CHIMERIC ANTIGEN RECEPTOR AND NATURAL KILLER CELLS EXPRESSING SAME

Abstract

The present invention provides a chimeric antigen receptor and natural killer cells expressing the same, and particularly, a chimeric antigen receptor (CAR) which includes an intracellular signaling domain including the whole or a portion of an OX40 ligand (CD252), thereby having excellent effects of increasing anticancer activity of immune cells, and immune cells expressing the same.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a National Stage of International Application No. PCT/KR2017/015635 filed Dec. 28, 2017, claiming priority based on Korean Patent Application No. 10-2016-0181119 filed Dec. 28, 2016.

TECHNICAL FIELD

[0002] The present invention relates to a chimeric antigen receptor and natural killer cells expressing the same.

BACKGROUND ART

[0003] Human natural killer cells (NK cells) play an important role in innate immune defense against malignant lymphoma cells, and thus may be suitable for adoptive immune therapy (i.e., adoptive cellular immunotherapy). However, due to difficulties in ex vivo cell expansion and differences in activities of NK cells in individual patients, it is difficult to use the NK cells.

[0004] In a field of immune cell therapy using T cells, that is, T cell therapy field, a chimeric antigen receptor for treatment of autoimmune cells has been developed. The chimeric antigen receptor has been known to help activation of T cells by reprogramming T cells in order to improve treatment effects to specific cancers or overcome resistance against T cell treatment.

[0005] OX40 ligand (CD252) is a protein belonging to a TNFR superfamily, and is known to be expressed in antigen-presenting cells (APCs), some of NK cells and some of B cells, as well as known to be expressed in several hours to a few days after activation of these cells.

[0006] As a receptor of OX40 ligand (CD252). OX40 (CD134) is known to be expressed in T cells, and also known to be expressed in 24 hours after activation of T cells by antigen and CD28. It is known that expression of CD134 may further enhance a response of T cells by CD28 activation, so as to increase proliferation of T cells, and cytokine secretion and survival.

[0007] However, a role of OX40 ligand in enhancement of anticancer activity of NK cells is not well known, and an attempt to use the OX40 ligand for the chimeric antigen receptor is still not yet reported.

[0008] In order to research functional roles of CD252 in antitumor response of NK cells, the present inventors have constructed two different kinds of NK receptors containing CAR. First, a variety of stimulatory cofactors known to increase cytotoxicity of NK cells in a signaling domain were genetically engineered to produce lentivirus encoding high affinity recombination FCRG3A V158 variants (CD16V). CD16V receptors including different intercellular signaling molecules were transduced in NK cells, followed by detecting expression thereof. Further, antitumor responses of the transduced NK cells were investigated using lymphoma cell lines in vitro in the presence of rituximab specific to CD20 molecules.

[0009] As a second approach, functional roles of CD252 in NKG2D-mediated antitumor activity was tested. NKG2D as a key receptor in NK cell activation can recognize MHC class 1 chain-related A (MICA), MICB and other diverse UL 16-binding proteins (ULBP), which are preferentially expressed after genotoxic stress, infection and/or cytotoxicity.

[0010] A variety of NKG2D chimeric antigen receptors containing different signaling molecules were genetically engineered, transduced, and expressed in NK92MI cells. Thereafter, the transduced cells were subjected to evaluation of antitumor effects in vitro.

[0011] The present inventors have found that, as one approach to overcome limitations of current immune cell therapy like NK cells, a chimeric antigen receptor containing OX40 ligand has significantly improved in anticancer activities of NK cells, as compared to the other signaling domains known, thereby completing the present invention.

DISCLOSURE

Technical Problem

[0012] Accordingly, it is an object of the present invention to provide a chimeric antigen receptor capable of increasing anticancer immune cell treatment efficiency using natural killer (NK) cells.

Technical Solution

[0013] 1. A chimeric antigen receptor, including: an intracellular signaling domain which includes the whole or a portion of OX40 ligand (CD252).

[0014] 2. The chimeric antigen receptor according to the above 1, further including: a transmembrane domain linked to the intracellular signaling domain; a spacer domain linked to the transmembrane domain; and an extracellular domain linked to the spacer domain.

[0015] 3. The chimeric antigen receptor according to the above 2, further comprising a signal sequence linked to the extracellular domain.

[0016] 4. The chimeric antigen receptor according to the above 3, wherein the signal sequence includes the whole or a portion of CD16 and the signal sequence includes the whole or a portion of CD8a, respectively.

[0017] 5. The chimeric antigen receptor according to the above 2, wherein the extracellular domain includes the whole or a portion of any one selected from the group consisting of an antigen-binding fragment of an antibody, an Fc receptor, a natural cytotoxicity receptor, NKG2D, 2B4 and DNAM-1.

[0018] 6. The chimeric antigen receptor according to the above 5, wherein the antigen-binding fragment is Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment.

[0019] 7. The chimeric antigen receptor according to the above 6, wherein the Fv fragment is a single-chain variable fragment (ScFv).

[0020] 8. The chimeric antigen receptor according to the above 5, wherein the Fc receptor is selected from the group consisting of CD16, CD32, CD64, CD23, CD89 and variants thereof.

[0021] 9. The chimeric antigen receptor according to the above 8, wherein the Fc receptor is CD16 or variants thereof.

[0022] 10. The chimeric antigen receptor according to the above 5, wherein the natural cytotoxicity receptor is selected from the group consisting of NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.

[0023] 11. The chimeric antigen receptor according to the above 2, wherein the spacer domain includes the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

[0024] 12. The chimeric antigen receptor according to the above 2, wherein the transmembrane domain includes the whole or a portion of any one selected from the group consisting of CD8a, and CD28.

[0025] 13. The chimeric antigen receptor according to the above 1, wherein the intracellular signaling domain further includes the whole or a portion of CD3-zeta.

[0026] 14. The chimeric antigen receptor according to the above 13, wherein the whole or a portion of CD3-zeta and the whole or a portion of OX40 ligand are arranged in order from a cell membrane toward an inside of the cell.

[0027] 15. The chimeric antigen receptor according to the above 2, further comprising an AAA linker between the extracellular domain and the spacer domain.

[0028] 16. A chimeric antigen receptor, comprising intracellular signaling domains which include: a first intracellular signaling domain including the whole or a portion of any one selected from the group consisting of CD28 and 4-1BB; a second intracellular signaling domain including the whole or a portion of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB; and a third intracellular signaling domain including the whole or a portion of CD3-zeta, wherein the first, second and third intracellular signaling domains are arranged in order from a cell membrane toward an inside of the cell.

[0029] 17. The chimeric antigen receptor according to the above 16, further including: a transmembrane domain linked to the intracellular signaling domain; a spacer domain linked to the transmembrane domain; and an extracellular domain linked to the spacer domain.

[0030] 18. The chimeric antigen receptor according to the above 17, further comprising a signal sequence linked to the extracellular domain.

[0031] 19. The chimeric antigen receptor according to the above 18, wherein the signal sequence includes the whole or a portion of CD16 and the signal sequence includes the whole or a portion of CD8a, respectively.

[0032] 20. The chimeric antigen receptor according to the above 17, wherein the extracellular domain includes the whole or a portion of any one selected from the group consisting of an antigen-binding fragment of an antibody, an Fc receptor, a natural cytotoxicity receptor, NKG2D, 2B4 and DNAM-1.

[0033] 21. The chimeric antigen receptor according to the above 17, wherein the spacer domain includes the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

[0034] 22. The chimeric antigen receptor according to the above 17, wherein the transmembrane domain includes the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

[0035] 23. The chimeric antigen receptor according to the above 16, wherein the first intracellular signaling domain includes the whole or a portion of CD28; the second intracellular signaling domain includes the whole or a portion of the OX40 ligand; and the third intracellular signaling domain includes the whole or a portion of CD3-zeta.

[0036] 24. The chimeric antigen receptor according to the above 17, further comprising an AAA linker between the extracellular domain and the spacer domain. 25. An immune cell expressing the chimeric antigen receptor according to any one of the above 1 to 24.

[0037] 26. The immune cell according to the above 25, wherein the immune cell is a natural killer cell (NK cell).

[0038] 27. A pharmaceutical composition for treatment of tumor, comprising the immune cell according to the above 25 as an active ingredient.

[0039] 28. The pharmaceutical composition according to the above 27, further comprising an antibody as an active ingredient when the extracellular domain is an Fc receptor.

[0040] 29. A nucleic acid sequence encoding the chimeric antigen receptor according to any one of the above 1 to 24.

[0041] 30. The nucleic acid sequence according to the above 29, wherein the nucleic acid sequence encodes one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 33, 41, 43, 45, 47, 49, 51, 53, 55, 69, 71, 77, 81, 83, 85, 87, 89, 91 and 93, or variants thereof having a sequence identity of 80% or more.

[0042] 31. The nucleic acid sequence according to the above 30, wherein the nucleic acid sequence includes one or more nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32, 40, 42, 44, 46, 48, 50, 52, 54, 68, 70, 76, 80, 82, 84, 86, 88, 90 and 92, or variants thereof having a sequence identity of 80% or more.

[0043] 32. A method for treatment of tumor, comprising administering the immune cell according to the above 25 to a subject.

Advantageous Effects

[0044] The chimeric antigen receptor according to the present invention is excellent in NK cell activation efficiency.

[0045] The chimeric antigen receptor according to the present invention may be used along with various cancer-target antibodies depending on cancer types to be targeted.

[0046] The chimeric antigen receptor according to the present invention is applicable to diverse cancer types by applying different antigen recognition sites.

[0047] The NK cells expressing the chimeric antigen receptor according to the present invention are excellent in cytotoxicity on cancer cells.

[0048] The NK cells expressing the chimeric antigen receptor according to the present invention may be usefully used in immune cell treatment.

DESCRIPTION OF DRAWINGS

[0049] FIG. 1A shows CAR expression levels of NK92MI cells transduced with CD16V-Z CAR (1.sup.st generation). CD16V-28Z CAR (2.sup.nd generation), CD16V-BBZ CAR (2.sup.nd generation), CD16V-OX40Z CAR (2.sup.nd generation) or CD16V-28OX40LZ CAR (3.sup.rd generation), respectively, according to an embodiment of the present invention.

[0050] FIG. 1B shows intrinsic cell killing ability (i.e., `cytotoxicity`) of NK92MI cells transduced with CD16V-Z CAR (1.sup.st generation), CD16V-28Z CAR (2.sup.nd generation), CD16V-BBZ CAR (2.sup.nd generation), CD16V-OX40Z CAR (2.sup.nd generation) or CD16V-28OX40LZ CAR (3.sup.rd generation), respectively, which is obtained through assessment of cytotoxicity on K562 cells.

[0051] FIG. 1C shows assessment of NK cell-mediated cytotoxicity of NK92MI cells transduced with CD16V-Z CAR (1.sup.st generation), CD16V-28Z CAR (2.sup.nd generation), CD16V-BBZ CAR (2.sup.nd generation), CD16V-OX40Z CAR (2.sup.nd generation) or CD16V-28OX40LZ CAR (3.sup.rd generation), respectively, according to an embodiment of the present invention, when combined with an antibody to Ramos cells.

[0052] FIG. 1D shows CAR expression levels of NK92MI cells transduced with CD16V-Z CAR (1 s generation) and CD16V-OX40LZ CAR (2.sup.nd generation), respectively, according to an embodiment of the present invention.

[0053] FIG. 1E shows assessment of NK cell-mediated cytotoxicity of NK92MI cells transduced with CD16V-Z CAR (1.sup.st generation) and CD16V-OX40LZ CAR (2.sup.nd generation), respectively, according to an embodiment of the present invention, when combined with an antibody to Ramos cells.

[0054] FIG. 1F shows CAR expression levels of NK92MI cells transduced with CD16V-Z CAR (1.sup.st generation) and CD16V-ZOX40L CAR (2.sup.nd generation), respectively, according to an embodiment of the present invention.

[0055] FIG. 1G shows assessment of NK cell-mediated cytotoxicity of NK92MI cells transduced with CD16V-Z CAR (1.sup.st generation) or CD16V-ZOX40L CAR (2.sup.nd generation), respectively, according to an embodiment of the present invention, when combined with an antibody to Ramos cells.

[0056] FIG. 2A shows CAR expression levels of NK92MI cells transduced with CD16V-Z CAR (1.sup.st generation), CD16V-BBZ CAR (2.sup.nd generation) or CD16V-BBOX40LZ CAR (3.sup.rd generation), respectively, according to an embodiment of the present invention.

[0057] FIG. 2B shows assessment of NK cell-mediated cytotoxicity of NK92MI cells expressing CD16V-Z CAR (1.sup.st generation), CD16V-BBZ CAR (2.sup.nd generation) or CD16V-BBOX40LZ CAR (3.sup.rd generation), respectively, according to an embodiment of the present invention, when combined with an antibody to Ramos cells.

[0058] FIG. 3A shows CAR expression levels of NK92MI cells transduced with CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR or CD16V-28BBZ CAR, respectively, all of which are 3.sup.rd generation CARs, according to an embodiment of the present invention.

[0059] FIG. 3B shows assessment of NK cell-mediated cytotoxicity of NK92MI cells expressing CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR or CD16V-28BBZ CAR, respectively, all of which are 3.sup.rd generation CARs, according to an embodiment of the present, when combined with an antibody to Ramos cells.

[0060] FIG. 4A shows CAR expression levels of NK92MI cells transduced with CD16V-28(H)BBZ CAR, CD16V-28(H)OX40Z CAR, or CD16V-28(H)OX40LZ CAR, respectively, all of which are 3.sup.rd generation CARs each containing CD28 at a hinge, according to an embodiment of the present invention.

[0061] FIG. 4B shows assessment of NK cell-mediated cytotoxicity of NK91MI cells expressing CD16V-28(H)BBZ CAR, CD16V-28(H)OX40Z CAR or CD16V-28(H)OX40LZ CAR, respectively, all of which are 3.sup.rd generation CARs each having CD28 at a hinge, according to an embodiment of the present invention, when combined with an antibody to Ramos cells.

[0062] FIG. 5A shows CAR expression levels of NK92MI cells transduced with NKG2D-Z CAR (1.sup.st generation), NKG2D-28Z CAR (2.sup.nd generation), NKG2D-BBZ CAR (2.sup.nd generation) or NKG2D-OX40Z CAR (2.sup.nd generation), respectively, according to an embodiment of the present invention.

[0063] FIG. 5B shows assessment of NK cell-mediated cytotoxicity of NK92MI cells transduced with NKG2D-Z CAR (1.sup.st generation), NKG2D-28Z CAR (2.sup.nd generation), NKG2D-BBZ CAR (2.sup.nd generation) or NKG2D-OX40Z CAR (2.sup.nd generation), respectively, against human breast cancer cell lines MCF-7, according to an embodiment of the present invention.

[0064] FIG. 6A shows CAR expression levels of NK92MI cells transduced with NKG2D-Z CAR (1.sup.st generation), NKG2D-28BBZ CAR (3.sup.rd generation) including CD28 signaling domain or NKG2D-28OX40Z CAR (3.sup.rd generation), respectively, according to the present invention.

[0065] FIG. 6B shows assessment of NK cell-mediated cytotoxicity of NK92MI cells transduced with NKG2D-Z CAR (1.sup.st generation), NKG2D-28BBZ CAR including CD28 signaling domain (3.sup.rd generation) or NKG2D-28OX40Z CAR (3.sup.rd generation), respectively, against human breast cancer cell lines MCF-7, according to an embodiment of the present invention.

