtgc ggaaaccctt tacttcggcc ctgggacaag acttacagtt 1320ctagaggatc tgagaaatgt gactccaccc aaggtctcct tgtttgagcc atcaaaagca 1380gagattgcaa acaaacaaaa ggctaccctc gtgtgcttgg ccaggggctt cttccctgac 1440cacgtggagc tgagctggtg ggtgaatggc aaggaggtcc acagtggggt cagcacggac 1500cctcaggcct acaaggagag caattatagc tactgcctga gcagccgcct gagggtctct 1560gctaccttct ggcacaatcc tcgaaaccac ttccgctgcc aagtgcagtt ccatgggctt 1620tcagaggagg acaagtggcc agagggctca cccaaacctg tcacacagaa catcagtgca 1680gaggcctggg gccgagcaga ctgtggaatc acttcagcat cctatcatca gggggttctg 1740tctgcaacca tcctctatga gatcctactg gggaaggcca ccctatatgc tgtgctggtc 1800agtggcctgg tgctgatggc catggtcaag aaaaaaaatt cctag 1845259619PRTArtificial SequenceH-Y TCR alpha and beta 259Met Phe Pro Val Thr Ile Leu Leu Leu Ser Ala Phe Phe Ser Leu Arg1 5 10 15Gly Asn Ser Ala Gln Ser Val Asp Gln Pro Asp Ala His Val Thr Leu 20 25 30Ser Glu Gly Ala Ser Leu Glu Leu Arg Cys Ser Tyr Ser Tyr Ser Ala 35 40 45Ala Pro Tyr Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln Ser Leu Gln 50 55 60Phe Leu Leu Lys Tyr Ile Thr Gly Asp Thr Val Val Lys Gly Thr Lys65 70 75 80Gly Phe Glu Ala Glu Phe Arg Lys Ser Asn Ser Ser Phe Asn Leu Lys 85 90 95Lys Ser Pro Ala His Trp Ser Asp Ser Ala Lys Tyr Phe Cys Ala Leu 100 105 110Glu Gly Gln Asp Gln Gly Gly Ser Ala Lys Leu Ile Phe Gly Glu Gly 115 120 125Thr Lys Leu Thr Val Ser Ser Pro Tyr Ile Gln Asn Pro Asp Pro Ala 130 135 140Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu145 150 155 160Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp 180 185 190Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe225 230 235 240Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270Arg Leu Trp Ser Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 275 280 285Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Asn Thr Ala 290 295 300Phe Pro Asp Pro Ala Trp Asn Thr Thr Leu Leu Ser Trp Val Ala Leu305 310 315 320Phe Leu Leu Gly Thr Lys His Met Glu Ala Ala Val Thr Gln Ser Pro 325 330 335Arg Asn Lys Val Ala Val Thr Gly Gly Lys Val Thr Leu Ser Cys Asn 340 345 350Gln Thr Asn Asn His Asn Asn Met Tyr Trp Tyr Arg Gln Asp Thr Gly 355 360 365His Gly Leu Arg Leu Ile His Tyr Ser Tyr Gly Ala Gly Ser Thr Glu 370 375 380Lys Gly Asp Ile Pro Asp Gly Tyr Lys Ala Ser Arg Pro Ser Gln Glu385 390 395 400Asn Phe Ser Leu Ile Leu Glu Leu Ala Thr Pro Ser Gln Thr Ser Val 405 410 415Tyr Phe Cys Ala Ser Gly Asp Asn Ser Ala Glu Thr Leu Tyr Phe Gly 420 425 430Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro 435 440 445Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr 450 455 460Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His465 470 475 480Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val 485 490 495Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser 500 505 510Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln 515 520 525Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser 530 535 540Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile545 550 555 560Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu 565 570 575Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu 580 585 590Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu 595 600 605Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly 610 6152601860DNAArtificial SequenceH-Y TCR alpha and beta 260atgttccccg ttaccatatt gctcctgagt gctttcttca gtttgcgagg gaactctgcc 60caatctgtcg atcagccaga tgcacacgtt accctttccg agggtgcgtc acttgaactg 120aggtgttcct atagctactc agcagcccct tatctgtttt ggtacgtaca atacccaggg 180cagagtctcc aatttctcct taaatatata accggggaca ctgtagtgaa ggggactaaa 240ggttttgaag cagagtttag gaagagcaac agctccttca accttaagaa gtcacccgca 300cactggtcag actccgcgaa atacttctgt gctctggaag ggcaagacca gggtggaagt 360gccaaattga tatttggtga gggtactaaa ttgactgtta gctcaccgta tatccagaac 420cctgaccctg ccgtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta 480ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc 540acagacaaat gtgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc 600tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 660gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga gaaaagcttt 720gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga ttgggttccg aatcctcctc 780ctgaaagtgg ccgggtttaa tctgctcatg acgctgcggc tgtggtccag cggatccgga 840gccaccaact tcagcctgct gaagcaggcc ggcgacgtgg aggagaaccc cggccccatg 900tctaatactg catttccaga tccggcctgg aatactacac tccttagttg ggttgccctc 960ttcctccttg gcacgaagca catggaagcc gccgtcaccc agagtccgag aaacaaagta 1020gcggtcaccg gcggtaaggt aacattgagt tgtaaccaaa cgaacaacca taacaatatg 1080tactggtata gacaagacac aggccatggc ttgcgcctga ttcactacag ctatggagcg 1140gggagtacgg agaagggtga tattcccgat ggctataaag cctctcggcc tagccaggag 1200aattttagtt tgatcctgga gctcgcaacc cccagtcaga ctagcgtcta cttttgtgcc 1260tcaggcgaca atagtgcgga aaccctttac ttcggccctg ggacaagact tacagttctg 1320gaggacctga aaaacgtgtt cccacccgag gtcgctgtgt ttgagccatc agaagcagag 1380atctcccaca cccaaaaggc cacactggtg tgcctggcca caggcttcta ccccgaccac 1440gtggagctga gctggtgggt gaatgggaag gaggtgcaca gtggggtctg cacagacccg 1500cagcccctca aggagcagcc cgccctcaat gactccagat actgcctgag cagccgcctg 1560agggtgtcgg ccaccttctg gcagaacccc cgcaaccact tccgctgtca agtccagttc 1620tacgggctct cggagaatga cgagtggacc caggataggg ccaaacctgt cacccagatc 1680gtcagcgccg aggcctgggg tagagcagac tgtggcttca cctccgagtc ttaccagcaa 1740ggggtcctgt ctgccaccat cctctatgag atcttgctag ggaaggccac cttgtatgcc 1800gtgctggtca gtgccctcgt gctgatggcc atggtcaaga gaaaggattc cagaggctag 1860261614PRTArtificial SequenceH-Y TCR alpha and beta 261Met Phe Pro Val Thr Ile Leu Leu Leu Ser Ala Phe Phe Ser Leu Arg1 5 10 15Gly Asn Ser Ala Gln Ser Val Asp Gln Pro Asp Ala His Val Thr Leu 20 25 30Ser Glu Gly Ala Ser Leu Glu Leu Arg Cys Ser Tyr Ser Tyr Ser Ala 35 40 45Ala Pro Tyr Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln Ser Leu Gln 50 55 60Phe Leu Leu Lys Tyr Ile Thr Gly Asp Thr Val Val Lys Gly Thr Lys65 70 75 80Gly Phe Glu Ala Glu Phe Arg Lys Ser Asn Ser Ser Phe Asn Leu Lys 85 90 95Lys Ser Pro Ala His Trp Ser Asp Ser Ala Lys Tyr Phe Cys Ala Leu 100 105 110Glu Gly Gln Asp Gln Gly Gly Ser Ala Lys Leu Ile Phe Gly Glu Gly 115 120 125Thr Lys Leu Thr Val Ser Ser Pro Asp Ile Gln Asn Pro Glu Pro Ala 130 135 140Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu145 150 155 160Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser 165 170 175Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp 180 185 190Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr 195 200 205Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp 210 215 220Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met225 230 235 240Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu 245 250 255Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 275 280 285Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Asn Thr 290 295 300Ala Phe Pro Asp Pro Ala Trp Asn Thr Thr Leu Leu Ser Trp Val Ala305 310 315 320Leu Phe Leu Leu Gly Thr Lys His Met Glu Ala Ala Val Thr Gln Ser 325 330 335Pro Arg Asn Lys