[0066] FIG. 7A shows CAR expression levels of NK92MI cells transduced with NKG2D-Z CAR (1.sup.st generation). NKG2D-28Z CAR (2.sup.nd generation), NKG2D-28(H)OX40LZ CAR (3.sup.rd generation) or NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd generation) including AAA sequence between NKG2D extracellular domain and CD28 hinge, respectively, according to an embodiment of the present invention.

[0067] FIG. 7B shows assessment of NK cell-mediated cytotoxicity of NK92MI cells transduced with NKG2D-Z CAR (1.sup.st generation), NKG2D-28Z CAR (2.sup.nd generation), NKG2D-28(H)OX40LZ CAR (3.sup.rd generation) or NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd generation) including AAA sequence between NKG2D extracellular domain and CD28 hinge, respectively, against human breast cell lines MCF-7, according to an embodiment of the present invention.

[0068] FIG. 8A shows CAR expression levels of NK92MI cells transduced with NKG2D-Z CAR (1.sup.st generation) or NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd generation) including AAA sequence between NKG2D extracellular domain and CD28 hinge, respectively, according to an embodiment of the present invention.

[0069] FIG. 8B shows expression levels of different NKG2D ligands in human lung cancer cell lines H, 1299 and H, 1944 according to an embodiment of the present invention.

[0070] FIG. 8C shows assessment of NK-mediated cytotoxicity of NK92MI cells transduced with NKG2D-Z CAR (1.sup.st generation) or NKG2D-AAA-28(H)OX40LZ (3.sup.rd generation) including AAA sequence between NKG2D extracellular domain and CD28 hinge, respectively, against human lung cancer cell lines H, 1299 and H, 1944, according to an embodiment of the present invention.

BEST MODE

[0071] The present invention discloses a chimeric antigen receptor and NK cells expressing the same, and more particularly, a chimeric antigen receptor (CAR) with excellent effects of increasing anticancer activity of immune cells, by including an intracellular signaling domain which contains the whole or a portion of OX40 ligand (CD252), as well as immune cells expressing the same.

[0072] Hereinafter, the present invention will be described in detail.

[0073] The chimeric antigen receptor of the present invention may include an intracellular signaling domain which contains the whole or a portion of OX40 ligand (CD252).

[0074] According to one embodiment of the present invention, the chimeric antigen receptor may be one further including a transmembrane domain linked to the intracellular signaling domain; a spacer domain linked to the transmembrane domain; and an extracellular domain linked to the spacer domain. In addition, according to one embodiment of the present invention, the chimeric antigen receptor may be one further including a signal sequence linked to a terminal of the extracellular domain that is not linked with the spacer domain. According to one embodiment of the present invention, the above-described domains may be directly linked to one another or may be linked by a linker.

[0075] According to one embodiment of the present invention, the signal sequence may allow the extracellular domain to be positioned outside a cell membrane of immune cells (e.g., NK cells) when the chimeric antigen receptor is expressed. For example, the signal sequence may include the whole or a portion of CD16.

[0076] According to one embodiment of the present invention, the extracellular domain is a domain to be specifically linked to an antibody or to specifically recognize an antigen, and may include, for example, an Fc receptor, an antigen-binding fragment such as a single-chain variable fragment (ScFv) in an antibody, a natural cytotoxicity receptor, NKG2D, 2B4 or DNAM-1. Accordingly, in the present disclosure, the term "extracellular domain" is used with the same meanings as "antigenic recognition site," "antigen-binding fragment" and/or "antibody binding site."

[0077] The chimeric antigen receptor according to one embodiment of the present invention may include an Fc receptor as the extracellular domain and thus be used associated with different antibodies depending on cell types of cancer to be treated. According to one embodiment, the Fc receptor may be any one selected from the group consisting of CD16, CD32. CD64, CD23 and CD89. According to a more specific embodiment, the Fc receptor may be one including the whole or a portion of the CD16 V158 variant (CD16V).

[0078] According to another embodiment, the chimeric antigen receptor of the present invention may further include an antigen-binding fragment of an antibody, as an extracellular domain, which can directly recognize the antigen without co-administration along with the antibody. According to one embodiment, the antigen-binding fragment may be an Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment. According to one embodiment of the present invention, the antibody may be any one of various types of antibodies capable of binding antigen-specifically, briefly, having antigen-specific binding ability. For example, the antibody may be one in which one light chain and one heavy chain are bonded with each other, or one in which two light chains and two heavy chains are bonded with each other. For example, when two light chains and two heavy chains are bonded with each other, the antibody may be one in which a first unit including a first light chain and a first heavy chain bonded with each other and a second unit including a second light chain and a second heavy chain bonded with each other are combined with each other. Herein, the bond may be a disulfide bond, but it is not limited thereto. According to embodiments of the present invention, the above two units may be the same as or different from each other. For example, the first unit including the first light chain and the first heavy chain and the second unit including the second light chain and the second heavy chain may be the same as or different from each other. As such, an antibody prepared to recognize two different antigens by the first unit and the second unit, respectively, is typically referred to as a `bispecific antibody.` In addition, for example, the antibody may be one in which three or more of the above-described units are combined with one another. The antigen-binding fragment of the present invention may be derived from various types of antibodies as described above, but it is not limited thereto.

[0079] According to another embodiment of the present invention, the extracellular domain used herein may be a natural killer receptor (also referred to as a `natural cytotoxicity receptor`). According to a specific embodiment, the NK receptor may include NKp46, NKp30, NKp44, NKp80 and NKp65 receptors, but it is not limited thereto.

[0080] According to one embodiment of the present invention, the transmembrane domain penetrates through the cell membrane, and may be any one without limitation as long as the domain can penetrate through the cell membrane without interfering with functions of the extracellular domain and the intracellular signaling domain. For example, the transmembrane domain may include the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

[0081] According to one embodiment of the present invention, the extracellular domain and the transmembrane domain may be linked to each other by a spacer domain. For example, the spacer domain may be a hinge domain. According to a specific embodiment, the spacer domain may include the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

[0082] According to one embodiment of the present invention, the intracellular signaling domain is a portion located on an inner side of the cell membrane of NK cell, that is, on the cytoplasm thereof, and may include a sequence that can transmit a signal to activate NK cells when an antibody linked to the extracellular domain is combined with a target antigen.

[0083] According to one embodiment of the present invention, the chimeric antigen receptor may include one or more intracellular signaling domain(s). When including two or more intracellular signaling domains, the intracellular signaling domains may be linked in series with each other. For example, when including three intracellular signaling domains, a terminal of a first intracellular signaling domain may be linked to a terminal of the transmembrane domain that is not linked with the spacer domain, a terminal of a second intracellular signaling domain may be linked to a terminal of the first intracellular signaling domain that is not linked with the transmembrane domain, and a terminal of a third intracellular signaling domain may be linked to a terminal of the second intracellular signaling domain that is not linked with the first intracellular signaling domain. That is, the first, second and third intracellular signaling domains may be arranged in order from a cell membrane toward an inside of the cell. Further, even when including two, four or more intracellular signaling domains, these domains may be linked to one another in the same manner as described above. According to one embodiment of the present invention, such the respective domains may be directly linked to one another or may be linked by a linker.

[0084] According to one embodiment of the present invention, the chimeric antigen receptor may include two intracellular signaling domains. For example, the chimeric antigen receptor may include a first intracellular signaling domain linked to the transmembrane domain and a second intracellular signaling domain linked to a terminal of the first intracellular signaling domain that is not linked with the transmembrane domain. According to a more specific embodiment, the first intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 (CD134), OX40 ligand (OX40L, CD252), 4-1BB (CD137), CD28, DAP10, CD3-zeta (CD3) and DAP12, and the second intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12. In this case, at least one of the first intracellular signaling domain and the second intracellular signaling domain includes the whole or a portion of OX40 ligand. For example, the chimeric antigen receptor may include a first intracellular signaling domain containing the whole or a portion of OX40 ligand and a second intracellular signaling domain containing the whole or a portion of any one selected from CD3-zeta and DAP12. Further, for example, the chimeric antigen receptor may include a first intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of CD3-zeta and DAP12 and a second intracellular signaling domain containing the whole or a portion of OX40 ligand.

[0085] According to another embodiment of the present invention, the chimeric antigen receptor may include three intracellular signaling domains. For example, the chimeric antigen receptor may include: a tirst intracellular signaling domain linked to the transmembrane domain; a second intracellular signaling domain linked to a terminal of the first intracellular signaling domain that is not linked with the transmembrane domain; and a third intracellular signaling domain linked to a terminal of the second intracellular signaling domain that is not linked with the first intracellular signaling domain. According to a more specific embodiment, the first intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of 4-1BB, OX40, OX40 ligand, CD28 and DAP10, the second intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB, and the third intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12. In such a case, at least one of the first intracellular signaling domain, the second intracellular signaling domain and the third intracellular signaling domain may include the whole or a portion of OX40 ligand.

[0086] In another aspect, the present invention may provide a chimeric antigen receptor, which includes: a first intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of CD28 and 4-1BB; a second intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB; and a third intracellular signaling domain containing the whole or a portion of CD3-zeta, wherein the first, second and third intracellular signaling domains are arranged in order from the cell membrane toward the inside of the cell. According to one embodiment of the present invention, the above respective domains may be directly linked to one another or may be linked by a linker.

[0087] According to one embodiment of the present invention, the chimeric antigen receptor may further include: a transmembrane domain linked to the first intracellular signaling domain; a spacer domain linked to the transmembrane domain; and an extracellular domain linked to the spacer domain. In addition, the chimeric antigen receptor may further include a signal sequence linked to the extracellular domain. According to one embodiment of the present invention, the above respective domains may be directly linked to one another or may be linked by a linker.

[0088] According to one embodiment of the present invention, the extracellular domain is a domain for specifically binding with an antibody or specifically recognizing an antigen, for example, an Fc receptor, an antigen-binding fragment of an antibody such as a single-chain variable fragment (ScFv), NK receptor (natural cytotoxicity receptor), NKG2D, 2B4 or DNAM-1, etc. Thus, in the present disclosure, the term "extracellular domain" is used with the same meanings as the "antigenic recognition site", "antigen-binding fragment" and/or "antibody binding site."

[0089] The chimeric antigen receptor according to an embodiment of the present invention may include an Fc receptor as the extracellular domain, and therefore, can be used along with a variety of antibodies depending on cell types of cancer to be treated. According to one embodiment, the Fc receptor may include any one selected from the group consisting of CD16, CD32, CD64, CD23 and CD89, and variants thereof. According to a more specific embodiment, the Fc receptor may include CD16 or variants thereof, and most specifically, may include the whole or a portion of CD16 V158 variant (CD16V).

[0090] According to another embodiment, the chimeric antigen receptor of the present invention may include, as the extracellular domain, an antigen-binding fragment of an antibody which directly recognizes the antigen without co-administration along with the antibody. According to one embodiment, the antigen-binding fragment may be an Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment. According to one embodiment of the present invention, the antibody may be any one of various types of antibodies capable of binding antigen-specifically. For example, the antibody may be one in which one light chain and one heavy chain are bonded with each other, or one in which two light chains and two heavy chains are bonded with each other. For example, when two light chains and two heavy chains are bonded with each other, the antibody may be one in which the first unit including the first light chain and the first heavy chain bonded with each other and the second unit including the second light chain and the second heavy chain bonded with each other are combined with each other. The bond may be a disulfide bond, but it is not limited thereto. According to an embodiment of the present invention, the above two units may be the same as or different from each other. For example, the first unit including the first light chain and the first heavy chain and the second unit including the second light chain and the second heavy chain may be the same as or different from each other. As such, an antibody prepared to recognize two different antigens by the first unit and the second unit, respectively, is commonly referred to as a `bispecific antibody` in the related art. In addition, for example, the antibody may be one in which the above three or more units are combined with one another. The antigen-binding fragment of the present invention may be derived from various types of antibodies as described above, but it is not limited thereto.

[0091] According to another embodiment of the present invention, the extracellular domain used herein may be a NK receptor (natural cytotoxicity receptor). According to a specific embodiment, the NK receptor may include NKp46, NKp30, NKp44, NKp80 and NKp65 receptors, but it is not limited thereto.

[0092] According to one embodiment, the signal sequence may include the whole or a portion of CD16. According to another embodiment, the extracellular domain may include the whole or a portion of CD16 V158 variant (CD16V). According to another embodiment, the spacer domain may include the whole or a portion of any one selected from the group consisting of CD8.alpha. (CD8-alpha) and CD28. According to another embodiment, the transmembrane domain may include the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

[0093] According to a specific embodiment, the chimeric antigen receptor may include one or more amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 41, 43, 45, 47, 49, 51, 53, 55, 69, 71, 77, 81, 83, 85, 87, 89, 91 and 93 or variants thereof having a sequence identity of 80% or more.

[0094] According to another aspect, the present invention provides immune cells (e.g., NK cells) to express the above-described chimeric antigen receptor according to the present invention.

[0095] The immune cells of the present invention may exhibit toxicity to tumor cells. It is determined that the chimeric antigen receptor according to the invention exhibits specific toxicity to what types of tumor cells depending on what types of antibodies are combined with the extracellular domains. Therefore, the types of tumor cells, to which the immune cells expressing the chimeric antigen receptor according to the present invention may exhibit specific toxicity, are not particularly limited. According to one embodiment, when the immune cells (e.g., NK cells) of the present invention are used along with rituximab, the cells may exhibit toxicity to malignant lymphoma cells. For example, the malignant lymphoma cells may express CD20. Further, for example, the malignant lymphoma may be B-cell lymphoma.

[0096] According to another aspect, the invention further provides a pharmaceutical composition for prevention or treatment of tumor or tumor metastasis, which includes the immune cells (e.g., NK cells) expressing the above-described chimeric antigen receptor according to the present invention, in the number of 2 to 7.5 times the number of tumor cells (e.g., malignant lymphoma cells) in a subject to be treated (`treatment target`).

[0097] According to one embodiment of the present invention, the number of immune cells (e.g., NK cells) included in the pharmaceutical composition of the present invention in a single dose may range from 0.75 to 10 times the number of tumor cells (e.g., malignant lymphoma cells) in the treatment target. For example, the number of the immune cells (e.g., NK cells) in a single dose may range from 2 to 7.5 times the number of tumor cells (e.g., malignant lymphoma cells) in the treatment target.

[0098] According to another aspect, the present invention further provides a nucleic acid sequence encoding the above-described chimeric antigen receptor according to the present invention.

[0099] According to one embodiment of the present invention, the nucleic acid sequence may include one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 32, 40, 42, 44, 46, 48, 50, 52, 54, 68, 70, 76, 80, 82, 84, 86, 88, 90 and 92 or variants thereof having a sequence identity of 80% or more.

[0100] According to another aspect, the present invention further provides a vector including the above-described nucleic acid sequence according to the present invention.

[0101] According to another aspect, the present invention further provides a method of treating tumor which includes administering the above-described immune cells to a subject.

[0102] According to another aspect, the present invention further provides a method of preventing tumor metastases which includes administering the above-described immune cells to a subject.

[0103] The subject may be a mammal having a tumor, specifically, a human, but it is not limited thereto.

[0104] Administration may be performed in such a way that the number of immune cells (e.g., NK cells) expressing the chimeric antigen receptor according to the present invention may be in a range of 2 to 7.5 times the number of tumor cells (e.g., malignant lymphoma cells) in the treatment target.

[0105] An administration method is not particularly limited and, for example, may include administration through an oral or parenteral route.

[0106] Tumor is not particularly limited but may include, for example, malignant lymphoma, leukemia, breast cancer, lung cancer, etc., and more specifically, B-cell lymphoma.

[0107] Hereinafter, the present invention will be described in more detail by way of the following examples. These examples are proposed only for describing the present invention in detail and the scope of the present invention is not limited to the contents illustrated in the examples.

Example 1: Methods and Reagents

[0108] Cell Lines

[0109] The human B-series cell line Ramos, human erythroleukemic cell line K562, human breast cancer cell line MCF-7, H1299 and H1944 of human lung cancer cell lines, as well as NK-92MI are supplied from American Type Culture Collection (ATCC, Manassas, Va., USA). K562 was maintained in RPMI-1640 containing 10% FBS (Gibco, Grand Island, N.Y., USA). Ramos was maintained in RPMI-1640 (ATCC) (Manassas, Va.) containing 10% FBS (fetal bovine serum; Gibco, Grand Island, N.Y., USA). MCF-7 was maintained in EMEM (ATCC)+10% FBS (Gibco) medium, while H1299 and H1944 cell lines were maintained in RPMI-1640 (ATCC)+10% FBS (Gibco). In addition, NK-92MI and transduced NK-92MI cells were maintained in CellGro.RTM. serum-free medium containing 1% human plasma. The human embryonic kidney fibroblast, that is, 293T cell line was supplied from the ATCC. This cell line was maintained in DMEM (Gibco, Grand Island, N.Y., USA) containing 10% FBS (Gibco, Grand Island, N.Y. USA).

[0110] Plasmid

[0111] The signal sequence and the extracellular domain of the FCRG3A V158 mutant (CD16V); the extracellular domain of NKG2D; the signal sequence of CD8a, the hinge and transmembrane domains of CD8a; the hinge and transmembrane domains of CD28; and the intracellular signaling domains of 4-1BB, OX40, OX40 ligand (OX40L) and CD3.zeta., respectively, were synthesized artificially. The above listed materials were assembled in various combinations through splicing by overlapping extension by PCR (`SOE-PCR`). The PCR products were confirmed by direct sequencing. Each PCR product was cut into NheI and EcoRI, and then inserted into (i.e., ligated to) NheI and EcoRI sites of a 3.sup.rd generation self-inactivating lentiviral expression vector such as MSCV-EF1.alpha.-GFP vector or EF1.alpha.-MCS vector.

[0112] Chimeric antigen receptors (CAR) according to embodiments of the present invention have been summarized in Table 1 below. The domains of all CARs according to the embodiments of the present invention have been linked in series (in tandem) to one another and also linked in frame.

TABLE-US-00001 TABLE 1 Type of CAR Serial Signal (generation) No. Abbreviation sequence ECD Hinge TM Signal-1 Signal-2 Signal-3 -- C1 NC CD16 CD16V CD8.alpha. CD8.alpha. 1st C2 CD16V-Z CD16 CD16V CD8.alpha. CD8.alpha. CD3.zeta. 2nd C3 CD16V-BBZ CD16 CD16V CD8.alpha. CD8.alpha. 4-1BB CD3.zeta. 2nd C4 CD16V-OX40Z CD16 CD16V CD8.alpha. CD8.alpha. OX40 CD3.zeta. 2nd C5 CD16V-OX40LZ CD16 CD16V CD8.alpha. CD8.alpha. OX40L CD3.zeta. 2nd C5-1 CD16V-OX40L CD16 CD16V CD8.alpha. CD8.alpha. CD3.zeta. OX40L 2nd C6 CD16V-28Z CD16 CD16V CD8.alpha. CD28 CD28 CD3.zeta. 2nd C7 CD16V-28(H)Z CD16 CD16V CD28 CD28 CD28 CD3.zeta. 3rd C8 CD16V-BBOX40Z CD16 CD16V CD8.alpha. CD8.alpha. 4-1BB OX40 CD3.zeta. 3rd C9 CD16V-OX40BBZ CD16 CD16V CD8.alpha. CD8.alpha. OX40 4-1BB CD3.zeta. 3rd C10 CD16V-28BBZ CD16 CD16V CD8.alpha. CD28 CD28 4-1BB CD3.zeta. 3rd C11 CD16V-28OX40Z CD16 CD16V CD8.alpha. CD28 CD28 OX40 CD3.zeta. 3rd C12 CD16V-28OX40LZ CD16 CD16V CD8.alpha. CD28 CD28 OX40L CD3.zeta. 3rd C13 CD16V-28(H)BBZ CD16 CD16V CD28 CD28 CD28 4-1BB CD3.zeta. 3rd C14 CD16V-28(H)OX40Z CD16 CD16V CD28 CD28 CD28 OX40 CD3.zeta. 3rd C15 CD16V-28(H)OX40LZ CD16 CD16V CD28 CD28 CD28 OX40L CD3.zeta. 3rd C16 CD16V-BBOX40LZ CD16 CD16V CD8.alpha. CD8.alpha. 41BB OX40L CD3.zeta.

[0113] CD16V-Z CAR (1.sup.st generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A Vi 58) (nucleotides 85-651. G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0114] CD16V-BBZ CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651. G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0115] CD16V-OX40Z CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0116] CD16V-OX40LZ CAR (2.sup.nd generation) is provided by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD252-derived intracellular signaling domain (nucleotides 141-206, GenBank NM 003326.4); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0117] CD16V-ZOX40L CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); CD252-derived intracellular signaling domain (nucleotides 141-206, GenBank NM 003326.4); and stop codon TGA. CD16V-28Z CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge domain (nucleotides 1292-1435. GenBank NM 001768.6); CD28-derived transmembrane and intracellular signaling domains (nucleotides 679-882, GenBank NM 006139.3); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0118] CD16V-28(H)Z CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645): CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882, GenBank NM 006139.3); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0119] CD16V-BBOX40Z CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0120] CD16V-OX40BBZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0121] CD16V-28BBZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); CD8.alpha.-derived hinge domain (nucleotides 1292-1435, GenBank NM 001768.6); CD28-derived transmembrane and intracellular signaling domains (nucleotides 679-882, GenBank NM 006139.3); CD137-derived intracellular signaling domain (nucleotides 901-1026. GenBank NM 001561.5); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0122] CD16V-28OX40Z CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); CD8.alpha.-derived hinge domain (nucleotides 1292-1435, GenBank NM 001768.6); CD28-derived transmembrane and intracellular signaling domains (nucleotides 679-882, GenBank NM 006139.3); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0123] CD16V-28OX40LZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84. GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); CD8.alpha.-derived hinge domain (nucleotides 1292-1435, GenBank NM 001768.6): CD28-derived transmembrane and intracellular signaling domains (nucleotides 679-882, GenBank NM 006139.3); CD252-derived intracellular signaling domain (nucleotides 141-206, GenBank NM 003326.4); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0124] CD16V-28(H)BBZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882. GenBank NM 006139.3); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0125] CD16V-28(H)OX40Z CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645): CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882, GenBank NM 006139.3); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0126] CD16V-28(H)OX40LZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84. GenBank Accession No. X52645); the extracellular domain of CD16V (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882, GenBank NM 006139.3); CD252-derived intracellular signaling domain (nucleotides 141-206, GenBank NM 003326.4); and CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3) and stop codon TGA.

[0127] CD16V-BBOX40LZ (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD16 (nucleotides 34-84, GenBank Accession No. X52645); the extracellular domain of CD16V (FCRG3A V158) (nucleotides 85-651, G mutation of nucleotide No. 559 in GenBank Accession No. X52645); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD137-derived intracellular signaling domain (nucleotides 901-1026. GenBank NM 001561.5); CD252-derived intracellular signaling domain (nucleotides 141-206, GenBank NM 003326.4); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634. GenBank NM 000734.3); and stop codon TGA.

[0128] The sequence listings of the domains used for the chimeric antigen receptors (CARs) and a preparation method thereof according to the embodiments of the present invention are summarized in Table 2 below.

TABLE-US-00002 TABLE 2 Sequence No. Name of sequence Details of sequence 1 CD16 nucleotide Signal sequence and extracellular domain of CD16 (34-651 nucleotides, GenBank Accession No. X52645) 2 CD16 amino acid Amino acid sequence corresponding to SEQ ID NO. 1 3 CD16V nucleotide G mutation of nucleotide No. 559 among signal sequence and extracellular domain of CD16V (34-651 nucleotides, GenBank Accession No. X52645) 4 CD16V nucleotide codon Codon optimized sequence of SEQ ID NO. 3 optimization 5 CD16V amino acid Amino acid sequence corresponding to SEQ ID NOs. 3 and 4 6 CD8.alpha. nucleotide Human CD8.alpha.-derived hinge and transmembrane domains (1292-1507 nucleotides, GenBank NM 001768.6) 7 CD8.alpha. nucleotide codon Codon optimized sequence of SEQ ID NO. 6 optimization 8 CD8.alpha. amino acid Amino acid sequence corresponding to SEQ ID NOs. 6 and 7 9 CD28 nucleotide CD28-derived hinge, transmembrane and intracellular signaling domains (562-882 nucleotides, GenBank MM 006139.3) 10 CD28 nucleotide codon Codon optimized sequence of SEQ ID NO. 9 optimization 11 CD28 amino acid Amino acid sequence corresponding to SEQ ID NOs. 9 and 10 12 CD3.zeta. nucleotide CD3.zeta.-derived intracellular signaling domain (299-634 nucleotides, GenBank NM 000734.3) 13 CD3.zeta. nucleotide codon Codon optimized sequence of SEQ ID NO. 12 optimization 14 CD3.zeta. amino acid Amino acid sequence corresponding to SEQ ID NOs. 12 and 13 15 OX40L (CD252) nucleotide OX40L (CD252)-derived intracellular signaling domain (141-206 nucleotides, GenBank NM 003326.4) 16 OX40L (CD252) nucleotide Codon optimized sequence of SEQ ID NO. 15 codon optimization 17 OX40L (CD252) amino acid Amino acid sequence corresponding to SEQ ID NOs. 15 and 16 18 OX40 (CD134) nucleotide OX40 (CD134)-derived intracellular signaling domain (733-840 nucleotides, GenBank AB 590584.1) 19 OX40 (CD134) nucleotide Codon optimized sequence of SEQ ID NO. 18 codon optimization 20 OX40 (CD134) amino acid Amino acid sequence corresponding to SEQ ID NOs. 18 and 19 21 4-1BB (CD137) nucleotide 4-1BB (CD137)-derived intracellular signaling domain (901-1026 nucleotides, GenBank NM 001561.5) 22 4-1BB (CD137) nucleotide Codon optimized sequence of SEQ ID NO. 21 codon optimization 23 4-1BB (CD137) amino acid Amino acid sequence corresponding to SEQ ID NOs. 21 and 22 24 CD16V-Z nucleotide Codon optimized nucleotide sequence of CD16V-Z CAR 25 CD16V-Z amino acid Amino acid sequence corresponding to SEQ ID NO. 24 26 CD16V-BBZ nucleotide Codon optimized nucleotide sequence of CD16V-BBZ CAR 27 CD16V-BBZ amino acid Amino acid sequence corresponding to SEQ ID NO. 26 28 CD16V-OX40Z nucleotide Codon optimized nucleotide sequence Of CD16V-OX40Z CAR 29 CD16V-OX40Z amino acid Amino acid sequence corresponding to SEQ ID NO. 28 30 CD16V-OX40LZ nucleotide Codon optimized nucleotide sequence of CD16V-OX30LZ CAR 31 CD16V-OX40LZ amino acid Amino acid sequence corresponding to SEQ ID NO. 30 32 CD16V-28Z nucleotide Codon optimized nucleotide sequence of CD16V-28Z CAR 33 CD16V-28Z amino acid Amino acid sequence corresponding to SEQ ID NO. 32 34 CD16V-28(H)Z nucleotide Codon optimized nucleotide sequence of CD16V-28(H)Z CAR 35 CD16V-28(H)Z amino acid Amino acid sequence corresponding to SEQ ID NO. 34 36 CD16V-BBOX40Z nucleotide Codon optimized nucleotide sequence of CD16V-BBOX40Z CAR 37 CDI6V-BBOX40Z amino acid Amino acid sequence cotresponding to SEQ ID NO. 36 38 CD16V-OX40BBZ nucleotide Codon optimized nucleotide sequence of CD16V-OX40BBZ CAR 39 CD16V-OX40BBZ amino acid Amino acid sequence corresponding to SEQ ID NO. 38 40 CD16V-28BBZ nucleotide Codon optimized nucleotide sequence of CD16V-28BBZ CAR 41 CD16V-28BBZ amino acid Amino acid sequence corresponding to SEQ ID NO. 40 42 CD16V-28OX40Z nucleotide Codon optimized nucleotide sequence of CD16V-28OX40Z CAR 43 CD16V-28OX40Z amino acid Amino acid sequence corresponding to SEQ ID NO. 42 44 CD16V-28OX40LZ nucleotide Codon optimized nucleotide sequence of CD16V-28OX40LZ CAR 45 CD16V-28OX40LZ amino acid Amino acid sequence corresponding to SEQ ID NO. 44 46 CD16V-28(H)BBZ nucleotide Codon optimized nucleotide sequence of CD16V-28(H)BBZ CAR 47 CD16V-28(H)BBZ amino acid Amino acid sequence corresponding to SEQ ID NO. 46 48 CD16V-28(H)OX40Z Codon optimized nucleotide sequence of nucleotide CD16V-28(H)OX40Z CAR 49 CD16V-28(H)OX40Z amino Amino acid sequence corresponding to SEQ ID NO. 48 acid 50 CD16V-28(H)OX40LZ Codon optimized nucleotide sequence of nucleotide CD16V-28(H)OX40LZ CAR 51 CD16V-28(H)OX40LZ amino Amino acid sequence corresponding to SEQ ID NO. 50 acid 52 CD16V-BBOX40LZ nucleotide Codon optimized nucleotide sequence of CD16V-BBOX40LZ CAR 53 CD16V-BBOX40LZ amino Amino acid sequence corresponding to SEQ ID NO. 52 acid 54 CD16V-ZOX40L nucleotide Codon optimized nucleotide sequence of CD16V-ZOX40L CAR 55 CD16V-ZOX40L amino acid Amino acid sequence corresponding to SEQ ID NO. 54

[0129] In addition, the chimeric antigen receptors (CARs) according to the embodiments of the present invention are summarized in Table 3 below. Domains of each of all CARs according to the embodiments of the present invention are linked in series (in tandem) to one other and also linked in the frame.