Val Ala Val Thr Gly Gly Lys Val Thr Leu Ser Cys 340 345 350Asn Gln Thr Asn Asn His Asn Asn Met Tyr Trp Tyr Arg Gln Asp Thr 355 360 365Gly His Gly Leu Arg Leu Ile His Tyr Ser Tyr Gly Ala Gly Ser Thr 370 375 380Glu Lys Gly Asp Ile Pro Asp Gly Tyr Lys Ala Ser Arg Pro Ser Gln385 390 395 400Glu Asn Phe Ser Leu Ile Leu Glu Leu Ala Thr Pro Ser Gln Thr Ser 405 410 415Val Tyr Phe Cys Ala Ser Gly Asp Asn Ser Ala Glu Thr Leu Tyr Phe 420 425 430Gly Pro Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Arg Asn Val Thr 435 440 445Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn 450 455 460Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp465 470 475 480His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly 485 490 495Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys 500 505 510Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg 515 520 525Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp 530 535 540Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala545 550 555 560Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His 565 570 575Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys 580 585 590Ala Thr Leu Tyr Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met 595 600 605Val Lys Lys Lys Asn Ser 6102621845DNAArtificial SequenceH-Y TCR alpha and beta 262atgttccccg ttaccatatt gctcctgagt gctttcttca gtttgcgagg gaactctgcc 60caatctgtcg atcagccaga tgcacacgtt accctttccg agggtgcgtc acttgaactg 120aggtgttcct atagctactc agcagcccct tatctgtttt ggtacgtaca atacccaggg 180cagagtctcc aatttctcct taaatatata accggggaca ctgtagtgaa ggggactaaa 240ggttttgaag cagagtttag gaagagcaac agctccttca accttaagaa gtcacccgca 300cactggtcag actccgcgaa atacttctgt gctctggaag ggcaagacca gggtggaagt 360gccaaattga tatttggtga gggtactaaa ttgactgtta gctcaccaga catccagaac 420ccagaacctg ctgtgtacca gttaaaagat cctcggtctc aggacagcac cctctgcctg 480ttcaccgact ttgactccca aatcaatgtg ccgaaaacca tggaatctgg aacgttcatc 540actgacaaaa ctgtgctgga catgaaagct atggattcca agagcaatgg ggccattgcc 600tggagcaacc agacaagctt cacctgccaa gatatcttca aagagaccaa cgccacctac 660cccagttcag acgttccctg tgatgccacg ttgaccgaga aaagctttga aacagatatg 720aacctaaact ttcaaaacct gtcagttatg ggactccgaa tcctcctgct gaaagtagcg 780ggatttaacc tgctcatgac gctgaggctg tggtccagtc gggccaagcg gtccggatcc 840ggagccacca acttcagcct gctgaagcag gccggcgacg tggaggagaa ccccggcccc 900atgtctaata ctgcatttcc agatccggcc tggaatacta cactccttag ttgggttgcc 960ctcttcctcc ttggcacgaa gcacatggaa gccgccgtca cccagagtcc gagaaacaaa 1020gtagcggtca ccggcggtaa ggtaacattg agttgtaacc aaacgaacaa ccataacaat 1080atgtactggt atagacaaga cacaggccat ggcttgcgcc tgattcacta cagctatgga 1140gcggggagta cggagaaggg tgatattccc gatggctata aagcctctcg gcctagccag 1200gagaatttta gtttgatcct ggagctcgca acccccagtc agactagcgt ctacttttgt 1260gcctcaggcg acaatagtgc ggaaaccctt tacttcggcc ctgggacaag acttttggtt 1320ctagaggatc tgagaaatgt gactccaccc aaggtctcct tgtttgagcc atcaaaagca 1380gagattgcaa acaaacaaaa ggctaccctc gtgtgcttgg ccaggggctt cttccctgac 1440cacgtggagc tgagctggtg ggtgaatggc aaggaggtcc acagtggggt cagcacggac 1500cctcaggcct acaaggagag caattatagc tactgcctga gcagccgcct gagggtctct 1560gctaccttct ggcacaatcc tcgaaaccac ttccgctgcc aagtgcagtt ccatgggctt 1620tcagaggagg acaagtggcc agagggctca cccaaacctg tcacacagaa catcagtgca 1680gaggcctggg gccgagcaga ctgtggaatc acttcagcat cctatcatca gggggttctg 1740tctgcaacca tcctctatga gatcctactg gggaaggcca ccctatatgc tgtgctggtc 1800agtggcctgg tgctgatggc catggtcaag aaaaaaaatt cctag 1845263619PRTArtificial SequenceH-Y TCR alpha and beta 263Met Phe Pro Val Thr Ile Leu Leu Leu Ser Ala Phe Phe Ser Leu Arg1 5 10 15Gly Asn Ser Ala Gln Ser Val Asp Gln Pro Asp Ala His Val Thr Leu 20 25 30Ser Glu Gly Ala Ser Leu Glu Leu Arg Cys Ser Tyr Ser Tyr Ser Ala 35 40 45Ala Pro Tyr Leu Phe Trp Tyr Val Gln Tyr Pro Gly Gln Ser Leu Gln 50 55 60Phe Leu Leu Lys Tyr Ile Thr Gly Asp Thr Val Val Lys Gly Thr Lys65 70 75 80Gly Phe Glu Ala Glu Phe Arg Lys Ser Asn Ser Ser Phe Asn Leu Lys 85 90 95Lys Ser Pro Ala His Trp Ser Asp Ser Ala Lys Tyr Phe Cys Ala Leu 100 105 110Glu Gly Gln Asp Gln Gly Gly Ser Ala Lys Leu Ile Phe Gly Glu Gly 115 120 125Thr Lys Leu Thr Val Ser Ser Pro Tyr Ile Gln Asn Pro Asp Pro Ala 130 135 140Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu145 150 155 160Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp 180 185 190Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe225 230 235 240Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270Arg Leu Trp Ser Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 275 280 285Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Asn Thr Ala 290 295 300Phe Pro Asp Pro Ala Trp Asn Thr Thr Leu Leu Ser Trp Val Ala Leu305 310 315 320Phe Leu Leu Gly Thr Lys His Met Glu Ala Ala Val Thr Gln Ser Pro 325 330 335Arg Asn Lys Val Ala Val Thr Gly Gly Lys Val Thr Leu Ser Cys Asn 340 345 350Gln Thr Asn Asn His Asn Asn Met Tyr Trp Tyr Arg Gln Asp Thr Gly 355 360 365His Gly Leu Arg Leu Ile His Tyr Ser Tyr Gly Ala Gly Ser Thr Glu 370 375 380Lys Gly Asp Ile Pro Asp Gly Tyr Lys Ala Ser Arg Pro Ser Gln Glu385 390 395 400Asn Phe Ser Leu Ile Leu Glu Leu Ala Thr Pro Ser Gln Thr Ser Val 405 410 415Tyr Phe Cys Ala Ser Gly Asp Asn Ser Ala Glu Thr Leu Tyr Phe Gly 420 425 430Pro Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Lys Asn Val Phe Pro 435 440 445Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr 450 455 460Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His465 470 475 480Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val 485 490 495Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser 500 505 510Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln 515 520 525Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser 530 535 540Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile545 550 555 560Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu 565 570 575Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu 580 585 590Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu 595 600 605Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly 610 6152641860DNAArtificial SequenceH-Y TCR alpha and beta 264atgttccccg ttaccatatt gctcctgagt gctttcttca gtttgcgagg gaactctgcc 60caatctgtcg atcagccaga tgcacacgtt accctttccg agggtgcgtc acttgaactg 120aggtgttcct atagctactc agcagcccct tatctgtttt ggtacgtaca atacccaggg 180cagagtctcc aatttctcct taaatatata accggggaca ctgtagtgaa ggggactaaa 240ggttttgaag cagagtttag gaagagcaac agctccttca accttaagaa gtcacccgca 