TABLE-US-00003 TABLE 3 Type of CAR Serial Signal (generation) No. Abbreviation sequence ECD Hinge TM Signal-1 Signal-2 Signal-3 -- C17 NC CD8.alpha. NKG2D CD8.alpha. CD8.alpha. 1st C18 NKG2D-Z CD8.alpha. NKG2D CD8.alpha. CD8.alpha. CD3.zeta. 2nd C19 NKG2D-BBZ CD8.alpha. NKG2D CD8.alpha. CD8.alpha. 4-1BB CD3.zeta. 2nd C20 NKG2D-OX40Z CD8.alpha. NKG2D CD8.alpha. CD8.alpha. OX40 CD3.zeta. 2nd C21 NKG2D-ZOX40L CD8.alpha. NKG2D CD8.alpha. CD8.alpha. CD3.zeta. OX40L 2nd C22 NKG2D-28Z CD8.alpha. NKG2D CD8.alpha. CD28 CD28 CD3.zeta. 2nd C23 NKG2D-28(H)Z CD8.alpha. NKG2D CD28 CD28 CD28 CD3.zeta. 3rd C24 NKG2D-BBOX40Z CD8.alpha. NKG2D CD8.alpha. CD8.alpha. 4-1BB OX40 CD3.zeta. 3rd C25 NKG2D-BBOX40LZ CD8.alpha. NKG2D CD8.alpha. CD8.alpha. 4-1BB OX40L CD3.zeta. 3rd C26 NKG2D-OX40BBZ CD8.alpha. NKG2D CD8.alpha. CD8.alpha. OX40 4-1BB CD3.zeta. 3rd C27 NKG2D-28BBZ CD8.alpha. NKG2D CD8.alpha. CD28 CD28 4-1BB CD3.zeta. 3rd C28 NKG2D-28OX40Z CD8.alpha. NKG2D CD8.alpha. CD28 CD28 OX40 CD3.zeta. 3rd C29 NKG2D-28OX40LZ CD8.alpha. NKG2D CD8.alpha. CD28 CD28 OX40L CD3.zeta. 3rd C30 NKG2D-28(H)BBZ CD8.alpha. NKG2D CD28 CD28 CD28 4-1BB CD3.zeta. 3rd C31 NKG2D-28(H)OX40Z CD8.alpha. NKG2D CD28 CD28 CD28 OX40 CD3.zeta. 3rd C32 NKG2D-28(H)OX40LZ CD8.alpha. NKG2D CD28 CD28 CD28 OX40L CD3.zeta. 3rd C33 NKG2D-AAA-28 CD8.alpha. NKG2D-AAA CD28 CD28 CD28 OX40L CD3.zeta. (H)OX40LZ

[0130] NKG2D-Z CAR (1.sup.st generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507. GenBank NM 001768.6); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0131] NKG2D-BBZ CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD137-derived intracellular signaling domain (nucleotides 901-1026. GenBank NM 001561.5); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0132] NKG2D-OX40Z CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507. GenBank NM 001768.6); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0133] NKG2D-ZOX40L CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); CD252-derived intracellular signaling domain (nucleotides 141-206, GenBank NM 003326.4); and CD3 stop codon TGA.

[0134] NKG2D-28Z CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); human CD8.alpha.-derived hinge domain (nucleotides 1292-1435, GenBank NM 001768.6); CD28-derived transmembrane and intracellular signaling domains (nucleotides 679-882. GenBank NM 006139.3); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0135] NKG2D-28(H)Z CAR (2.sup.nd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882, GenBank NM 006139.3); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0136] NKG2D-BBOX40Z CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0137] NKG2D-BBOX40LZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507, GenBank NM 001768.6); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD252-derived intracellular signaling domain (nucleotides 141-206. GenBank NM 003326.4); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0138] NKG2D-OX40BBZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); human CD8.alpha.-derived hinge and transmembrane domains (nucleotides 1292-1507. GenBank NM 001768.6); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD36-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0139] NKG2D-28BBZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); CD8.alpha.-derived hinge domain (nucleotides 1292-1435, GenBank NM 001768.6); CD28-derived transmembrane and intracellular signaling domains (nucleotides 679-882, GenBank NM 006139.3); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0140] NKG2D-28OX40Z CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); CD8.alpha.-derived hinge domain (nucleotides 1292-1435, GenBank NM 001768.6); CD28-derived transmembrane and intracellular signaling domains (nucleotides 679-882, GenBank NM 006139.3); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0141] NKG2D-28OX40LZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); CD8.alpha.-derived hinge domain (nucleotides 1292-1435, GenBank NM 001768.6); CD28-derived transmembrane and intracellular signaling domains (nucleotides 679-882, GenBank NM 006139.3); CD252-derived intracellular signaling domain (nucleotides 141-206. GenBank NM 003326.4); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0142] NKG2D-28(H)BBZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882, GenBank NM 006139.3); CD137-derived intracellular signaling domain (nucleotides 901-1026, GenBank NM 001561.5); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0143] NKG2D-28(H)OX40Z CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882, GenBank NM 006139.3); CD134-derived intracellular signaling domain (nucleotides 733-840, GenBank AB590584.1); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0144] NKG2D-28(H)OX40LZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1): CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882, GenBank NM 006139.3); CD252-derived intracellular signaling domain (nucleotides 141-206, GenBank NM 003326.4); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0145] NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd generation) is produced by connecting: the signal sequence domain of CD8.alpha. (nucleotides 890-952, GenBank NM 001768.6); the extracellular domain of NKG2D (nucleotides 788-1192, GenBank ID: AF461811.1); AAA (Triple alanine); CD28-derived hinge, transmembrane and intracellular signaling domains (nucleotides 562-882, GenBank NM 006139.3); CD252-derived intracellular signaling domain (nucleotides 141-206, GenBank NM 003326.4); CD3.zeta.-derived intracellular signaling domain (nucleotides 299-634, GenBank NM 000734.3); and stop codon TGA.

[0146] The sequence listings of the domains used for the chimeric antigen receptors (CARs) and a method for preparation thereof according to the embodiments of the present invention are summarized in Table 4 below.

TABLE-US-00004 TABLE 4 Sequence No. Name of sequence Details of sequence 56 CD8.alpha. nucleotide Signal sequence domain of CD8.alpha. (890-952 nucleotides, GenBank NM 001768.6) 57 CD8.alpha. nucleotide codon Codon optimized sequence of SEQ ID NO. 56 optimization 58 CD8.alpha. amino acid Amino acid sequence corresponding to SEQ ID NOs. 56 and 57 59 NKG2D nucleotide Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF 461811.1) 60 NKG2D nucleotide codon Codon optimized sequence of SEQ ID NO. 59 optimization 61 NKG2D amino acid Amino acid sequence corresponding to SEQ ID NOs. 59 and 60 62 NKG2D-Z nucleotide Codon optimized nucleotide sequence of NKG2D-Z CAR 63 NKG2D-Z amino acid Amino acid sequence corresponding to SEQ ID NO. 62 64 NKG2D-BBZ nucleotide Codon optimized nucleotide sequence of NKG2D-BBZ CAR 65 NKG2D-BBZ amino acid Amino acid sequence corresponding to SEQ ID NO. 64 66 NKG2D-OX40Z nucleotide Codon optimized nucleotide sequence of NKG2D-OX40Z CAR 67 NKG2D-OX40Z amino acid Amino acid sequence corresponding to SEQ ID NO. 66 68 NKG2D-ZOX40L nucleotide Codon optimized nucleotide sequence of NKG2D-ZOX40L CAR 69 NKG2D-ZOX40L amino acid Amino acid sequence corresponding to SEQ ID NO. 68 70 NKG2D-28Z nucleotide Codon optimized nucleotide sequence of NKG2D-28Z CAR 71 NKG2D-28Z amino acid Amino acid sequence corresponding to SEQ ID NO. 70 72 NKG2D-28(H)Z nucleotide Codon optimized nucleotide sequence of NKG2D-28(H)Z CAR 73 NKG2D-28(H)Z amino acid Amino acid sequence corresponding to SEQ ID NO. 72 74 NKG2D-BBOX40Z nucleotide Codon optimized nucleotide sequence of NKG2D-BBOX40Z CAR 75 NKG2D-BBOX40Z amino acid Amino acid sequence corresponding to SEQ ID NO. 74 76 NKG2D-BBOX40LZ nucleotide Codon optimized nucleotide sequence of NKG2D- BBOX40LZ CAR 77 NKG2D-BBOX40LZ amino Amino acid sequence corresponding to SEQ ID NO. 76 acid 78 NKG2D-OX40BBZ nucleotide Codon optimized nucleotide sequence of NKG2D-OX40BBZ CAR 79 NKG2D-OX40BBZ amino acid Amino acid sequence corresponding to SEQ ID NO. 78 80 NKG2D-28BBZ nucleotide Codon optimized nucleotide sequence of NKG2D-28BBZ CAR 81 NKG2D-28BBZ amino acid Amino acid sequence corresponding to SEQ ID NO. 80 82 NKG2D-28OX40Z nucleotide Codon optimized nucleotide sequence of NKG2D-28OX40Z CAR 83 NKG2D-28OX40Z amino acid Amino acid sequence corresponding to SEQ ID NO. 82 84 NKG2D-28OX40LZ nucleotide Codon optimized nucleotide sequence of NKG2D-28OX40LZ CAR 85 NKG2D-28OX40LZ amino Amino acid sequence corresponding to SEQ ID NO. 84 acid 86 NKG2D-28(H)BBZ nucleotide Codon optimized nucleotide sequence of NKG2D-28(H)BBZ CAR 87 NKG2D-28(H)BBZ amino acid Amino acid sequence corresponding to SEQ ID NO. 86 88 NKG2D-28(H)OX40Z Codon optimized nucleotide sequence of NKG2D- nucleotide 28(H)OX40Z CAR 89 NKG2D-28(H)OX40Z amino Amino acid sequence corresponding to SEQ ID NO. 88 acid 90 NKG2D-28(H)OX40LZ Codon optimized nucleotide sequence of NKG2D- nucleotide 28(H)OX40LZ CAR 91 NKG2D-28(H)OX40LZ amino Amino acid sequence corresponding to SEQ ID NO. 90 acid 92 NKG2D-AAA-28(H)OX40LZ Codon optimized nucleotide sequence of NKG2D-AAA- Nucleotide 28(H)OX40LZ CAR 93 NKG2D-AAA-28(H)OX40LZ Amino acid sequence corresponding to SEQ ID NO. 92 amino acid

[0147] Virus Production and Gene Transfer

[0148] In order to prepare VSVG-pseudotyped lentivirus, 293T cells cultured in a DMEM medium were co-transfected with various types of vectors such as PCDH1-MSCV-CD16-construct-EF1-copGFP vector, EF1.alpha.-NKG2D-construct vector, PCDH1-MSCV-EF1-copGFP control vector, or EF1a-GFP control vector (for production of Mock infection virus using empty vector) together with HIV-based pPACKH1 lentivirus Package Kit (System Biosciences). For this purpose, Lipofectamine 2000 reagent (Invitrogen. Carlsbad, Calif.) was used. Various types of CD16V constructs are as follows; CD16V-Z CAR, CD16V-BBZ CAR, CD16V-OX40Z CAR, CD16V-OX40LZ CAR, CD16V-ZOX40L, CD16V-28Z CAR, CD16V-28(H)Z CAR, CD16V-BBOX40Z CAR, CD16V-BBOX40LZ CAR, CD16V-OX40BBZ CAR, CD16V-28BBZ CAR, CD16V-28OX40Z CAR, CD16V-28OX40LZ CAR. CD16V-28(H)BBZ CAR, CD16V-28(H)OX40Z CAR, CD16V-28(H)OX40LZ CAR. Further, various types of NKG2D constructs are as follows; NKG2D-Z CAR, NKG2D-BBZ CAR, NKG2D-OX40Z CAR, NKG2D-ZOX40L CAR, NKG2D-28Z CAR, NKG2D-28(H)Z CAR. NKG2D-BBOX40Z CAR, NKG2D-BBOX40LZ CAR, NKG2D-OX40BBZ CAR, NKG2D-28BBZ CAR, NKG2D-28OX40Z CAR, NKG2D-28OX40LZ CAR, NKG2D-28(H)BBZ CAR, NKG2D-28(H)OX40Z CAR, NKG2D-28(H)OX40LZ CAR. NKG2D-AAA-28(H)OX40LZ. Each lentivirus was prepared by transfection of 80% dense HEK293T cells in a flask with: various types of CD16V construct expression vectors, various types of NKG2D construct expression vectors or a control plasmid together with pPACKH1 lentivirus packaging plasmids. After 6 hours, the medium was replaced by a DMEM medium containing 10% FBS. The conditioned medium containing lentivirus was collected after 48 hours of transfection, followed by filtering with a 0.45 .mu.m filter unit (Milliopore, Billerica, Mass. USA) in order to remove cell debris. A viral supernatant containing the virus was concentrated about 50 times by centrifugation at 3000 rpm and 4.degree. C. for 20 minutes using Amicon Filter (Millipore). The concentrated virus was stored at -80.degree. C.

[0149] For the lentiviral infection, NK92MI cells in an exponential growth phase was adjusted to a concentration of 1.times.10.sup.6 cells/ml using Cellgro (Cellgenix) including 1% human plasma, and then a lentiviral supernatant in 50 to 100 MOI was added in the presence of 8 .mu.g/ml polybrene, followed by centrifugation at 1800 g for 90 minutes. After centrifugation, the cells were left in a humidified incubator at 37.quadrature. and 5% CO.sub.2 conditions for 48 hours. Thereafter, the cells were washed twice with RPMI-1640, and then left in RPMI-1640 including 10% FBS for future use. Control cells were transduced with a vector only.

[0150] Detection of Expression of Receptor Including CD16V or NKG2D

[0151] CD16V CAR-transduced NK92MI cells, NKG2D CAR-transduced NK92MI cells, the control vector-transduced NK92MI (NK92MI-Mock) or NK-92MI parent cells were washed twice with FACS buffer, and the washed cells were stained using 7-AAD (Beckman coulter), anti-CD3, anti-CD56 and anti-CD16 (BD Biosciences) mAbs. An expression ratio and a mean fluorescence intensity (MFI) of the stained cells were measured using a BD LSRFortessa.

[0152] The transduction efficiency using the NKG2D construct was determined by flow cytometric analysis of cells expressing NKG2D among CD3-CD56+ cells. First, NK92MI cells were gated in regard to singlet, and then gated in regard to 7AAD- and CD3-CD56+. The transformation efficiency using the CD16 constructs was determined by flow cytometric analysis of cells expressing GFP and CD16 among CD3-CD56+ cells.

[0153] Calcein Releasing Cytotoxicity Assay

[0154] Target cells were labeled at 37.degree. C. for 1 hour with 30 .mu.M calcein-acetoxymethyl ester (Calcein-AM: Molecular probes). After washing, the labeled target cells were dispensed to 1.times.10.sup.4 cells per well in 96-well plates. NK92MI cells were harvested, washed, and then were added at different E/T (effector-to-target) ratios under conditions with or without rituximab at various concentrations. As a control group independent of the rituximab, an anti-human antibody (Sigma aldrich) was used. After 2 hours, the plates were centrifuged at 2000 rpm for 3 minutes, and a supernatant of 100 .mu.L was collected and subjected to measurement of calcein release using a fluorescence microplate reader (Victor3, PerkinElmer) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Specific calcein release amount was calculated by the following equation: percent specific lysis=(test release-spontaneous release).times.100/(maximal release-spontaneous release). For maximal lysis, a 1% Triton X-100 was used.