300cactggtcag actccgcgaa atacttctgt gctctggaag ggcaagacca gggtggaagt 360gccaaattga tatttggtga gggtactaaa ttgactgtta gctcaccgta tatccagaac 420cctgaccctg ccgtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta 480ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc 540acagacaaat gtgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc 600tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 660gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga gaaaagcttt 720gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga ttgggttccg aatcctcctc 780ctgaaagtgg ccgggtttaa tctgctcatg acgctgcggc tgtggtccag cggatccgga 840gccaccaact tcagcctgct gaagcaggcc ggcgacgtgg aggagaaccc cggccccatg 900tctaatactg catttccaga tccggcctgg aatactacac tccttagttg ggttgccctc 960ttcctccttg gcacgaagca catggaagcc gccgtcaccc agagtccgag aaacaaagta 1020gcggtcaccg gcggtaaggt aacattgagt tgtaaccaaa cgaacaacca taacaatatg 1080tactggtata gacaagacac aggccatggc ttgcgcctga ttcactacag ctatggagcg 1140gggagtacgg agaagggtga tattcccgat ggctataaag cctctcggcc tagccaggag 1200aattttagtt tgatcctgga gctcgcaacc cccagtcaga ctagcgtcta cttttgtgcc 1260tcaggcgaca atagtgcgga aaccctttac ttcggccctg ggacaagact tttggttctg 1320gaggacctga aaaacgtgtt cccacccgag gtcgctgtgt ttgagccatc agaagcagag 1380atctcccaca cccaaaaggc cacactggtg tgcctggcca caggcttcta ccccgaccac 1440gtggagctga gctggtgggt gaatgggaag gaggtgcaca gtggggtctg cacagacccg 1500cagcccctca aggagcagcc cgccctcaat gactccagat actgcctgag cagccgcctg 1560agggtgtcgg ccaccttctg gcagaacccc cgcaaccact tccgctgtca agtccagttc 1620tacgggctct cggagaatga cgagtggacc caggataggg ccaaacctgt cacccagatc 1680gtcagcgccg aggcctgggg tagagcagac tgtggcttca cctccgagtc ttaccagcaa 1740ggggtcctgt ctgccaccat cctctatgag atcttgctag ggaaggccac cttgtatgcc 1800gtgctggtca gtgccctcgt gctgatggcc atggtcaaga gaaaggattc cagaggctag 18602659PRTArtificial SequenceNY-ESO-1 265Ser Leu Leu Met Trp Ile Thr Gln Val1 52669PRTArtificial SequenceHA-1(R) peptide 266Val Leu Arg Asp Asp Leu Leu Glu Ala1 5267607PRTArtificial SequenceNY-ESO-1 TCR alpha and beta 267Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp1 5 10 15Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val 20 25 30Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala 35 40 45Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr 50 55 60Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg65 70 75 80Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile 85 90 95Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg 100 105 110Pro Leu Tyr Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser 115 120 125Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp145 150 155 160Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp225 230 235 240Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270Ser Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly 275 280 285Asp Val Glu Glu Asn Pro Gly Pro Met Ser Ile Gly Leu Leu Cys Cys 290 295 300Ala Ala Leu Ser Leu Leu Trp Ala Gly Pro Val Asn Ala Gly Val Thr305 310 315 320Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln Ser Met Thr Leu 325 330 335Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser Trp Tyr Arg Gln 340 345 350Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser Val Gly Ala Gly 355 360 365Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr Asn Val Ser Arg Ser 370 375 380Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser Ala Ala Pro Ser Gln385 390 395 400Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Val Gly Asn Thr Gly Glu 405 410 415Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys 420 425 430Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu 435 440 445Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe 450 455 460Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val465 470 475 480His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala 485 490 495Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 500 505 510Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe 515 520 525Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro 530 535 540Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly545 550 555 560Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu 565 570 575Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 580 585 590Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly 595 600 6052681824DNAArtificial SequenceNY-ESO-1 TCR alpha and beta 268atggagacac tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagcaaa 60caggaggtga cgcagattcc tgcagctctg agtgtcccag aaggagaaaa cttggttctg 120aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca ggaccctggg 180aaaggactca catctctgtt gcttattcag tcaagtcaga gagagcaaac aagtggaaga 240cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc agcttctcag 300cctggtgact cagccaccta cctctgtgct gtgaggcccc tctacggagg aagctacata 360cctacatttg gaagaggaac cagccttatt gttcatccgt atatccagaa ccctgaccct 420gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 480tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 540tgtgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 600aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 660ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 720acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 780gccgggttta atctgctcat gacgctgcgg ctgtggtcca gcggatccgg agccaccaac 840ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccat gagcatcggc 900ctcctgtgct gtgcagcctt gtctctcctg tgggcaggtc cagtgaatgc tggtgtcact 960cagaccccaa aattccaggt cctgaagaca ggacagagca tgacactgca gtgtgcccag 1020gatatgaacc atgaatacat gtcctggtat cgacaagacc caggcatggg gctgaggctg 1080attcattact cagttggtgc tggtatcact gaccaaggag aagtccccaa tggctacaat 1140gtctccagat caaccacaga ggatttcccg ctcaggctgc tgtcggctgc tccctcccag 1200acatctgtgt acttctgtgc cagcagttac gtcgggaaca ccggggagct gttttttgga 1260gaaggctcta ggctgaccgt actggaggac ctgaaaaacg tgttcccacc cgaggtcgct 1320gtgtttgagc catcagaagc agagatctcc cacacccaaa aggccacact ggtgtgcctg 1380gccacaggct tctaccccga ccacgtggag ctgagctggt gggtgaatgg gaaggaggtg 1440cacagtgggg tctgcacaga cccgcagccc ctcaaggagc agcccgccct caatgactcc 1500agatactgcc tgagcagccg cctgagggtg tcggccacct tctggcagaa cccccgcaac 1560cacttccgct gtcaagtcca gttctacggg ctctcggaga atgacgagtg gacccaggat 1620agggccaaac ctgtcaccca gatcgtcagc gccgaggcct ggggtagagc agactgtggc 1680ttcacctccg agtcttacca gcaaggggtc ctgtctgcca ccatcctcta tgagatcttg 1740ctagggaagg ccaccttgta tgccgtgctg gtcagtgccc tcgtgctgat ggccatggtc 1800aagagaaagg