Example 2: Assessment of Cytotoxicity of NK92MI Cells Expressing OX40 Ligand (CD252)-Containing Chimeric Antigen Receptor (CAR) Against CD20-Positive Lymphoma Cells

[0155] Transduction and Expression of Chimeric Antigen Receptor Containing CD16V Associated with the Co-Stimulating Motif

[0156] V158 variant (polymorphism) of FCRG3A (CD16) is a high affinity immunoglobulin Fc receptor and is considered to exhibit good effects in antibody treatment. The present inventors have prepared the V158 variant of FCRG3A (CD16) and combined the prepared variant with: the hinge and transmembrane domains of CD8.alpha.; a T cell stimulatory molecule, that is, CD3.zeta.; and intracellular domains of different costimulatory molecules such as CD28, 4-1BB, OX-40 and OX-40 ligand in various combinations thereof (Table 1). The prepared CD16V-containing chimeric antigen receptors (e.g., CD16V-Z CAR (1.sup.st generation), CD16V-28Z CAR (2.sup.nd generation), CD16V-BBZ CAR (2.sup.nd generation), CD16V-OX40Z CAR (2.sup.nd generation) or CD16V-28OX40LZ CAR (3.sup.rd generation)) were expressed in NK92MI cells by a lentiviral vector including a MSCV promoter. We confirmed surface expression of each CAR on NK92MI cells by detection of the human CD16, using a monoclonal mouse anti-human antibody. By repetitive experiments involving flow cytometric analysis, it was demonstrated that CARs were transduced with efficiency more than 90% in NK92MI cells (FIG. 1A). An amount of lentivirus used herein is equal to the multiple of infection (MOI) of 50 or more.

[0157] Increase of Tumor Killing Effect of NK92MI Cells Expressing CD16V Receptor Combined with CD252 (OX40 Ligand) Against CD20-Positive Lymphoma

[0158] In order to determine whether genetic modification according to the invention induces an increase in cancer cell death, cytotoxicity of NK92MI cells transduced with an empty vector and NK92MI cells expressing a CD16-containing receptor on CD20-positive lymphoma cells (Ramos cells) was evaluated by calcein-AM release assay.

[0159] Before test for lymphoma cells, K562, a human erythroleukemic cell line, was used as a standard control to assess intrinsic cell killing ability (i.e., cytotoxicity) of the transduced NK92MI cells. By transduction using lentivirus having multiple of infection (MOI) of 50 or more, each of CARs according to the present invention (CD16V-Z CAR (1.sup.st generation), CD16V-28Z CAR (2.sup.nd generation), CD16V-BBZ CAR (2.sup.nd generation), CD16V-OX40Z CAR (2.sup.nd generation) or CD16V-28OX40LZ CAR (3.sup.rd generation)) was expressed at a high level in 90% or more NK92MI cells (FIG. 1A).

[0160] It was demonstrated that cytotoxicity of NK92MI cells expressing CARs according to the invention on K562 was similar to cytotoxicity of a control group (Mock) transduced with an empty vector. This means that the intrinsic cytotoxicity of NK92MI cells on a target was not increased by side effects due to the genetic modification itself (FIG. 1B). In FIG. 1B, a ratio of the number of NK92MI cells as effector cells to the number of K562 cells as target cells is indicated by 5:1, 2.5:1, 1:1 and 0.5:1.

[0161] In order to assess how diverse co-stimulatory molecules affect in vitro on antigen-specific antitumor activity of NK92MI cells expressing CD16V-containing receptor, transduced NK92MI cells and cancer cells (B-cell lymphoma cell line, that is, Ramos) were subjected to co-incubation, followed by assessment of lysis of the cancer cells according to calcein-AM release assay (FIG. 1C). In FIG. 1C, ratios of the number of NK92MI cells (effector cells) to the number of Ramos cells (target cells) are indicated by 5:1, 2.5:1, 1:1 and 0.5:1. It has been shown that rituximab does not increase NK-92MI mediated cytotoxicity significantly because NK92MI does not express CD16.

[0162] As shown in FIG. 1C, we found that expression of various CARs according to the present invention without rituximab did not increase significantly NK-92MI mediated cytotoxicity over that cytotoxicity induced by the control (Mock), NK-92MI cells transduced with the vector containing only GFP.

[0163] CD16V-Z CAR (first generation), which was previously shown to enhance cytotoxicity in various CAR applications, served as positive control. In order to increase cytotoxicity of the CD16V-containing receptor, the present inventors have introduced CD28, CD134 (OX40), CD137 (4-1BB) or CD252 (OX40 ligand) into CD16V-Z-CAR. In 2 hour in vitro cytotoxicity assays, NK-92MI cells expressing CD16V-Z CAR (1.sup.st generation), CD16V-28Z CAR (2.sup.nd generation), CD16V-BBZ CAR (2.sup.nd generation), CD16V-OX40Z CAR (2.sup.nd generation) or CD16V-28OX40LZ CAR (3.sup.rd generation) exhibited high cytotoxicity in the presence of rituximab at effector cells to target cells ratio of 5:1 and 2.5:1. Among them, in particular, 3.sup.rd generation CAR including OX40 ligand, that is, CD16V-28OX40LZ CAR exhibited the highest cytotoxicity, compared to the other CARs (FIG. 1C).

[0164] In order to investigate whether 2.sup.nd generation CAR containing OX40 ligand can enhance anticancer activity of NK92MI cells. CD16V-OX40LZ CAR was prepared and subjected to evaluation of cytotoxicity. When NK92MI cells were transduced with CD16-OX40LZ using a lentiviral vector, CD16V-OX40LZ CAR was not expressed (FIG. 1D). Even in the presence of rituximab, cytotoxicity on Ramos cells mediated by CD16V-OX40LZ CAR was not exhibited (FIG. 1E). Accordingly, the present inventors noticed a fact that OX40 ligand is type II protein and CD3 linked to the OX40 ligand is type I protein, and has prepared CD16V-ZOX40L CAR having CD3c at N-terminal and OX40 ligand at C-terminal by changing the order of the above two domains. When NK92MI cells were transduced with CD16V-ZOX40L using a lentiviral vector having an MSCV promoter, NK92MI cells effectively expressed CD16V-OX40LZ CAR compared with CD16V-OX40LZ (FIG. 1F). Further, as a result of assessing anticancer activity of 2.sup.nd generation CAR having CD16V-ZOX40L introduced therein, high cytotoxicity on Ramos cells was demonstrated at ratios of effector cells to target cells of 10:1, 5:1 and 2.5:1 in the presence of rituximab (FIG. 1G). It was determined that, in addition to CD16V-28OX40LZ CAR (3'3 generation) containing OX40 ligand, CD16V-ZOX40L CAR (2.sup.nd generation) also has anticancer activity superior to CD16V-Z CAR used as a positive control.

[0165] Assessment of Efficacy of CD16V-BBOX40LZ CAR

[0166] By introducing OX40 ligand into CD137 (41BB) signaling domain-based CAR, NK92MI cells were subjected to assessment of expression and cytotoxicity. Both of CD16V-BBZ CAR (2.sup.nd generation) and CD16V-BBOX40LZ CAR (3.sup.rd generation) were expressed at high levels in NK92MI cells (FIG. 2A). When rituximab is not present in an in vitro test for cytotoxicity, all of CD16V-Z, CD16V-BBZ and CD16V-BBOX40LZ exhibited low cytotoxicity to Ramos cells. On the other hand, in the presence of rituximab, CD16V-BBOX40LZ in combination with OX40 ligand exhibited highest cytotoxicity at even any ratio of effector cells to target cells (FIG. 2B).

[0167] Comparison of Efficacies Between CD16V-28OX40LZ CAR and Other CARs

[0168] Next, the present inventors have compared the cytotoxicity of NK92MI cells expressing CD16V-28OX40LZ CAR and the cytotoxicity of NK92MI cells expressing 3.sup.rd generation CAR that has other intracellular signaling domains. The 3.sup.rd generation CARs used in the present experiment (CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR and CD16V-28BBZ CAR) were all proven to be expressed at high levels in NK92MI cells (FIG. 3A).

[0169] After comparing NK cell activating efficacies of various 3.sup.rd generation CARs (that is, 3.sup.rd generation chimeric antigen receptors) according to the present invention, results thereof are summarized in FIG. 3B. As shown in FIG. 3B, all of these CARs show low cytotoxicity to Ramos cells when rituximab is not present, and an extent of the cytotoxicity is all similar. On the other hand, in the presence of rituximab, all of CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR and CAR CD16V-28BBZ showed high cytotoxicity to Ramos cells. Among them, in particular, CD16V-28OX40LZ CAR containing OX40 ligand exhibited the most excellent cytotoxicity (FIG. 3B).

[0170] Assessment of Efficacy of 3.sup.rd Generation CAR Including OX40 Ligand Having CD28 at Hinge

[0171] In order for the CAR expressing NK cells to recognize a cancer cell antigen in an optimized condition, not only cancer cell antigen-recognizing receptor itself, but also hinge sequence and composition between an antigen-specific receptor and a cell membrane are also important. The hinge sequence and composition may need to be designed differently depending on target molecules.

[0172] The CARs used in the experiments described above use the fragment of CD8.alpha. as spacer domains (`hinge`). As compared to this, variation of cytotoxicity of CARs when the fragment of CD28 is used at the hinge, was assessed and results thereof are summarized in FIGS. 4A and 4B. The present inventors have produced a lentiviral vector including an extracellular domain (ectodomain) of CD16V and the hinge of CD28. In 3.sup.rd generation CARs containing CD28 fragment as spacer, CD28-derived hinge, transmembrane and intracellular signaling domains were linked to a signaling module of CD134 (OX40), CD137 (4-1BB) or OX40 ligand (CD252). Such produced 3.sup.rd generation CARs were expressed on NK92MI cells using the lentiviral vector. It was demonstrated that the transduced NK92MI cells could express the above 3.sup.rd generation CARs, that is, CD16V-28(H)BBZ CAR. CD16V-28(H)OX40Z CAR or CD16V-28(H)OX40LZ CAR) at high levels, respectively (FIG. 4A).

[0173] As shown in FIG. 4B, when rituximab is not present, all of CD16V-28(H)BBZ CAR, CD16V-28(H)OX40Z CAR and CD16V-28(H)OX40LZ CAR do not exhibit cytotoxicity. On the other hand, in the presence of rituximab, all of CD16V-28(H)BBZ CAR, CD16V-28(H)OX40Z CAR and CD16V-28(H)OX40LZ CAR exhibit high cytotoxicity. Among them, in particular, CD16V-28(H)OX40LZ CAR including OX40 ligand has the highest cytotoxicity.

[0174] The experimental results described above demonstrate that, when a novel CAR particularly including OX40 ligand as an intracellular signaling domain discovered by the present inventors is expressed in natural killer cells, excellent antitumor effects may be attained.

Example 3: Assessment of Cytotoxicity of NK92MI Cells Expressing OX40 Ligand (CD252)-Containing NKG2D Chimeric Antigen Receptor (NKG2D-CAR) on Human Breast Cancer Cells and Lung Cancer Cells

[0175] Transduction and Expression of a Chimeric Antigen Receptor Including NKG2D Associated with Co-Stimulatory Motif

[0176] The present inventors synthesized a human NKG2D gene and combined the same with diverse combinations of: hinge and transmembrane domains of CD8.alpha.; and intracellular domains of T-cell stimulatory molecules CD3.zeta. and cofactors including CD28, 4-1BB, OX-40 and OX-40 ligand to significantly enhance activity of T or NK cells (Table 3). Such NKG2D CAR constructs were expressed in NK92MI cells using a lentiviral vector. The present inventors have identified surface expression of each NKG2D CAR in NK92MI cells by means of detection of human NKG2D using a monoclonal mouse anti-human antibody. By repetitive experiments involving flow cytometric analysis, it was demonstrated that CARs were transduced with 70% or more efficiency in NK92MI cells.

[0177] Increase of Tumor Killing Effect of NK92MI Cells Expressing NKG2D Receptor, which Contains Various Co-Stimulatory Signaling Domains, Against MCF7 Breast Cancer Cell Line

[0178] In order to investigate variation in killing activity due to genetic modification, cytotoxicity of NK-92MI cells, which express NKG2D CAR, on MCF7 breast cancer cells was compared by calcein-AM release assay.

[0179] In order to assess in vitro effects of various co-stimulatory signaling domains on antigen-specific antitumor functions of NK-92MI cells expressing NKG2D CAR, transduced NK-92MI cells and cancer cells were co-cultured, followed by measuring lysis of tumor cells through calcein-AM release. According to previous studies, it was known that adding the co-stimulatory signaling domains to the chimeric receptor exhibits increased cytotoxicity of T and NK lymphocytes. In order to increase the cytotoxicity of NKG2D CAR, the present inventors have introduced signaling domains of the most well-known three cofactors, that is, CD28, CD134 (OX-40) and CD137 (4-1BB) into the NKG2D CAR. The NK92MI cells containing the introduced NKG2D-Z CAR (1.sup.st generation), NKG2D-28Z CAR, NKG2D-BBZ CAR, NKG2D-OX40Z CAR (2.sup.nd generation) showed efficient expression of NKG2D (FIG. 5A). In vitro cytotoxicity analysis for two hours, it was demonstrated that NK-92MI cells expressing NKG2D-Z CAR (1.sup.st generation), NKG2D-28Z CAR, NKG2D-BBZ CAR and NKG2D-OX40Z CAR (2.sup.nd generation) exhibited cytotoxicity to MCF7 breast cancer cells. However, addition of CD134 (OX-40) or CD137 (4-1BB) signaling domain to a backbone of NKG2D-Z 1.sup.st generation CAR did not exhibit higher cytotoxicity than the cytotoxicity of NKG2D-Z CAR (1.sup.st generation). Cancer cell killing by NK-92MI cells including NKG2D-28Z CAR (2.sup.nd generation) was superior to the positive control, NK-92MI cells expressing NKG2D-Z CAR (1.sup.st generation) (FIG. 5B).

[0180] Increase of Tumor Killing Effect of NK92MI Cells Expressing NKG2D Receptor, which Contains CD28 Intracellular Domain, Against MCF7 Breast Cancer Cell Line

[0181] 3.sup.rd generation CAR is known to increase antitumor activity. In physiological T cell response, the optimal lymphocyte activation needs one or more cofactor receptors combined with a co-stimulatory molecule such as CD28. Among the cofactor receptors, the most important receptors are CD137 (4-1BB) and OX40 (CD134) which are members of tumor necrosis factor (TNFR).

[0182] In order to investigate effects of different cofactors in NK-92MI cells expressing NKG2D CAR (3.sup.rd generation), the present inventors have produced NKG2D-28OX40Z CAR (3.sup.rd generation) and NKG2D-28BBZ CAR (3.sup.rd generation). The NKG2D CAR (3.sup.rd generation) constructs were expressed in NK92MI cells using a lentiviral vector. The transduced NK92MI cells efficiently expressed various NKG2D receptors containing 3.sup.rd generation CAR (FIG. 6A). As compared to NK92MI cells transduced with a control vector, NK92MI cells expressing various NKG2D CARs (3.sup.rd generation) have efficiently killed MCF7 cells in vitro. However, 3.sup.rd generation NKG2D-28OX40Z CAR and NKG2D-28BBZ CAR, each of which includes CD134 or CD137 co-stimulatory signaling domain in a backbone of NKG2D-28Z CAR (2.sup.nd generation), did not exhibit higher cytotoxicity than NKG2D-Z CAR (1.sup.st generation) (FIG. 6B).

[0183] Comparison of NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd Generation) and Other Receptors

[0184] In order to assess CD252 (OX40 ligand) co-stimulatory signaling domain effects, the signaling domain of the OX40 ligand was fused to the backbone of NKG2D-28Z CAR (2.sup.nd generation), so as to build NKG2D-28(H)OX40LZ CAR (3.sup.rd generation). In the tumor killing assay, tumor specific cytotoxicity to MCF7 cells, which was induced by NKG2D-28(H)OX40LZ CAR (3.sup.rd generation), is greatly enhanced compared to NKG2D-Z CAR (1.sup.st generation), however, is substantially similar to NKG2D-28Z CAR (2.sup.nd generation) (FIG. 7B).