attccagagg ctag 1824269612PRTArtificial SequenceKRAS TCR 269Met Gln Arg Asn Leu Gly Ala Val Leu Gly Ile Leu Trp Val Gln Ile1 5 10 15Cys Trp Val Arg Gly Asp Gln Val Glu Gln Ser Pro Ser Ala Leu Ser 20 25 30Leu His Glu Gly Thr Asp Ser Ala Leu Arg Cys Asn Phe Thr Thr Thr 35 40 45Met Arg Ser Val Gln Trp Phe Arg Gln Asn Ser Arg Gly Ser Leu Ile 50 55 60Ser Leu Phe Tyr Leu Ala Ser Gly Thr Lys Glu Asn Gly Arg Leu Lys65 70 75 80Ser Ala Phe Asp Ser Lys Glu Arg Arg Tyr Ser Thr Leu His Ile Arg 85 90 95Asp Ala Gln Leu Glu Asp Ser Gly Thr Tyr Phe Cys Ala Ala Asp Ser 100 105 110Ser Asn Thr Gly Tyr Gln Asn Phe Tyr Phe Gly Lys Gly Thr Ser Leu 115 120 125Thr Val Ile Pro Asn Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu 130 135 140Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe145 150 155 160Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile 165 170 175Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn 180 185 190Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile 195 200 205Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp 210 215 220Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe225 230 235 240Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala 245 250 255Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser Arg Ala Lys 260 265 270Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly 275 280 285Asp Val Glu Glu Asn Pro Gly Pro Met Ser Asn Thr Ala Phe Pro Asp 290 295 300Pro Ala Trp Asn Thr Thr Leu Leu Ser Trp Val Ala Leu Phe Leu Leu305 310 315 320Gly Thr Ser Ser Ala Asn Ser Gly Val Val Gln Ser Pro Arg Tyr Ile 325 330 335Ile Lys Gly Lys Gly Glu Arg Ser Ile Leu Lys Cys Ile Pro Ile Ser 340 345 350Gly His Leu Ser Val Ala Trp Tyr Gln Gln Thr Gln Gly Gln Glu Leu 355 360 365Lys Phe Phe Ile Gln His Tyr Asp Lys Met Glu Arg Asp Lys Gly Asn 370 375 380Leu Pro Ser Arg Phe Ser Val Gln Gln Phe Asp Asp Tyr His Ser Glu385 390 395 400Met Asn Met Ser Ala Leu Glu Leu Glu Asp Ser Ala Val Tyr Phe Cys 405 410 415Ala Ser Ser Leu Thr Asp Pro Leu Asp Ser Asp Tyr Thr Phe Gly Ser 420 425 430Gly Thr Arg Leu Leu Val Ile Glu Asp Leu Arg Asn Val Thr Pro Pro 435 440 445Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln 450 455 460Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val465 470 475 480Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 485 490 495Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser 500 505 510Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His 515 520 525Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp 530 535 540Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala545 550 555 560Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly 565 570 575Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 580 585 590Leu Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys 595 600 605Arg Lys Asn Ser 6102701839DNAArtificial SequenceKRAS TCR 270atgcagagga acctgggagc tgtgctgggg attctgtggg tgcagatttg ctgggtgaga 60ggggatcagg tggagcagag tccttcagcc ctgagcctcc acgagggaac cgattctgct 120ctgagatgca attttacgac caccatgagg agtgtgcagt ggttccgaca gaattccagg 180ggcagcctca tcagtttgtt ctacttggct tcaggaacaa aggagaatgg gaggctaaag 240tcagcatttg attctaagga gcggcgctac agcaccctgc acatcaggga tgcccagctg 300gaggactcag gcacttactt ctgtgctgct gactcttcga acacgggtta ccagaacttc 360tattttggga aaggaacaag tttgactgtc attccaaaca tccagaaccc agaacctgct 420gtgtaccagt taaaagatcc tcggtctcag gacagcaccc tctgcctgtt caccgacttt 480gactcccaaa tcaatgtgcc gaaaaccatg gaatctggaa cgttcatcac tgacaaaact 540gtgctggaca tgaaagctat ggattccaag agcaatgggg ccattgcctg gagcaaccag 600acaagcttca cctgccaaga tatcttcaaa gagaccaacg ccacctaccc cagttcagac 660gttccctgtg atgccacgtt gaccgagaaa agctttgaaa cagatatgaa cctgaacttt 720caaaacctgt cagttatggg actccgaatc ctcctgctga aagtagcggg atttaacctg 780ctcatgacgc tgaggctgtg gtccagtcgg gccaagcggt ccggatccgg agccaccaac 840ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccat gtctaacact 900gccttccctg accccgcctg gaacaccacc ctgctatctt gggttgctct ctttctcctg 960ggaacaagtt cagcaaattc tggggttgtc cagtctccaa gatacataat caaaggaaag 1020ggagaaaggt ccattctaaa atgtattccc atctctggac atctctctgt ggcctggtat 1080caacagactc aggggcagga actaaagttc ttcattcagc attatgataa aatggagaga 1140gataaaggaa acctgcccag cagattctca gtccaacagt ttgatgacta tcactctgag 1200atgaacatga gtgccttgga gctagaggac tctgccgtgt acttctgtgc cagctctctc 1260acagatccgc tagactccga ctacaccttc ggctcaggga ccaggctttt ggtaatagag 1320gatctgagaa atgtgactcc acccaaggtc tccttgtttg agccatcaaa agcagagatt 1380gcaaacaaac aaaaggctac cctcgtgtgc ttggccaggg gcttcttccc tgaccacgtg 1440gagctgagct ggtgggtgaa tggcaaggag gtccacagtg gggtcagcac ggaccctcag 1500gcctacaagg agagcaatta tagctactgc ctgagcagcc gcctgagggt ctctgctacc 1560ttctggcaca atcctcgcaa ccacttccgc tgccaagtgc agttccatgg gctttcagag 1620gaggacaagt ggccagaggg ctcacccaaa cctgtcacac agaacatcag tgcagaggcc 1680tggggccgag cagactgtgg gattacctca gcatcctatc aacaaggggt cttgtctgcc 1740accatcctct atgagatcct gctagggaaa gccaccctgt atgctgtgct tgtcagtaca 1800ctggtggtga tggctatggt caaaagaaag aattcatag 183927118PRTArtificial SequenceT2A self cleaving peptide 271Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro1 5 10 15Gly Pro27220PRTArtificial SequenceE2A self cleaving peptide 272Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser1 5 10 15Asn Pro Gly Pro 2027322PRTArtificial SequenceF2A self cleaving peptide 273Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val1 5 10 15Glu Ser Asn Pro Gly Pro 202746PRTArtificial SequenceITIMZ(1)..(1)Z is S, I V or LX(2)..(2)X is any amino acidX(4)..(5)X is any amino acidZ(6)..(6)Z is I, V or L 274Glx Xaa Tyr Xaa Xaa Glx1 5275242PRTArtificial SequenceCD19 ScFv 275Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg145 150 155 160Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val225 230 235 240Ser Ser276726DNAArtificial SequenceCD19 ScFv 276gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtg 720tcctca 726277244PRTArtificial SequenceCD19 ScFv 277Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Val Lys Leu 115 120 125Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val 130 135 140Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp145 150 155 160Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp 165 170 175Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr 180 185 190Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser 195 200 205Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr 210 215 220Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val225 230 235 240Thr Val Ser Ser278732DNAArtificial SequenceCD19 ScFv 278gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300gggaccaagc tggagatcac aggcggaggt ggaagcggag ggggaggatc tggcggcgga 360ggaagcggag gcgaggtgaa actgcaggag tcaggacctg gcctggtggc gccctcacag 420agcctgtccg tcacatgcac tgtctcaggg gtctcattac ccgactatgg tgtaagctgg 480attcgccagc ctccacgaaa gggtctggag tggctgggag taatatgggg tagtgaaacc 540acatactata attcagctct caaatccaga ctgaccatca tcaaggacaa ctccaagagc 600caagttttct taaaaatgaa cagtctgcaa actgatgaca cagccattta ctactgtgcc 660aaacattatt actacggtgg tagctatgct atggactact ggggccaagg aacctcagtc 720accgtgtcct ca 732279509PRTArtificial SequenceCD19 CAR 279Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg145 150 155 160Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val225 230 235 240Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 245 250 255Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 260 265 270Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe 275 280 285Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 290 295 300Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg305 310 315 320Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 325 330 335Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 340 345 350Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 355 360 365Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 370 375 380Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys385 390 395 400Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln 405 410 415Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 420 425 430Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 435 440 445Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 450 455 460Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly465 470 475 480Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 485 490 495Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 5052801527DNAArtificial SequenceCD19 CAR 280gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtg 720tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840gggctggact tcgcctgtga tttctgggtg ctggtcgttg tgggcggcgt gctggcctgc

900tacagcctgc tggtgacagt ggccttcatc atcttttggg tgaggagcaa gcggagcaga 960ctgctgcaca gcgactacat gaacatgacc ccccggaggc ctggccccac ccggaagcac 1020taccagccct acgcccctcc cagggatttc gccgcctacc ggagcaaacg gggcagaaag 1080aaactcctgt atatattcaa acaaccattt atgaggccag tacaaactac tcaagaggaa 1140gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1200ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1260ctcaatctag gacgaagaga ggagtacgat gttttggaca agcgtagagg ccgggaccct 1320gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1380aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1440aaggggcacg atggccttta ccagggactc agtacagcca ccaaggacac ctacgacgcc 1500cttcacatgc aggccctgcc ccctcgc 1527281509PRTArtificial SequenceCD19 CAR 281Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg145 150 155 160Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val225 230 235 240Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 245 250 255Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 260 265 270Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe 275 280 285Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 290 295 300Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg305 310 315 320Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 325 330 335Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 340 345 350Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 355 360 365Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 370 375 380Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys385 390 395 400Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln 405 410 415Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 420 425 430Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 435 440 445Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 450 455 460Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly465 470 475 480Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 485 490 495Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505282246PRTArtificial SequenceCEA ScFv 282Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln 130 135 140Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr145 150 155 160Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln 165 170 175Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg 180 185 190Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 210 215 220Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr Phe Gly Gln Gly225 230 235 240Thr Lys Leu Glu Ile Lys 2452831542DNAArtificial SequenceCEA ScFv 283caggtgcagc tggtgcaatc tgggtctgag ttgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg cttctggata caccttcact gagtttggaa tgaactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg ataaacacca aaactggaga ggcaacatat 180gttgaagagt ttaagggacg gtttgtcttc tccttggaca cctctgtcag cacggcatat 240ctgcagatca gcagcctaaa ggctgaagac actgccgtgt attactgtgc gagatgggac 300ttcgcttatt acgtggaggc tatggactac tggggccaag ggaccacggt caccgtctcc 360tcaggcggag gtggaagcgg agggggagga tctggcggcg gaggaagcgg aggcgatatc 420cagatgaccc agtctccatc ctccctgtct gcatctgtgg gagacagagt caccatcact 480tgcaaggcca gtcagaatgt gggtactaat gttgcctggt atcagcagaa accagggaaa 540gcacctaagc tcctgatcta ttcggcatcc taccgctaca gtggagtccc atcaaggttc 600agtggcagtg gatctgggac agatttcact ctcaccatca gcagtctgca acctgaagat 660ttcgcaactt actactgtca ccaatattac acctatcctc tattcacgtt tggccagggc 720accaagctcg agatcaagat ggacatgagg gtccccgctc agctcctggg gctcctgcta 780ctctggctcc gaggtgccag atgtcaggtg cagctggtgc aatctgggtc tgagttgaag 840aagcctgggg cctcagtgaa ggtttcctgc aaggcttctg gatacacctt cactgagttt 900ggaatgaact gggtgcgaca ggcccctgga caagggcttg agtggatggg atggataaac 960accaaaactg gagaggcaac atatgttgaa gagtttaagg gacggtttgt cttctccttg 1020gacacctctg tcagcacggc atatctgcag atcagcagcc taaaggctga agacactgcc 1080gtgtattact gtgcgagatg ggacttcgct tattacgtgg aggctatgga ctactggggc 1140caagggacca cggtcaccgt ctcctcaggc ggaggtggaa gcggaggggg aggatctggc 1200ggcggaggaa gcggaggcga tatccagatg acccagtctc catcctccct gtctgcatct 1260gtgggagaca gagtcaccat cacttgcaag gccagtcaga atgtgggtac taatgttgcc 1320tggtatcagc agaaaccagg gaaagcacct aagctcctga tctattcggc atcctaccgc 1380tacagtggag tcccatcaag gttcagtggc agtggatctg ggacagattt cactctcacc 1440atcagcagtc tgcaacctga agatttcgca acttactact gtcaccaata ttacacctat 1500cctctattca cgtttggcca gggcaccaag ctcgagatca ag 1542284246PRTArtificial SequenceCEA ScFv 284Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln 130 135 140Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr145 150 155 160Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala Trp Tyr Gln Gln 165 170 175Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg 180 185 190Lys Arg Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 210 215 220Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr Phe Gly Gln Gly225 230 235 240Thr Lys Leu Glu Ile Lys 245285738DNAArtificial SequenceCEA ScFv 285caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggagctag tgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc gagttcggca tgaactgggt ccgacaggct 120ccaggccagg gcctcgaatg gatgggctgg atcaacacca agaccggcga ggccacctac 180gtggaagagt tcaagggcag agtgaccttc accacggaca ccagcaccag caccgcctac 240atggaactgc ggagcctgag aagcgacgac accgccgtgt actactgcgc cagatgggac 300ttcgcttatt acgtggaagc catggactac tggggccagg gcaccaccgt gaccgtgtct 360agcggcggag gtggaagcgg agggggagga tctggcggcg gaggaagcgg aggcgatatc 420cagatgaccc agtctccatc ctccctgtct gcatctgtgg gagacagagt caccatcact 480tgcaaggcca gtgcggctgt gggtacgtat gttgcgtggt atcagcagaa accagggaaa 540gcacctaagc tcctgatcta ttcggcatcc taccgcaaaa ggggagtccc atcaaggttc 600agtggcagtg gatctgggac agatttcact ctcaccatca gcagtctgca acctgaagat 660ttcgcaactt actactgtca ccaatattac acctatcctc tattcacgtt tggccagggc 720accaagctcg agatcaag 738286246PRTArtificial SequenceCEA ScFv 286Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala His Tyr Phe Gln Thr Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln 130 135 140Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr145 150 155 160Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala Trp Tyr Gln Gln 165 170 175Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg 180 185 190Lys Arg Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 210 215 220Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr Phe Gly Gln Gly225 230 235 240Thr Lys Leu Glu Ile Lys 245287738DNAArtificial SequenceCEA ScFv 287caggtgcagc tggtgcaatc tgggtctgag ttgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg cttctggata caccttcact gagtttggaa tgaactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg ataaacacca aaactggaga ggcaacatat 180gttgaagagt ttaagggacg gtttgtcttc tccttggaca cctctgtcag cacggcatat 240ctgcagatca gcagcctaaa ggctgaagac actgccgtgt attactgtgc gagatgggac 300tttgctcatt actttcagac tatggactac tggggccaag ggaccacggt caccgtctcc 360tcaggcggag gtggaagcgg agggggagga tctggcggcg gaggaagcgg aggcgatatc 420cagatgaccc agtctccatc ctccctgtct gcatctgtgg gagacagagt caccatcact 480tgcaaggcca gtgcggctgt gggtacgtat gttgcgtggt atcagcagaa accagggaaa 540gcacctaagc tcctgatcta ttcggcatcc taccgcaaaa ggggagtccc atcaaggttc 600agtggcagtg gatctgggac agatttcact ctcaccatca gcagtctgca acctgaagat 660ttcgcaactt actactgtca ccaatattac acctatcctc tattcacgtt tggccagggc 720accaagctcg agatcaag 738288535PRTArtificial SequenceCEA CAR 288Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ser Glu 20 25 30Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45Tyr Thr Phe Thr Glu Phe Gly Met Asn Trp Val Arg Gln Ala Pro Gly 50 55 60Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala65 70 75 80Thr Tyr Val Glu Glu Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr 85 90 95Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp 100 105 110Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu 115 120 125Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly145 150 155 160Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 165 170 175Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 180 185 190Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 195 200 205Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 210 215 220Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro225 230 235 240Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu 245 250 255Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro 260 265 270Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val305 310 315 320Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile 325 330 335Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr 340 345 350Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln 355 360 365Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly 370 375 380Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val385 390 395 400Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 405 410 415Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 420 425 430Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 435 440 445Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 450 455 460Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly465 470 475 480Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 485 490 495Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 500 505 510Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 515 520 525Met Gln Ala Leu Pro Pro Arg 530 5352891608DNAArtificial SequenceCEA CAR 289atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60agatgtcagg tgcagctggt gcaatctggg tctgagttga agaagcctgg ggcctcagtg 120aaggtttcct gcaaggcttc tggatacacc ttcactgagt ttggaatgaa