[0185] For optimal CAR T-cell recognition, it was determined that, not only tumor antigen recognition receptor itself, but also hinge and linker sequences between the antigen-specific receptor and the cell membrane are also important. In the present experiment, additional effects of the spacer to the function of NKG2D-28(H)OX40LZ CAR (3.sup.rd generation) were analyzed. The present inventors have introduced an AAA (triple alanine) linker between the NKG2D extracellular domain and CD28 hinge. NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd generation) containing AAA linker introduced therein was expressed in NK92MI cells using a lentiviral vector (FIG. 7A). In the present experiment, MCF7 cell killing by NK-92MI cells expressing NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd generation) was compared with MCF7 cell killing by NK-92MI cells expressing NKG2D-Z CAR (1.sup.st generation), NKG2D-28Z CAR (2.sup.nd generation) or NKG2D-28(H)OX40LZ CAR (3.sup.rd generation). In particular, introduction of the AAA linker between NKG2D extracellular domain and CD28 hinge of NKG2D-28(H)OX40LZ CAR (3.sup.rd generation) has resulted in better target cell killing than parental NKG2D-28(H)OX40LZ CAR (3.sup.rd generation) without AAA, NKG2D-Z CAR (1.sup.st generation) and/or NKG2D-28Z CAR (2.sup.nd generation) (FIG. 7B).

[0186] Increase of Tumor Killing Effect of NK92MI Cells Expressing NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd Generation) Against the Lung Cancer Cell Line

[0187] In order to assess whether NK92MI cells transduced with NKG2D-AAA-28(H)OX40LZ CAR (3.sup.rd generation) can recognize NKG2D ligand in the lung cancer cells, NKG2D ligand-positive tumor cells (H1299 and H1944) were used as a target of NK92MI cells including NKG2D-AAA-28(H)OX40L CAR (3.sup.rd generation). The transduced NK92MI cells efficiently expressed NKG2D-Z CAR (1.sup.st generation) or NKG2D-AAA-28(H)OX40L CAR (3.sup.rd generation) (FIG. 8A). Next, the NKG2D ligand expression in H1299 and H1944 cells was assessed. The present inventors have identified that the NKG2D ligand expression was detected in H1299 and H1944 cells by a flow cytometric analysis (FIG. 8B). As shown in FIG. 8C, NK92MI cells including NKG2D-AAA-28(H)OX40L CAR (3.sup.rd generation) could more efficiently killed the target cells expressing NKG2D ligand in vitro than the NK92MI cells as a control group and NK92MI cells expressing NKG2D-Z CAR (1.sup.st generation). Furthermore, specificity of target cell killing was obviously confirmed because H1299 and H1944 cells are not killed by NKG2D non-expressing NK92MI cells transduced by a control empty vector.

Sequence CWU 1

1

931618DNAArtificial SequenceCD16 nucleotide 1atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600tcattctttc cacctggg 6182206PRTArtificial SequenceCD16 amino acid 2Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly 195 200 2053618DNAArtificial SequenceCD16V nucleotide 3atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480aaagccacac tcaaagacag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat 540gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600tcattctttc cacctggg 6184618DNAArtificial SequenceCD16V nucleotide codon optimization 4atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggg 6185206PRTArtificial SequenceCD16V amino acid 5Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly 195 200 2056216DNAArtificial SequenceCD8a nucleotide 6gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 60cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 120agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 180gtccttctcc tgtcactggt tatcaccctt tactgc 2167216DNAArtificial SequenceCD8a nucleotide codon optimization 7gccaaaccta ccacaactcc tgctccaaga ccacccacac ccgctcctac tattgcatct 60cagccactga gtctgcgacc agaggcctgc cggcccgccg ccggcggggc cgtgcatacc 120aggggcctgg acttcgcctg tgatatctac atttgggctc cactggctgg gacttgcggc 180gtgctgctgc tgtctctggt catcactctg tattgt 216872PRTArtificial SequenceCD8a amino acid 8Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro1 5 10 15Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 20 25 30Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 50 55 60Ser Leu Val Ile Thr Leu Tyr Cys65 709321DNAArtificial SequenceCD28 nucleotide 9attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 180tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300gacttcgcag cctatcgctc c 32110321DNAArtificial SequenceCD28 nucleotide codon optimization 10atcgaggtca tgtacccccc tccatatctg gacaacgaaa agtccaatgg aactatcatt 60cacgtgaagg gcaaacatct gtgcccatct cccctgttcc ctgggccaag taaacctttt 120tgggtcctgg tggtcgtggg aggggtgctg gcatgttact cactgctggt caccgtggcc 180ttcatcatct tctgggtgcg gagcaagagg tcccgcctgc tgcacagcga ctatatgaac 240atgaccccac ggagacccgg ccctacacgg aaacattacc agccctatgc tccaccccgg 300gacttcgcag cttacagaag t 32111107PRTArtificial SequenceCD28 amino acid 11Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn1 5 10 15Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 35 40 45Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 50 55 60Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn65 70 75 80Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 85 90 95Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 100 10512336DNAArtificial SequenceCD3z nucleotide 12agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300tacgacgccc ttcacatgca ggccctgccc cctcgc 33613336DNAArtificial SequenceCD3z nucleotide codon optimization 13cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 60tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 120cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 180gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 240aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 300tatgatgcac tgcacatgca ggccctgccc cctcgg 33614112PRTArtificial SequenceCD3z amino acid 14Arg 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 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 1101566DNAArtificial SequenceOX40L (CD252) nucleotide 15gaaagggtcc aacccctgga agagaatgtg ggaaatgcag ccaggccaag attcgagagg 60aacaag 661666DNAArtificial SequenceOX40L (CD252) nucleotide codon optimization 16gaaagagtgc agcccctgga agagaatgtc gggaatgccg ctcgcccaag atttgaaagg 60aacaaa 661722PRTArtificial SequenceOX40L (CD252) amino acid 17Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg Pro1 5 10 15Arg Phe Glu Arg Asn Lys 2018108DNAArtificial SequenceOX40 (CD134) nucleotide 18agggaccaga ggctgccccc cgatgcccac aagccccctg ggggaggcag tttccggacc 60cccatccaag aggagcaggc cgacgcccac tccaccctgg ccaagatc 10819108DNAArtificial SequenceOX40 (CD134) nucleotide codon optimization 19cgggaccaga gactgccacc tgatgcacac aagccaccag gaggaggcag cttcaggacc 60cccatccagg aggaacaggc cgacgctcat tccacactgg ccaaaatt 1082036PRTArtificial SequenceOX40 (CD134) amino acid 20Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly1 5 10 15Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 20 25 30Leu Ala Lys Ile 3521126DNAArtificial Sequence4-1BB (CD137) nucleotide 21aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg 12622126DNAArtificial Sequence4-1BB (CD137) nucleotide codon optimization 22aagcggggaa gaaagaaact gctgtacatc ttcaaacagc cctttatgag gcctgtgcag 60accacacagg aggaagacgg ctgctcctgc cggttccccg aggaagagga aggcgggtgc 120gagctg 1262342PRTArtificial Sequence4-1BB (CD137) amino acid 23Lys 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 40241173DNAArtificial SequenceCD16V-Z nucleotide 24atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtcgagtg 840aagttcagca ggtccgccga cgctcctgca taccagcagg gacagaacca gctgtataac 900gagctgaatc tgggccggag agaggaatac gacgtgctgg acaaaaggcg gggccgggac 960cccgaaatgg gagggaagcc acgacggaaa aacccccagg agggcctgta caatgagctg 1020caaaaggaca aaatggccga ggcttattct gaaatcggga tgaagggaga gagaaggcgc 1080ggaaaaggcc acgatggcct gtaccagggg ctgagcaccg ctacaaagga cacctatgat 1140gcactgcaca tgcaggccct gccccctcgg tga 117325390PRTArtificial SequenceCD16V-Z amino acid 25Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 245 250 255Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 260 265 270Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 275 280 285Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 290 295 300Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp305 310 315 320Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 325 330 335Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 340 345 350Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 355 360 365Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 370 375 380Gln Ala Leu Pro Pro Arg385 390261299DNAArtificial SequenceCD16V-BBZ nucleotide 26atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta

ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggcctgt gcagaccaca 900caggaggaag acggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 1020tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1080cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1140gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1200aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1260tatgatgcac tgcacatgca ggccctgccc cctcggtga 129927432PRTArtificial SequenceCD16V-BBZ amino acid 27Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 245 250 255Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 260 265 270Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 275 280 285Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 290 295 300Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu305 310 315 320Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 325 330 335Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 340 345 350Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 355 360 365Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 370 375 380Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg385 390 395 400Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 405 410 415Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 420 425 430281281DNAArtificial SequenceCD16V-OX40Z nucleotide 28atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780ctggctggga cttgcggcgt gctgctgctg tccctggtca tcactctgta ttgtcgggac 840cagagactgc cacctgatgc acacaagcca ccaggaggag gcagcttcag gacccccatc 900caggaggaac aggccgacgc tcattccaca ctggccaaaa ttcgagtgaa gttcagcagg 960tccgccgacg ctcctgcata ccagcaggga cagaaccagc tgtataacga gctgaatctg 1020ggccggagag aggaatacga cgtgctggac aaaaggcggg gccgggaccc cgaaatggga 1080gggaagccac gacggaaaaa cccccaggag ggcctgtaca atgagctgca aaaggacaaa 1140atggccgagg cttattctga aatcgggatg aagggagaga gaaggcgcgg aaaaggccac 1200gatggcctgt accaggggct gagcaccgct acaaaggaca cctatgatgc actgcacatg 1260caggccctgc cccctcggtg a 128129426PRTArtificial SequenceCD16V-OX40Z amino acid 29Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 245 250 255Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 260 265 270Val Ile Thr Leu Tyr Cys Arg Asp Gln Arg Leu Pro Pro Asp Ala His 275 280 285Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln 290 295 300Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg305 310 315 320Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 325 330 335Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 340 345 350Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 355 360 365Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 370 375 380Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His385 390 395 400Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 405 410 415Ala Leu His Met Gln Ala Leu Pro Pro Arg 420 425301239DNAArtificial SequenceCD16V-OX40LZ nucleotide 30atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtgaaaga 840gtgcagcccc tggaagagaa tgtcgggaat gccgctcgcc caagatttga aaggaacaaa 900cgagtgaagt tcagcaggtc cgccgacgct cctgcatacc agcagggaca gaaccagctg 960tataacgagc tgaatctggg ccggagagag gaatacgacg tgctggacaa aaggcggggc 1020cgggaccccg aaatgggagg gaagccacga cggaaaaacc cccaggaggg cctgtacaat 1080gagctgcaaa aggacaaaat ggccgaggct tattctgaaa tcgggatgaa gggagagaga 1140aggcgcggaa aaggccacga tggcctgtac caggggctga gcaccgctac aaaggacacc 1200tatgatgcac tgcacatgca ggccctgccc cctcggtga 123931412PRTArtificial SequenceCD16V-OX40LZ amino acid 31Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 245 250 255Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 260 265 270Val Ile Thr Leu Tyr Cys Glu Arg Val Gln Pro Leu Glu Glu Asn Val 275 280 285Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys Arg Val Lys Phe 290 295 300Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu305 310 315 320Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 325 330 335Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 340 345 350Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 355 360 365Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 370 375 380Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr385 390 395 400Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 405 410321305DNAArtificial SequenceCD16V-28Z nucleotide 32atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atttttgggt cctggtggtc 780gtgggagggg tgctggcatg ttactcactg ctggtcaccg tggccttcat catcttctgg 840gtgcggagca agaggtcccg cctgctgcac agcgactata tgaacatgac cccacggaga 900cccggcccta cacggaaaca ttaccagccc tatgctccac cccgggactt cgcagcttac 960agaagtcgag tgaagttcag caggtccgcc gacgctcctg cataccagca gggacagaac 1020cagctgtata acgagctgaa tctgggccgg agagaggaat acgacgtgct ggacaaaagg 1080cggggccggg accccgaaat gggagggaag ccacgacgga aaaaccccca ggagggcctg 1140tacaatgagc tgcaaaagga caaaatggcc gaggcttatt ctgaaatcgg gatgaaggga 1200gagagaaggc gcggaaaagg ccacgatggc ctgtaccagg ggctgagcac cgctacaaag 1260gacacctatg atgcactgca catgcaggcc ctgccccctc ggtga 130533434PRTArtificial SequenceCD16V-28Z amino acid 33Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp 245 250 255Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 260 265 270Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu 275 280 285Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr 290 295 300Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr305 310 315 320Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 325 330 335Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 340 345 350Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 355 360 365Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 370 375 380Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly385 390 395 400Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