ctgggtgcga 180caggcccctg gacaagggct tgagtggatg ggatggataa acaccaaaac tggagaggca 240acatatgttg aagagtttaa gggacggttt gtcttctcct tggacacctc tgtcagcacg 300gcatatctgc agatcagcag cctaaaggct gaagacactg ccgtgtatta ctgtgcgaga 360tgggacttcg cttattacgt ggaggctatg gactactggg gccaagggac cacggtcacc 420gtctcctcag gcggaggtgg aagcggaggg ggaggatctg gcggcggagg aagcggaggc 480gatatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 540atcacttgca aggccagtca gaatgtgggt actaatgttg cctggtatca gcagaaacca 600gggaaagcac ctaagctcct gatctattcg gcatcctacc gctacagtgg agtcccatca 660aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 720gaagatttcg caacttacta ctgtcaccaa tattacacct atcctctatt cacgtttggc 780cagggcacca agctcgagat caagacaacg acgccagctc cccgcccgcc aacccctgca 840cctacgattg catcacaacc gctgtccctg cggcctgaag cttgtcgccc agccgcaggt 900ggcgccgtac atacacgggg gctggatttt gcctgtgatt tctgggtgct ggtcgttgtg 960ggcggcgtgc tggcctgcta cagcctgctg gtgacagtgg ccttcatcat cttttgggtg 1020aggagcaagc ggagtcgact gctgcacagc gactacatga acatgacccc ccggaggcct 1080ggccccaccc ggaagcacta ccagccctac gcccctccca gggatttcgc cgcctaccgg 1140agcaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gaggccagta 1200caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 1260tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacaa gcagggccag 1320aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1380cgtagaggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1440ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1500ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggactcag tacagccacc 1560aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgctag 1608290537PRTArtificial SequenceCEA CAR 290Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45Tyr Thr Phe Thr Glu Phe Gly Met Asn Trp Val Arg Gln Ala Pro Gly 50 55 60Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala65 70 75 80Thr Tyr Val Glu Glu Phe Lys Gly Arg Val Thr Phe Thr Thr Asp Thr 85 90 95Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp 100 105 110Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu 115 120 125Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly145 150 155 160Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 165 170 175Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr 180 185 190Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 195 200 205Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly 210 215 220Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro225 230 235 240Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu 245 250 255Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr 260 265 270Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val305 310 315 320Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 325 330 335Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser 340 345 350Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 355 360 365Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys 370 375 380Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg385 390 395 400Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 405 410 415Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 420 425 430Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu 435 440 445Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 450 455 460Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln465 470 475 480Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 485 490 495Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 500 505 510Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 515 520 525Leu His Met Gln Ala Leu Pro Pro Arg 530 5352911614DNAArtificial SequenceCEA CAR 291atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60agatgtcagg tgcagctggt gcagtctggc gccgaagtga agaaacctgg agctagtgtg 120aaggtgtcct gcaaggccag cggctacacc ttcaccgagt tcggcatgaa ctgggtccga 180caggctccag gccagggcct cgaatggatg ggctggatca acaccaagac cggcgaggcc 240acctacgtgg aagagttcaa gggcagagtg accttcacca cggacaccag caccagcacc 300gcctacatgg aactgcggag cctgagaagc gacgacaccg ccgtgtacta ctgcgccaga 360tgggacttcg cttattacgt ggaagccatg gactactggg gccagggcac caccgtgacc 420gtgtctagcg gcggaggtgg aagcggaggg ggaggatctg gcggcggagg aagcggaggc 480gatatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 540atcacttgca aggccagtgc ggctgtgggt acgtatgttg cgtggtatca gcagaaacca 600gggaaagcac ctaagctcct gatctattcg gcatcctacc gcaaaagggg agtcccatca 660aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 720gaagatttcg caacttacta ctgtcaccaa tattacacct atcctctatt cacgtttggc 780cagggcacca agctcgagat caagcgtacg acaacgacgc cagctccccg cccgccaacc 840cctgcaccta cgattgcatc acaaccgctg tccctgcggc ctgaagcttg tcgcccagcc 900gcaggtggcg ccgtacatac acgggggctg gattttgcct gtgatttctg ggtgctggtc 960gttgtgggcg gcgtgctggc ctgctacagc ctgctggtga cagtggcctt catcatcttt 1020tgggtgagga gcaagcggag tcgactgctg cacagcgact acatgaacat gaccccccgg 1080aggcctggcc ccacccggaa gcactaccag ccctacgccc ctcccaggga tttcgccgcc 1140taccggagca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgagg 1200ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1260ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1320ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1380gacaagcgta gaggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1440gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1500atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg actcagtaca 1560gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctag 1614292536PRTArtificial SequenceCEA CAR 292Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ser Glu 20 25 30Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45Tyr Thr Phe Thr Glu Phe Gly Met Asn Trp Val Arg Gln Ala Pro Gly 50 55 60Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala65 70 75 80Thr Tyr Val Glu Glu Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr 85 90 95Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp 100 105 110Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asp Phe Ala His Tyr Phe Gln 115 120 125Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly145 150 155 160Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 165 170 175Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr 180 185 190Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 195 200 205Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly 210 215 220Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro225 230 235 240Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu 245 250 255Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr 260 265 270Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val305 310 315 320Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 325 330 335Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp 340 345 350Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 355 360 365Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg 370 375 380Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro385 390 395 400Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 405 410 415Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 420 425 430Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 435 440 445Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 450 455 460Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu465 470 475 480Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 485 490 495Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 500 505 510Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 515 520 525His Met Gln Ala Leu Pro Pro Arg 530 5352931611DNAArtificial SequenceCEA CAR 293atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60agatgtcagg tgcagctggt gcaatctggg tctgagttga agaagcctgg ggcctcagtg 120aaggtttcct gcaaggcttc tggatacacc ttcactgagt ttggaatgaa ctgggtgcga 180caggcccctg gacaagggct tgagtggatg ggatggataa acaccaaaac tggagaggca 240acatatgttg aagagtttaa gggacggttt gtcttctcct tggacacctc tgtcagcacg 300gcatatctgc agatcagcag cctaaaggct gaagacactg ccgtgtatta ctgtgcgaga 360tgggactttg ctcattactt tcagactatg gactactggg gccaagggac cacggtcacc 420gtctcctcag gcggaggtgg aagcggaggg ggaggatctg gcggcggagg aagcggaggc 480gatatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 540atcacttgca aggccagtgc ggctgtgggt acgtatgttg cgtggtatca gcagaaacca 600gggaaagcac ctaagctcct gatctattcg gcatcctacc gcaaaagggg agtcccatca 660aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 720gaagatttcg caacttacta ctgtcaccaa tattacacct atcctctatt cacgtttggc 780cagggcacca agctcgagat caagcgtaca acgacgccag ctccccgccc gccaacccct 840gcacctacga ttgcatcaca accgctgtcc ctgcggcctg aagcttgtcg cccagccgca 900ggtggcgccg tacatacacg ggggctggat tttgcctgtg atttctgggt gctggtcgtt 960gtgggcggcg tgctggcctg ctacagcctg ctggtgacag tggccttcat catcttttgg 1020gtgaggagca agcggagtcg actgctgcac agcgactaca tgaacatgac cccccggagg 1080cctggcccca cccggaagca ctaccagccc tacgcccctc ccagggattt cgccgcctac 1140cggagcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgaggcca 1200gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1260ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1320cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1380aagcgtagag gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1440ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1500aaaggcgagc gccggagggg caaggggcac gatggccttt accagggact cagtacagcc 1560accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta g 16112945PRTArtificial SequenceCEA CDR-H1 294Glu Phe Gly Met Asn1 529517PRTArtificial SequenceCEA CDR-H2 295Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe Lys1 5 10 15Gly29612PRTArtificial SequenceCEA CDR-H3 296Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr1 5 1029712PRTArtificial SequenceCEA CDR-H3 297Trp Asp Phe Ala His Tyr Phe Gln Thr Met Asp Tyr1 5 1029811PRTArtificial SequenceCEA CDR-L1 298Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala1 5 1029911PRTArtificial SequenceCEA CDR-L1 299Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala1 5 103007PRTArtificial SequenceCEA CDR-L2 300Ser Ala Ser Tyr Arg Tyr Ser1 53017PRTArtificial SequenceCEA CDR-L2 301Ser Ala Ser Tyr Arg Lys Arg1 530210PRTArtificial SequenceCEA CDR-L3 302His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr1 5 1030310PRTArtificial SequenceLB-CYBA-1Y peptideX(10)..(10)X is Y or H 303Ser Thr Met Glu Arg Trp Gly Gln Lys Xaa1 5 1030411PRTArtificial SequenceLB-OAS1-1RX(7)..(7)X is R or T 304Glu Thr Asp Asp Pro Arg Xaa Tyr Gln Lys Tyr1 5 103059PRTArtificial SequenceHA-2 peptideX(9)..(9)X is V or M 305Tyr Ile Gly Glu Val Leu Val Ser Xaa1 53069PRTArtificial SequenceHA-8 peptideX(1)..(1)X is R or P 306Xaa Thr Leu Asp Lys Val Leu Glu Val1 53079PRTArtificial SequenceHA-3 peptideX(2)..(2)X is T or M 307Val Xaa Glu Pro Gly Thr Ala Gln Tyr1 530811PRTArtificial SequenceHwA11-S peptideX(6)..(6)X is S or T 308Cys Ile Pro Pro Asp Xaa Leu Leu Phe Pro Ala1 5 1030911PRTArtificial SequenceLB-ADIR-1F peptideX(9)..(9)X is F or S 309Ser Val Ala Pro Ala Leu Ala Leu Xaa Pro Ala1 5 103109PRTArtificial SequenceLB-HIVEP1-1S peptideX(6)..(6)X is S or N 310Ser Leu Pro Lys His Xaa Val Thr Ile1 53119PRTArtificial SequenceLB-NISCH-1A peptideX(7)..(7)X is A or V 311Ala Leu Ala Pro Ala Pro Xaa Glu Val1 53129PRTArtificial SequenceLB-SSR1-1S peptideX(1)..(1)X is S or L 312Xaa Leu Ala Val Ala Gln Asp Leu Thr1 531310PRTArtificial SequenceLB-WNK1-1I peptideX(7)..(7)X is I or M 313Arg Thr Leu Ser Pro Glu Xaa Ile Thr Val1 5 1031410PRTArtificial SequenceT4A peptideX(10)..(10)X is A or E 314Gly Leu Tyr Thr Tyr Trp Ser Ala Gly Xaa1 5 103159PRTArtificial SequenceUTA2-1 peptideX(3)..(3)X is L or P 315Gln Leu Xaa Asn Ser Val Leu Thr Leu1 53169PRTArtificial SequenceLB-CLYBL-1Y peptideX(5)..(5)X is Y or D 316Ser Leu Ala Ala Xaa Ile Pro Arg Leu1 531710PRTArtificial SequenceTRIM22 peptideX(7)..(7)X is C or R 317Met Ala Val Pro Pro Cys Xaa Ile Gly Val1 5 1031811PRTArtificial SequencePARP10-1L peptideX(3)..(3)X is L or P 318Gly Leu Xaa Gly Gln Glu Gly Leu Val Glu Ile1 5 103199PRTArtificial SequenceFAM119A-1T peptideX(8)..(8)X is T or I 319Ala Met Leu Glu Arg Gln Phe Xaa Val1 53209PRTArtificial SequenceGLRX3-1S peptideX(3)..(3)X is S or P 320Phe Leu Xaa Ser Ala Asn Glu His Leu1 53219PRTArtificial SequenceHNF4G-1M peptideX(2)..(2)X is M or I 321Met Xaa Tyr Lys Asp Ile Leu Leu Leu1 53229PRTArtificial SequenceHMMR-1V peptideX(6)..(6)X is V or A 322Ser Leu Gln Glu Lys Xaa Ala Lys Ala1 53239PRTArtificial SequenceBCL2A1 peptideX(4)..(4)X is N or K 323Val Leu Gln Xaa Val Ala Phe Ser Val1 532410PRTArtificial SequenceCDC26-1F peptideX(1)..(1)X is F or S 324Xaa Val Ala Gly Thr Gln Glu Val Phe Val1 5 1032510PRTArtificial SequenceAPOBEC3F-1S/A peptideX(3)..(3)X is S or A 325Phe Leu Xaa Glu His Pro Asn Val Thr Leu1 5 103269PRTArtificial SequenceLB-PRCP-1D peptideX(8)..(8)X is D or E 326Phe Met Trp Asp Val Ala Glu Xaa Leu1 532711PRTArtificial SequenceLB-PRCP-1D peptideX(8)..(8)X is D or E 327Phe Met Trp Asp Val Ala Glu Xaa Leu Lys Ala1 5

103289PRTArtificial SequenceLB-CCL4-1T peptideX(7)..(7)X is T or S 328Cys Ala Asp Pro Ser Glu Xaa Trp Val1 53299PRTArtificial SequenceLB-NCAPD3-1Q peptideX(3)..(3)X is Q or R 329Trp Leu Xaa Gly Val Val Pro Val Val1 53309PRTArtificial SequenceLB-NDC80-1P peptideX(7)..(7)X is P or A 330His Leu Glu Glu Gln Ile Xaa Lys Val1 53319PRTArtificial SequenceLB-TTK-1D peptideX(4)..(4)X is D or E 331Arg Leu His Xaa Gly Arg Val Phe Val1 53329PRTArtificial SequenceWDR27-1L peptideX(2)..(2)X is L or P 332Ser Xaa Asp Asp His Val Val Ala Val1 533311PRTArtificial SequenceMIIP peptideX(8)..(8)X is K or E 333Ser Glu Glu Ser Ala Val Pro Xaa Arg Ser Trp1 5 1033410PRTArtificial SequenceMIIP peptideX(7)..(7)X is K or E 334Glu Glu Ser Ala Val Pro Xaa Arg Ser Trp1 5 103359PRTArtificial SequenceLB-DHX33-1C peptideX(9)..(9)X is C or R 335Tyr Leu Tyr Glu Gly Gly Ile Ser Xaa1 533610PRTArtificial SequencePANE1 peptide 336Arg Val Trp Asp Leu Pro Gly Val Leu Lys1 5 1033710PRTArtificial SequenceSP110 peptideX(4)..(4)X is R or G 337Ser Leu Pro Xaa Gly Thr Ser Thr Pro Lys1 5 103389PRTArtificial SequenceACC-1C/YX(5)..(5)X is Y or C 338Asp Tyr Leu Gln Xaa Val Leu Gln Ile1 53399PRTArtificial SequenceP2RX7 peptideX(6)..(6)X is H or R 339Trp Phe His His Cys Xaa Pro Lys Tyr1 53409PRTArtificial SequenceACC-4 peptideX(9)..(9)X is R or G 340Ala Thr Leu Pro Leu Leu Cys Ala Xaa1 534110PRTArtificial SequenceACC-5X(10)..(10)X is R or G 341Trp Ala Thr Leu Pro Leu Leu Cys Ala Xaa1 5 103429PRTArtificial SequenceAKAP13 peptideX(9)..(9)X is M or T 342Ala Pro Ala Gly Val Arg Glu Val Xaa1 534310PRTArtificial SequenceLB-APOBEC3B-1K peptideX(1)..(1)X is K or E 343Xaa Pro Gln Tyr His Ala Glu Met Cys Phe1 5 103449PRTArtificial SequenceAPOBEC3H peptideX(5)..(5)X is K or E 344Lys Pro Gln Gln Xaa Gly Leu Arg Leu1 53458PRTArtificial SequenceLB-ARHGDIB-1R peptideX(6)..(6)X is R or P 345Pro Arg Ala Cys Trp Xaa Glu Ala1 534610PRTArtificial SequenceLB-BCAT2-1R peptideX(3)..(3)X is R or T 346Gln Pro Xaa Arg Ala Leu Leu Phe Val Ile1 5 1034711PRTArtificial SequenceBFAR peptideX(11)..(11)X is M or R 347Ala Pro Asn Thr Gly Arg Ala Asn Gln Gln Xaa1 5 1034811PRTArtificial SequenceC14orf169 peptideX(4)..(4)X is A or V 348Arg Pro Arg Xaa Pro Thr Glu Glu Leu Ala Leu1 5 1034911PRTArtificial SequenceLB-C16ORF-1R peptideX(1)..(1)X is R or W 349Xaa Pro Cys Pro Ser Val Gly Leu Ser Phe Leu1 5 103509PRTArtificial SequenceC18orf21 peptideX(6)..(6)X is A or T 350Asn Pro Ala Thr Pro Xaa Ser Lys Leu1 535110PRTArtificial SequenceLB-EBI3-1I peptideX(8)..(8)X is I or V 351Arg Pro Arg Ala Arg Tyr Tyr Xaa Gln Val1 5 103529PRTArtificial SequencePOP1 peptideX(7)..(7)X is N or K 352Leu Pro Gln Lys Lys Ser Xaa Ala Leu1 53539PRTArtificial SequenceSCRIB peptideX(7)..(7)X is L or P 353Leu Pro Gln Gln Pro Pro Xaa Ser Leu1 53549PRTArtificial SequenceMTRR peptideX(5)..(5)X is S or L 354Ser Pro Ala Ser Xaa Arg Thr Asp Leu1 53559PRTArtificial SequenceLLGL2X(5)..(5)X is R or H 355Ser Pro Ser Leu Xaa Ile Leu Ala Ile1 535611PRTArtificial SequenceLB-ECGF-1H peptideX(3)..(3)X is H or R 356Arg Pro Xaa Ala Ile Arg Arg Pro Leu Ala Leu1 5 1035711PRTArtificial SequenceLB-ERAP1-1R peptideX(3)..(3)X is R or P 357His Pro Xaa Gln Glu Gln Ile Ala Leu Leu Ala1 5 103589PRTArtificial SequenceLB-ERAP1-1R peptideX(3)..(3)X is R or P 358His Pro Xaa Gln Glu Gln Ile Ala Leu1 53599PRTArtificial SequenceLB-FUCA2-1V peptideX(5)..(5)X is V or M 359Arg Leu Arg Gln Xaa Gly Ser Trp Leu1 53609PRTArtificial SequenceLB-GEMIN4-1V peptideX(9)..(9)X is V or E 360Phe Pro Ala Leu Arg Phe Val Glu Xaa1 536111PRTArtificial SequenceHDGF peptideX(11)..(11)X is L or P 361Leu Pro Met Glu Val Glu Lys Asn Ser Thr Xaa1 5 1036211PRTArtificial SequenceLB-PDCD11-1F peptideX(8)..(8)X is F or L 362Gly Pro Asp Ser Ser Lys Thr Xaa Leu Cys Leu1 5 103639PRTArtificial SequenceLB-PFAS-1P peptideX(2)..(2)X is P or S 363Ala Xaa Gly His Thr Arg Arg Lys Leu1 536410PRTArtificial SequenceLB-TEP1-1S peptideX(9)..(9)X is S or P 364Ala Pro Asp Gly Ala Lys Val Ala Xaa Leu1 5 1036511PRTArtificial SequenceLB-TMEM8A-1I peptideX(7)..(7)X is I or V 365Arg Pro Arg Ser Val Thr Xaa Gln Pro Leu Leu1 5 1036610PRTArtificial SequenceLB-USP15-1I peptideX(8)..(8)X is I or T 366Met Pro Ser His Leu Arg Asn Xaa Leu Leu1 5 103679PRTArtificial SequenceLRH-1 peptide 367Thr Pro Asn Gln Arg Gln Asn Val Cys1 53689PRTArtificial SequenceLB-MOB3A-1C peptideX(1)..(1)X is C or S 368Xaa Pro Arg Pro Gly Thr Trp Thr Cys1 536911PRTArtificial SequenceLB-ZDHHC6-1Y peptideX(4)..(4)X is Y or H 369Arg Pro Arg Xaa Trp Ile Leu Leu Val Lys Ile1 5 1037010PRTArtificial SequenceZAPHIR peptide 370Ile Pro Arg Asp Ser Trp Trp Val Glu Leu1 5 103719PRTArtificial SequenceHEATR1 peptideX(7)..(7)X is E or G 371Ile Ser Lys Glu Arg Ala Xaa Ala Leu1 53729PRTArtificial SequenceLB-GSTP1-1V peptideX(7)..(7)X is V or I 372Asp Leu Arg Cys Lys Tyr Xaa Ser Leu1 53739PRTArtificial SequenceHA-1/B60 peptideX(6)..(6)X is H or R 373Lys Glu Cys Val Leu Xaa Asp Asp Leu1 53749PRTArtificial SequenceLB-SON-1R peptideX(6)..(6)X is R or C 374Ser Glu Thr Lys Gln Xaa Thr Val Leu1 53759PRTArtificial SequenceLB-SWAP70-1Q peptideX(6)..(6)X is Q or E 375Met Glu Gln Leu Glu Xaa Leu Glu Leu1 53769PRTArtificial SequenceLB-TRIP10-1EPC peptideX(2)..(2)X is E or GX(3)..(3)X is P or SX(7)..(7)X is C or G 376Gly Xaa Xaa Gln Asp Leu Xaa Thr Leu1 53779PRTArtificial SequenceLB-NUP133-1R peptideX(8)..(8)X is R or Q 377Ser Glu Asp Leu Ile Leu Cys Xaa Leu1 537811PRTArtificial SequenceLB-ZNFX1-1Q peptideX(8)..(8)X is Q or H 378Asn Glu Ile Glu Asp Val Trp Xaa Leu Asp Leu1 5 1037911PRTArtificial SequenceSLC1A5 peptideX(3)..(3)X is A or P 379Ala Glu Xaa Thr Ala Asn Gly Gly Leu Ala Leu1 5 1038010PRTArtificial SequenceACC-2 peptideX(6)..(6)X is D or G 380Lys Glu Phe Glu Asp Xaa Ile Ile Asn Trp1 5 1038111PRTArtificial SequenceACC-6 peptide 381Met Glu Ile Phe Ile Glu Val Phe Ser His Phe1 5 1038210PRTArtificial SequenceHB-1H/Y peptideX(8)..(8)X is H or Y 382Glu Glu Lys Arg Gly Ser Leu Xaa Val Trp1 5 103839PRTArtificial SequenceDPH1 peptideX(2)..(2)X is V or L 383Ser Xaa Leu Pro Glu Val Asp Val Trp1 53849PRTArtificial SequenceUGT2B17/A02 peptide 384Cys Val Ala Thr Met Ile Phe Met Ile1 538510PRTArtificial SequenceUGT2B17/A29 385Ala Glu Leu Leu Asn Ile Pro Phe Leu Tyr1 5 1038610PRTArtificial SequenceUGT2B17/B44 peptide 386Ala Glu Leu Leu Asn Ile Pro Phe Leu Tyr1 5 103879PRTArtificial SequenceDFFRY peptideX(4)..(4)X is C or S 387Ile Val Asp Xaa Leu Thr Glu Met Tyr1 53889PRTArtificial SequenceSMCY peptide 388Phe Ile Asp Ser Tyr Ile Cys Gln Val1 538911PRTArtificial SequenceTMSB4Y peptide 389Glu Val Leu Leu Arg Pro Gly Leu His Phe Arg1 5 103901533DNAArtificial SequenceCD19 CAR 390gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300gggaccaagc tggagatcac aggcggaggt ggaagcggag ggggaggatc tggcggcgga 360ggaagcggag gcgaggtgaa actgcaggag tcaggacctg gcctggtggc gccctcacag 420agcctgtccg tcacatgcac tgtctcaggg gtctcattac ccgactatgg tgtaagctgg 480attcgccagc ctccacgaaa gggtctggag tggctgggag taatatgggg tagtgaaacc 540acatactata attcagctct caaatccaga ctgaccatca tcaaggacaa ctccaagagc 600caagttttct taaaaatgaa cagtctgcaa actgatgaca cagccattta ctactgtgcc 660aaacattatt actacggtgg tagctatgct atggactact ggggccaagg aacctcagtc 720accgtgtcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 780tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 840acgagggggc tggacttcgc ctgtgatttc tgggtgctgg tcgttgtggg cggcgtgctg 900gcctgctaca gcctgctggt gacagtggcc ttcatcatct tttgggtgag gagcaagcgg 960agcagactgc tgcacagcga ctacatgaac atgacccccc ggaggcctgg ccccacccgg 1020aagcactacc agccctacgc ccctcccagg gatttcgccg cctaccggag caaacggggc 1080agaaagaaac tcctgtatat attcaaacaa ccatttatga ggccagtaca aactactcaa 1140gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1200gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1260aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagcg tagaggccgg 1320gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1380ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1440aggggcaagg ggcacgatgg cctttaccag ggactcagta cagccaccaa ggacacctac 1500gacgcccttc acatgcaggc cctgccccct cgc 1533391245PRTArtificial SequenceEGFR ScFv 391Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75 80Ser Leu Lys Val Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Ile Val Met Thr Gln 130 135 140Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser145 150 155 160Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln 165 170 175Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg 180 185 190Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr 210 215 220Tyr Cys His Gln Tyr Gly Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr225 230 235 240Lys Ala Glu Ile Lys 245