Ser 405 410 415Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 420 425 430Pro Arg341278DNAArtificial SequenceCD16V-28(H)Z nucleotide 34atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctgggat cgaggtcatg tacccccctc catatctgga caacgaaaag 660tccaatggaa ctatcattca cgtgaagggc aaacatctgt gcccatctcc cctgttccct 720gggccaagta aacctttttg ggtcctggtg gtcgtgggag gggtgctggc atgttactca 780ctgctggtca ccgtggcctt catcatcttc tgggtgcgga gcaagaggtc ccgcctgctg 840cacagcgact atatgaacat gaccccacgg agacccggcc ctacacggaa acattaccag 900ccctatgctc caccccggga cttcgcagct tacagaagtc gagtgaagtt cagcaggtcc 960gccgacgctc ctgcatacca gcagggacag aaccagctgt ataacgagct gaatctgggc 1020cggagagagg aatacgacgt gctggacaaa aggcggggcc gggaccccga aatgggaggg 1080aagccacgac ggaaaaaccc ccaggagggc ctgtacaatg agctgcaaaa ggacaaaatg 1140gccgaggctt attctgaaat cgggatgaag ggagagagaa ggcgcggaaa aggccacgat 1200ggcctgtacc aggggctgag caccgctaca aaggacacct atgatgcact gcacatgcag 1260gccctgcccc ctcggtga 127835425PRTArtificial SequenceCD16V-28(H)Z amino acid 35Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ile Glu 195 200 205Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr 210 215 220Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro225 230 235 240Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu 245 250 255Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 260 265 270Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 275 280 285Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 290 295 300Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser305 310 315 320Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 325 330 335Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 340 345 350Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 355 360 365Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 370 375 380Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp385 390 395 400Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 405 410 415Leu His Met Gln Ala Leu Pro Pro Arg 420 425361407DNAArtificial SequenceCD16V-BBOX40Z nucleotide 36atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggcctgt gcagaccaca 900caggaggaag acggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960cgggaccaga gactgccacc tgatgcacac aagccaccag gaggaggcag cttcaggacc 1020cccatccagg aggaacaggc cgacgctcat tccacactgg ccaaaattcg agtgaagttc 1080agcaggtccg ccgacgctcc tgcataccag cagggacaga accagctgta taacgagctg 1140aatctgggcc ggagagagga atacgacgtg ctggacaaaa ggcggggccg ggaccccgaa 1200atgggaggga agccacgacg gaaaaacccc caggagggcc tgtacaatga gctgcaaaag 1260gacaaaatgg ccgaggctta ttctgaaatc gggatgaagg gagagagaag gcgcggaaaa 1320ggccacgatg gcctgtacca ggggctgagc accgctacaa aggacaccta tgatgcactg 1380cacatgcagg ccctgccccc tcggtga 140737468PRTArtificial SequenceCD16V-BBOX40Z amino acid 37Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 245 250 255Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 260 265 270Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 275 280 285Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 290 295 300Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu305 310 315 320Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 325 330 335Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 340 345 350Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 355 360 365Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 370 375 380Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu385 390 395 400Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 405 410 415Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 420 425 430Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 435 440 445Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 450 455 460Leu Pro Pro Arg465381407DNAArtificial SequenceCD16V-OX40BBZ nucleotide 38atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780ctggctggga cttgcggcgt gctgctgctg tccctggtca tcactctgta ttgtcgggac 840cagagactgc cacctgatgc acacaagcca ccaggaggag gcagcttcag gacccccatc 900caggaggaac aggccgacgc tcattccaca ctggccaaaa ttaagcgggg aagaaagaaa 960ctgctgtaca tcttcaaaca gccctttatg aggcctgtgc agaccacaca ggaggaagac 1020ggctgctcct gccggttccc cgaggaagag gaaggcgggt gcgagctgcg agtgaagttc 1080agcaggtccg ccgacgctcc tgcataccag cagggacaga accagctgta taacgagctg 1140aatctgggcc ggagagagga atacgacgtg ctggacaaaa ggcggggccg ggaccccgaa 1200atgggaggga agccacgacg gaaaaacccc caggagggcc tgtacaatga gctgcaaaag 1260gacaaaatgg ccgaggctta ttctgaaatc gggatgaagg gagagagaag gcgcggaaaa 1320ggccacgatg gcctgtacca ggggctgagc accgctacaa aggacaccta tgatgcactg 1380cacatgcagg ccctgccccc tcggtga 140739468PRTArtificial SequenceCD16V-OX40BBZ amino acid 39Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 245 250 255Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 260 265 270Val Ile Thr Leu Tyr Cys Arg Asp Gln Arg Leu Pro Pro Asp Ala His 275 280 285Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln 290 295 300Ala Asp Ala His Ser Thr Leu Ala Lys Ile Lys Arg Gly Arg Lys Lys305 310 315 320Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 325 330 335Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 340 345 350Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 355 360 365Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 370 375 380Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu385 390 395 400Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 405 410 415Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 420 425 430Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 435 440 445Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 450 455 460Leu Pro Pro Arg465401431DNAArtificial SequenceCD16V-28BBZ nucleotide 40atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac tttgcctgcg atttttgggt cctggtggtc 780gtgggagggg tgctggcatg ttactcactg ctggtcaccg tggccttcat catcttctgg 840gtgcggagca agaggtcccg cctgctgcac agcgactata tgaacatgac cccacggaga 900cccggcccta cacggaaaca ttaccagccc tatgctccac cccgggactt cgcagcttac 960agaagtaagc ggggaagaaa gaaactgctg tacatcttca aacagccctt tatgaggcct 1020gtgcagacca cacaggagga agacggctgc tcctgccggt tccccgagga agaggaaggc 1080gggtgcgagc tgcgagtgaa gttcagcagg tccgccgacg ctcctgcata ccagcaggga 1140cagaaccagc tgtataacga gctgaatctg ggccggagag aggaatacga cgtgctggac 1200aaaaggcggg gccgggaccc cgaaatggga gggaagccac gacggaaaaa cccccaggag 1260ggcctgtaca atgagctgca aaaggacaaa atggccgagg cttattctga aatcgggatg 1320aagggagaga gaaggcgcgg aaaaggccac gatggcctgt accaggggct gagcaccgct 1380acaaaggaca cctatgatgc actgcacatg caggccctgc cccctcggtg a 143141476PRTArtificial SequenceCD16V-28BBZ amino acid 41Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90

95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp 245 250 255Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 260 265 270Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu 275 280 285Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr 290 295 300Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr305 310 315 320Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 325 330 335Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 340 345 350Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 355 360 365Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 370 375 380Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp385 390 395 400Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 405 410 415Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 420 425 430Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 435 440 445Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 450 455 460Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg465 470 475421413DNAArtificial SequenceCD16V-28OX40Z nucleotide 42atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac tttgcctgcg atttttgggt cctggtggtc 780gtgggagggg tgctggcatg ttactcactg ctggtcaccg tggccttcat catcttctgg 840gtgcggagca agaggtcccg cctgctgcac agcgactata tgaacatgac cccacggaga 900cccggcccta cacggaaaca ttaccagccc tatgctccac cccgggactt cgcagcttac 960agaagtcggg accagagact gccacctgat gcacacaagc caccaggagg aggcagcttc 1020aggaccccca tccaggagga acaggccgac gctcattcca cactggccaa aattcgagtg 1080aagttcagca ggtccgccga cgctcctgca taccagcagg gacagaacca gctgtataac 1140gagctgaatc tgggccggag agaggaatac gacgtgctgg acaaaaggcg gggccgggac 1200cccgaaatgg gagggaagcc acgacggaaa aacccccagg agggcctgta caatgagctg 1260caaaaggaca aaatggccga ggcttattct gaaatcggga tgaagggaga gagaaggcgc 1320ggaaaaggcc acgatggcct gtaccagggg ctgagcaccg ctacaaagga cacctatgat 1380gcactgcaca tgcaggccct gccccctcgg tga 141343470PRTArtificial SequenceCD16V-28OX40Z amino acid 43Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp 245 250 255Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 260 265 270Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu 275 280 285Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr 290 295 300Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr305 310 315 320Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly 325 330 335Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His 340 345 350Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp385 390 395 400Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 405 410 415Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 420 425 430Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 435 440 445Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 450 455 460Gln Ala Leu Pro Pro Arg465 470441371DNAArtificial SequenceCD16V-28OX40LZ nucleotide 44atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atttttgggt cctggtggtc 780gtgggagggg tgctggcatg ttactcactg ctggtcaccg tggccttcat catcttctgg 840gtgcggagca agaggtcccg cctgctgcac agcgactata tgaacatgac cccacggaga 900cccggcccta cacggaaaca ttaccagccc tatgctccac cccgggactt cgcagcttac 960agaagtgaaa gagtgcagcc cctggaagag aatgtcggga atgccgctcg cccaagattt 1020gaaaggaaca aacgagtgaa gttcagcagg tccgccgacg ctcctgcata ccagcaggga 1080cagaaccagc tgtataacga gctgaatctg ggccggagag aggaatacga cgtgctggac 1140aaaaggcggg gccgggaccc cgaaatggga gggaagccac gacggaaaaa cccccaggag 1200ggcctgtaca atgagctgca aaaggacaaa atggccgagg cttattctga aatcgggatg 1260aagggagaga gaaggcgcgg aaaaggccac gatggcctgt accaggggct gagcaccgct 1320acaaaggaca cctatgatgc actgcacatg caggccctgc cccctcggtg a 137145456PRTArtificial SequenceCD16V-28OX40LZ amino acid 45Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp 245 250 255Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 260 265 270Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu 275 280 285Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr 290 295 300Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr305 310 315 320Arg Ser Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala 325 330 335Arg Pro Arg Phe Glu Arg Asn Lys Arg Val Lys Phe Ser Arg Ser Ala 340 345 350Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 355 360 365Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 370 375 380Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu385 390 395 400Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 405 410 415Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 420 425 430Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 435 440 445His Met Gln Ala Leu Pro Pro Arg 450 455461404DNAArtificial SequenceCD16V-28(H)BBZ nucleotide 46atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctgggat cgaggtcatg tacccccctc catatctgga caacgaaaag 660tccaatggaa ctatcattca cgtgaagggc aaacatctgt gcccatctcc cctgttccct 720gggccaagta aacctttttg ggtcctggtg gtcgtgggag gggtgctggc atgttactca 780ctgctggtca ccgtggcctt catcatcttc tgggtgcgga gcaagaggtc ccgcctgctg 840cacagcgact atatgaacat gaccccacgg agacccggcc ctacacggaa acattaccag 900ccctatgctc caccccggga cttcgcagct tacagaagta agcggggaag aaagaaactg 960ctgtacatct tcaaacagcc ctttatgagg cctgtgcaga ccacacagga ggaagacggc 1020tgctcctgcc ggttccccga ggaagaggaa ggcgggtgcg agctgcgagt gaagttcagc 1080aggtccgccg acgctcctgc ataccagcag ggacagaacc agctgtataa cgagctgaat 1140ctgggccgga gagaggaata cgacgtgctg gacaaaaggc ggggccggga ccccgaaatg 1200ggagggaagc cacgacggaa aaacccccag gagggcctgt acaatgagct gcaaaaggac 1260aaaatggccg aggcttattc tgaaatcggg atgaagggag agagaaggcg cggaaaaggc 1320cacgatggcc tgtaccaggg gctgagcacc gctacaaagg acacctatga tgcactgcac 1380atgcaggccc tgccccctcg gtga 140447467PRTArtificial SequenceCD16V-28(H)BBZ amino acid 47Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ile Glu 195 200 205Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr 210 215 220Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro225 230 235 240Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu 245 250 255Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 260 265 270Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 275 280 285Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 290 295 300Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu305 310 315 320Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 325 330 335Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 340 345 350Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 355 360 365Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 370 375 380Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met385 390 395 400Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 405 410 415Leu Gln Lys

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 420 425 430Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 435 440 445Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 450 455 460Pro Pro Arg465481386DNAArtificial SequenceCD16V-28(H)OX40Z nucleotide 48atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctgggat cgaggtcatg tacccccctc catatctgga caacgaaaag 660tccaatggaa ctatcattca cgtgaagggc aaacatctgt gcccatctcc cctgttccct 720gggccaagta aacctttttg ggtcctggtg gtcgtgggag gggtgctggc atgttactca 780ctgctggtca ccgtggcctt catcatcttc tgggtgcgga gcaagaggtc ccgcctgctg 840cacagcgact atatgaacat gaccccacgg agacccggcc ctacacggaa acattaccag 900ccctatgctc caccccggga cttcgcagct tacagaagtc gggaccagag actgccacct 960gatgcacaca agccaccagg aggaggcagc ttcaggaccc ccatccagga ggaacaggcc 1020gacgctcatt ccacactggc caaaattcga gtgaagttca gcaggtccgc cgacgctcct 1080gcataccagc agggacagaa ccagctgtat aacgagctga atctgggccg gagagaggaa 1140tacgacgtgc tggacaaaag gcggggccgg gaccccgaaa tgggagggaa gccacgacgg 1200aaaaaccccc aggagggcct gtacaatgag ctgcaaaagg acaaaatggc cgaggcttat 1260tctgaaatcg ggatgaaggg agagagaagg cgcggaaaag gccacgatgg cctgtaccag 1320gggctgagca ccgctacaaa ggacacctat gatgcactgc acatgcaggc cctgccccct 1380cggtga 138649461PRTArtificial SequenceCD16V-28(H)OX40Z amino acid 49Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ile Glu 195 200 205Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr 210 215 220Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro225 230 235 240Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu 245 250 255Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 260 265 270Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 275 280 285Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 290 295 300Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro305 310 315 320Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln 325 330 335Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys 340 345 350Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 355 360 365Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 370 375 380Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg385 390 395 400Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 405 410 415Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 420 425 430Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 435 440 445Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 450 455 460501344DNAArtificial SequenceCD16V-28(H)OX40LZ nucleotide 50atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctgggat cgaggtcatg tacccccctc catatctgga caacgaaaag 660tccaatggaa ctatcattca cgtgaagggc aaacatctgt gcccatctcc cctgttccct 720gggccaagta aacctttttg ggtcctggtg gtcgtgggag gggtgctggc atgttactca 780ctgctggtca ccgtggcctt catcatcttc tgggtgcgga gcaagaggtc ccgcctgctg 840cacagcgact atatgaacat gaccccacgg agacccggcc ctacacggaa acattaccag 900ccctatgctc caccccggga cttcgcagct tacagaagtg aaagagtgca gcccctggaa 960gagaatgtcg ggaatgccgc tcgcccaaga tttgaaagga acaaacgagt gaagttcagc 1020aggtccgccg acgctcctgc ataccagcag ggacagaacc agctgtataa cgagctgaat 1080ctgggccgga gagaggaata cgacgtgctg gacaaaaggc ggggccggga ccccgaaatg 1140ggagggaagc cacgacggaa aaacccccag gagggcctgt acaatgagct gcaaaaggac 1200aaaatggccg aggcttattc tgaaatcggg atgaagggag agagaaggcg cggaaaaggc 1260cacgatggcc tgtaccaggg gctgagcacc gctacaaagg acacctatga tgcactgcac 1320atgcaggccc tgccccctcg gtga 134451447PRTArtificial SequenceCD16V-28(H)OX40LZ amino acid 51Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ile Glu 195 200 205Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr 210 215 220Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro225 230 235 240Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu 245 250 255Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 260 265 270Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 275 280 285Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 290 295 300Pro Arg Asp Phe Ala Ala Tyr Arg Ser Glu Arg Val Gln Pro Leu Glu305 310 315 320Glu Asn Val Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys Arg 325 330 335Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 340 345 350Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 355 360 365Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 370 375 380Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp385 390 395 400Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 405 410 415Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 420 425 430Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 435 440 445521365DNAArtificial SequenceCD16V-BBOX40LZ nucleotide 52atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtaagcgg 840ggaagaaaga aactgctgta catcttcaaa cagcccttta tgaggcctgt gcagaccaca 900caggaggaag acggctgctc ctgccggttc cccgaggaag aggaaggcgg gtgcgagctg 960gaaagagtgc agcccctgga agagaatgtc gggaatgccg ctcgcccaag atttgaaagg 1020aacaaacgag tgaagttcag caggtccgcc gacgctcctg cataccagca gggacagaac 1080cagctgtata acgagctgaa tctgggccgg agagaggaat acgacgtgct ggacaaaagg 1140cggggccggg accccgaaat gggagggaag ccacgacgga aaaaccccca ggagggcctg 1200tacaatgagc tgcaaaagga caaaatggcc gaggcttatt ctgaaatcgg gatgaaggga 1260gagagaaggc gcggaaaagg ccacgatggc ctgtaccagg ggctgagcac cgctacaaag 1320gacacctatg atgcactgca catgcaggcc ctgccccctc ggtga 136553454PRTArtificial SequenceCD16V-BBOX40LZ amino acid 53Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 245 250 255Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 260 265 270Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 275 280 285Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 290 295 300Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu305 310 315 320Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg Pro 325 330 335Arg Phe Glu Arg Asn Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 340 345 350Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 355 360 365Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 370 375 380Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu385 390 395 400Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 405 410 415Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 420 425 430Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 435 440 445Gln Ala Leu Pro Pro Arg 450541239DNAArtificial SequenceCD16V-ZOX40L nucleotide 54atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tctccgctgg gatgcgaacc 60gaggatctgc ctaaagccgt ggtcttcctg gagcctcagt ggtacagagt gctggagaag 120gactctgtga cactgaaatg ccagggcgct tattcaccag aggataacag cactcagtgg 180ttccacaatg aatccctgat cagctcccag gcatctagtt actttattga cgccgctaca 240gtggacgatt ctggagagta tcgatgccag actaacctga gcaccctgtc cgatcccgtg 300cagctggaag tccacatcgg atggctgctg ctccaggcac caagatgggt cttcaaggag 360gaagacccca ttcacctgcg ctgtcatagc tggaagaata ccgctctgca taaagtgaca 420tacctccaga acggaaaggg ccgaaaatac ttccaccata attccgactt ttatatcccc 480aaggcaaccc tgaaagatag tgggtcatat ttttgtcggg ggctggtggg aagtaaaaac 540gtctcaagcg agactgtgaa tatcaccatt acacagggcc tggctgtcag caccatctcc 600tctttctttc cccctggggc caaacctacc acaactcctg ctccaagacc acccacaccc 660gctcctacta ttgcatctca gccactgagt ctgcgaccag aggcctgccg gcccgccgcc 720ggcggggccg tgcataccag gggcctggac ttcgcctgtg atatctacat ttgggctcca 780ctggctggga cttgcggcgt gctgctgctg tctctggtca ttactctgta ttgtcgagtg 840aagttcagca ggtccgccga cgctcctgca taccagcagg gacagaacca gctgtataac 900gagctgaatc tgggccggag agaggaatac gacgtgctgg acaaaaggcg gggccgggac 960cccgaaatgg gagggaagcc acgacggaaa aacccccagg agggcctgta caatgagctg 1020caaaaggaca aaatggccga ggcttattct gaaatcggga tgaagggaga gagaaggcgc 1080ggaaaaggcc acgatggcct gtaccagggg ctgagcaccg ctacaaagga cacctatgat 1140gcactgcaca tgcaggccct gccccctcgg gaaagagtgc agcccctgga agagaatgtc 1200gggaatgccg ctcgcccaag atttgaaagg aacaaatga 123955412PRTArtificial SequenceCD16V-ZOX40L amino acid 55Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100