Claims

1. An engineered immune cell comprising: activating and blocking receptors on a surface of the cell, wherein when the engineered immune cell encounters a tumor cell and a healthy cell: a first region of the activating and blocking receptors forms proximal to the healthy cell and blocking receptors in the first region inhibit cytotoxic effects on the healthy cell, while, simultaneously, a second region of the activating and blocking receptors forms proximal to the tumor cell and promotes a cytotoxic response by the engineered immune cell that exhibits cytotoxic effects on the tumor cell.

2. The engineered immune cell of claim 1, wherein the activating and blocking receptors in the first region bind to cognate activating and blocking ligands on the healthy cell, and the activating receptors in the second region bind to cognate activating ligands on the tumor cell.

3. The engineered immune cell of claim 2, wherein the activating and blocking receptors form a first micro-cluster in the first region, and the activating and blocking receptors form a second micro-cluster in the second region.

4. The engineered immune cell of claim 3, wherein binding of the blocking receptors in the first micro-cluster to the cognate blocking ligands on the healthy cell causes the engineered immune cell to exhibit reduced surface expression of the activating receptor in the first micro-cluster.

5. The engineered immune cell of claim 3, wherein binding of the blocking receptors in the first micro-cluster to the cognate blocking ligands on the healthy cell prevents breakup of the first micro-cluster.

6. The engineered immune cell of claim 2, wherein the immune cell expresses different concentrations of the activating and blocking receptors based on a ratio of a quantity of the activating ligand to a quantity of the blocking ligand expressed in a healthy cell.

7. The engineered immune cell of claim 6, wherein the ratio of the concentration of blocking receptors to activating receptors expressed by the immune cell is less than or equal to 1.

8. The engineered immune cell of claim 2, wherein the blocking receptors do not bind to the cognate blocking ligands until the activating receptors bind to cognate activating ligands.

9. The engineered immune cell of claim 1, wherein the cytotoxic response by the engineered immune cell that exhibits cytotoxic effects on the tumor cell is localized to the second region.

10. The engineered immune cell of claim 9, wherein the localized cytotoxic response does not exhibit cytotoxic effects on the healthy cell.

11. A method for treating cancer, the method comprising: providing an engineered immune cell to a patient, the engineered immune cell comprising activating and blocking cell-surface receptors, wherein when the engineered immune cell encounters a tumor cell and a healthy cell of the patient: a first set of the activating and blocking receptors collect into a first cell-surface region of the engineered immune cell proximal to the healthy cell in which the blocking receptors inhibit cytotoxic effects of the engineered immune cell on the healthy cell, simultaneously, a second set of the activating and blocking receptors collect into a second cell-surface region of the engineered immune cell proximal to the tumor cell in which the activating receptors promote a cytotoxic response by the engineered immune cell that kills the tumor cell.

12. The method of claim 11, wherein the activating and blocking receptors in the first cell-surface region bind to cognate activating and blocking ligands on the healthy cell, and the activating receptors in the second cell-surface region bind to cognate activating ligands on the tumor cell.

13. The method of claim 12, wherein the activating and blocking receptors form a first micro-cluster on the first cell-surface region, and the activating and blocking receptors form a second micro-cluster on the second cell-surface region.

14. The method of claim 13, wherein binding of the blocking receptors in the first micro-cluster to the cognate blocking ligands on the healthy cell causes the engineered immune cell to exhibit reduced surface expression of the activating receptor in the first micro-cluster.

15. The method of claim 13, wherein biding of the blocking receptors in the first micro-cluster to the cognate blocking ligands on the healthy cell prevents breakup of the first micro-cluster.

16. The method of claim 12, wherein the immune cell expresses different concentrations of the activating and blocking receptors based on a ratio of a quantity of the activating ligand to a quantity of a blocking ligand expressed in a healthy cell.

17. The method of claim 16, wherein the ratio of the concentration of blocking receptors to activating receptors expressed by the immune cell is less than or equal to 1.

18. The method of claim 12, wherein the blocking receptors do not bind to the cognate blocking ligands until the activating receptors bind to the cognate activating ligands.

19. The method of claim 12, wherein the cytotoxic response is localized to the second cell-surface region.

20. The method of claim 19, wherein the localized cytotoxic response does not exhibit cytotoxic effects on the healthy cell.