105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ala Lys 195 200 205Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 210 215 220Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala225 230 235 240Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 245 250 255Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 260 265 270Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 275 280 285Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 290 295 300Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp305 310 315 320Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 325 330 335Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 340 345 350Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 355 360 365Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 370 375 380Gln Ala Leu Pro Pro Arg Glu Arg Val Gln Pro Leu Glu Glu Asn Val385 390 395 400Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys 405 4105663DNAArtificial SequenceCD8 alpha nucleotide 56atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccg 635763DNAArtificial SequenceCD8 alpha nucleotide codon optimization 57atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cct 635821PRTArtificial SequenceCD8 alpha amino acid 58Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro 2059405DNAArtificial SequenceNKG2D nucleotide 59ttattcaacc aagaagttca aattcccttg accgaaagtt actgtggccc atgtcctaaa 60aactggatat gttacaaaaa taactgctac caattttttg atgagagtaa aaactggtat 120gagagccagg cttcttgtat gtctcaaaat gccagccttc tgaaagtata cagcaaagag 180gaccaggatt tacttaaact ggtgaagtca tatcattgga tgggactagt acacattcca 240acaaatggat cttggcagtg ggaagatggc tccattctct cacccaacct actaacaata 300attgaaatgc agaagggaga ctgtgcactc tatgcctcga gctttaaagg ctatatagaa 360aactgttcaa ctccaaatac atacatctgc atgcaaagga ctgtg 40560405DNAArtificial SequenceNKG2D nucleotide codon optimization 60ctgttcaacc aggaggtcca gatcccactg accgaaagtt actgcggacc atgtcccaag 60aactggatct gctacaagaa caactgttac cagttctttg acgagtctaa gaactggtat 120gaatctcagg ccagttgcat gtcacagaat gcttcactgc tgaaggtgta cagcaaagag 180gaccaggatc tgctgaagct ggtgaaatcc tatcactgga tgggcctggt ccatatccca 240accaacgggt cttggcagtg ggaggacgga agcattctgt cccccaatct gctgacaatc 300attgaaatgc agaagggcga ttgtgctctg tacgcaagct ccttcaaagg gtatatcgag 360aactgctcca cccccaatac atacatttgt atgcagagga cagtg 40561135PRTArtificial SequenceNKG2D amino acid 61Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly1 5 10 15Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe 20 25 30Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser 35 40 45Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu 50 55 60Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro65 70 75 80Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn 85 90 95Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala 100 105 110Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr 115 120 125Ile Cys Met Gln Arg Thr Val 130 135621023DNAArtificial SequenceNKG2D-Z nucleotide 62atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660tccctggtca ttactctgta ttgtcgagtg aagttcagca ggtccgccga cgctcctgca 720taccagcagg gacagaacca gctgtataac gagctgaatc tgggccggag agaggaatac 780gacgtgctgg acaaaaggcg gggccgggac cccgaaatgg gagggaagcc acgacggaaa 840aacccccagg agggcctgta caatgagctg caaaaggaca aaatggccga ggcttattct 900gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggcct gtaccagggg 960ctgagcaccg ctacaaagga cacctatgat gcactgcaca tgcaggccct gccccctcgg 1020tga 102363340PRTArtificial SequenceNKG2D-Z amino acid 63Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 195 200 205Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 210 215 220Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala225 230 235 240Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 245 250 255Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 260 265 270Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 275 280 285Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 290 295 300Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly305 310 315 320Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 325 330 335Leu Pro Pro Arg 340641149DNAArtificial SequenceNKG2D-BBZ nucleotide 64atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660tccctggtca ttactctgta ttgtaagcgg ggaagaaaga aactgctgta catcttcaaa 720cagcccttta tgaggcctgt gcagaccaca caggaggaag acggctgctc ctgccggttc 780cccgaggaag aggaaggcgg gtgcgagctg cgagtgaagt tcagcaggtc cgccgacgct 840cctgcatacc agcagggaca gaaccagctg tataacgagc tgaatctggg ccggagagag 900gaatacgacg tgctggacaa aaggcggggc cgggaccccg aaatgggagg gaagccacga 960cggaaaaacc cccaggaggg cctgtacaat gagctgcaaa aggacaaaat ggccgaggct 1020tattctgaaa tcgggatgaa gggagagaga aggcgcggaa aaggccacga tggcctgtac 1080caggggctga gcaccgctac aaaggacacc tatgatgcac tgcacatgca ggccctgccc 1140cctcggtga 114965382PRTArtificial SequenceNKG2D-BBZ amino acid 65Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 195 200 205Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 210 215 220Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys225 230 235 240Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 245 250 255Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 260 265 270Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 275 280 285Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 290 295 300Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg305 310 315 320Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 325 330 335Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 340 345 350Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 355 360 365Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 370 375 380661131DNAArtificial SequenceNKG2D-OX40Z nucleotide 66atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660tccctggtca ttactctgta ttgtcgggac cagagactgc cacctgatgc acacaagcca 720ccaggaggag gcagcttcag gacccccatc caggaggaac aggccgacgc tcattccaca 780ctggccaaaa ttcgagtgaa gttcagcagg tccgccgacg ctcctgcata ccagcaggga 840cagaaccagc tgtataacga gctgaatctg ggccggagag aggaatacga cgtgctggac 900aaaaggcggg gccgggaccc cgaaatggga gggaagccac gacggaaaaa cccccaggag 960ggcctgtaca atgagctgca aaaggacaaa atggccgagg cttattctga aatcgggatg 1020aagggagaga gaaggcgcgg aaaaggccac gatggcctgt accaggggct gagcaccgct 1080acaaaggaca cctatgatgc actgcacatg caggccctgc cccctcggtg a 113167376PRTArtificial SequenceNKG2D-OX40Z amino acid 67Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 195 200 205Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 210 215 220Thr Leu Tyr Cys Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro225 230 235 240Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp 245 250 255Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala 260 265 270Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 275 280 285Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 290 295 300Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu305 310 315 320Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 325 330 335Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 340 345 350Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 355 360 365His Met Gln Ala Leu Pro Pro Arg 370 375681089DNAArtificial SequenceNKG2D-ZOX40L nucleotide 68atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg

caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660tccctggtca ttactctgta ttgtcgagtg aagttcagca ggtccgccga cgctcctgca 720taccagcagg gacagaacca gctgtataac gagctgaatc tgggccggag agaggaatac 780gacgtgctgg acaaaaggcg gggccgggac cccgaaatgg gagggaagcc acgacggaaa 840aacccccagg agggcctgta caatgagctg caaaaggaca aaatggccga ggcttattct 900gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggcct gtaccagggg 960ctgagcaccg ctacaaagga cacctatgat gcactgcaca tgcaggccct gccccctcgg 1020gaaagagtgc agcccctgga agagaatgtc gggaatgccg ctcgcccaag atttgaaagg 1080aacaaatga 108969362PRTArtificial SequenceNKG2D-ZOX40L amino acid 69Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 195 200 205Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 210 215 220Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala225 230 235 240Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 245 250 255Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 260 265 270Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 275 280 285Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 290 295 300Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly305 310 315 320Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 325 330 335Leu Pro Pro Arg Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn 340 345 350Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys 355 360701155DNAArtificial SequenceNKG2D-28Z nucleotide 70atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atttttgggt cctggtggtc gtgggagggg tgctggcatg ttactcactg 660ctggtcaccg tggccttcat catcttctgg gtgcggagca agaggtcccg cctgctgcac 720agcgactata tgaacatgac cccacggaga cccggcccta cacggaaaca ttaccagccc 780tatgctccac cccgggactt cgcagcttac agaagtcgag tgaagttcag caggtccgcc 840gacgctcctg cataccagca gggacagaac cagctgtata acgagctgaa tctgggccgg 900agagaggaat acgacgtgct ggacaaaagg cggggccggg accccgaaat gggagggaag 960ccacgacgga aaaaccccca ggagggcctg tacaatgagc tgcaaaagga caaaatggcc 1020gaggcttatt ctgaaatcgg gatgaaggga gagagaaggc gcggaaaagg ccacgatggc 1080ctgtaccagg ggctgagcac cgctacaaag gacacctatg atgcactgca catgcaggcc 1140ctgccccctc ggtga 115571384PRTArtificial SequenceNKG2D-28Z amino acid 71Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu 195 200 205Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 210 215 220Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His225 230 235 240Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 245 250 255His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 260 265 270Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 275 280 285Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 290 295 300Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys305 310 315 320Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 325 330 335Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 340 345 350Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 355 360 365Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 370 375 380721128DNAArtificial SequenceNKG2D-28(H)Z nucleotide 72atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtgat cgaggtcatg 480tacccccctc catatctgga caacgaaaag tccaatggaa ctatcattca cgtgaagggc 540aaacatctgt gcccatctcc cctgttccct gggccaagta aacctttttg ggtcctggtg 600gtcgtgggag gggtgctggc atgttactca ctgctggtca ccgtggcctt catcatcttc 660tgggtgcgga gcaagaggtc ccgcctgctg cacagcgact atatgaacat gaccccacgg 720agacccggcc ctacacggaa acattaccag ccctatgctc caccccggga cttcgcagct 780tacagaagtc gagtgaagtt cagcaggtcc gccgacgctc ctgcatacca gcagggacag 840aaccagctgt ataacgagct gaatctgggc cggagagagg aatacgacgt gctggacaaa 900aggcggggcc gggaccccga aatgggaggg aagccacgac ggaaaaaccc ccaggagggc 960ctgtacaatg agctgcaaaa ggacaaaatg gccgaggctt attctgaaat cgggatgaag 1020ggagagagaa ggcgcggaaa aggccacgat ggcctgtacc aggggctgag caccgctaca 1080aaggacacct atgatgcact gcacatgcag gccctgcccc ctcggtga 112873375PRTArtificial SequenceNKG2D-28(H)Z amino acid 73Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ile Glu Val Met145 150 155 160Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile 165 170 175His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro 180 185 190Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 195 200 205Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 210 215 220Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg225 230 235 240Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 245 250 255Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp 260 265 270Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 275 280 285Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 290 295 300Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly305 310 315 320Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 325 330 335Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 340 345 350Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 355 360 365Met Gln Ala Leu Pro Pro Arg 370 375741257DNAArtificial SequenceNKG2D-BBOX40Z nucleotide 74atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660tccctggtca ttactctgta ttgtaagcgg ggaagaaaga aactgctgta catcttcaaa 720cagcccttta tgaggcctgt gcagaccaca caggaggaag acggctgctc ctgccggttc 780cccgaggaag aggaaggcgg gtgcgagctg cgggaccaga gactgccacc tgatgcacac 840aagccaccag gaggaggcag cttcaggacc cccatccagg aggaacaggc cgacgctcat 900tccacactgg ccaaaattcg agtgaagttc agcaggtccg ccgacgctcc tgcataccag 960cagggacaga accagctgta taacgagctg aatctgggcc ggagagagga atacgacgtg 1020ctggacaaaa ggcggggccg ggaccccgaa atgggaggga agccacgacg gaaaaacccc 1080caggagggcc tgtacaatga gctgcaaaag gacaaaatgg ccgaggctta ttctgaaatc 1140gggatgaagg gagagagaag gcgcggaaaa ggccacgatg gcctgtacca ggggctgagc 1200accgctacaa aggacaccta tgatgcactg cacatgcagg ccctgccccc tcggtga 125775418PRTArtificial SequenceNKG2D-BBOX40Z amino acid 75Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 195 200 205Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 210 215 220Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys225 230 235 240Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 245 250 255Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Asp 260 265 270Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe 275 280 285Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala 290 295 300Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln305 310 315 320Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 325 330 335Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 340 345 350Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 355 360 365Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 370 375 380Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser385 390 395 400Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 405 410 415Pro Arg761215DNAArtificial SequenceNKG2D-BBOX40LZ nucleotide 76atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660tccctggtca ttactctgta ttgtaagcgg ggaagaaaga aactgctgta catcttcaaa 720cagcccttta tgaggcctgt gcagaccaca caggaggaag acggctgctc ctgccggttc 780cccgaggaag aggaaggcgg gtgcgagctg gaaagagtgc agcccctgga agagaatgtc 840gggaatgccg ctcgcccaag atttgaaagg aacaaacgag tgaagttcag caggtccgcc 900gacgctcctg cataccagca gggacagaac cagctgtata acgagctgaa tctgggccgg 960agagaggaat acgacgtgct ggacaaaagg cggggccggg accccgaaat gggagggaag 1020ccacgacgga aaaaccccca ggagggcctg tacaatgagc tgcaaaagga caaaatggcc 1080gaggcttatt

ctgaaatcgg gatgaaggga gagagaaggc gcggaaaagg ccacgatggc 1140ctgtaccagg ggctgagcac cgctacaaag gacacctatg atgcactgca catgcaggcc 1200ctgccccctc ggtga 121577404PRTArtificial SequenceNKG2D-BBOX40LZ amino acid 77Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 195 200 205Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 210 215 220Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys225 230 235 240Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 245 250 255Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Glu Arg 260 265 270Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg Pro Arg Phe 275 280 285Glu Arg Asn Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 290 295 300Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg305 310 315 320Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 325 330 335Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 340 345 350Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 355 360 365Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 370 375 380Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala385 390 395 400Leu Pro Pro Arg781257DNAArtificial SequenceNKG2D-OX40BBZ nucleotide 78atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atatctacat ttgggcccct ctggctggaa catgtggcgt gctgctgctg 660tccctggtca ttactctgta ttgtcgggac cagagactgc cacctgatgc acacaagcca 720ccaggaggag gcagcttcag gacccccatc caggaggaac aggccgacgc tcattccaca 780ctggccaaaa ttaagcgggg aagaaagaaa ctgctgtaca tcttcaaaca gccctttatg 840aggcctgtgc agaccacaca ggaggaagac ggctgctcct gccggttccc cgaggaagag 900gaaggcgggt gcgagctgcg agtgaagttc agcaggtccg ccgacgctcc tgcataccag 960cagggacaga accagctgta taacgagctg aatctgggcc ggagagagga atacgacgtg 1020ctggacaaaa ggcggggccg ggaccccgaa atgggaggga agccacgacg gaaaaacccc 1080caggagggcc tgtacaatga gctgcaaaag gacaaaatgg ccgaggctta ttctgaaatc 1140gggatgaagg gagagagaag gcgcggaaaa ggccacgatg gcctgtacca ggggctgagc 1200accgctacaa aggacaccta tgatgcactg cacatgcagg ccctgccccc tcggtga 125779418PRTArtificial SequenceNKG2D-OX40BBZ amino acid 79Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 195 200 205Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 210 215 220Thr Leu Tyr Cys Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro225 230 235 240Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp 245 250 255Ala His Ser Thr Leu Ala Lys Ile Lys Arg Gly Arg Lys Lys Leu Leu 260 265 270Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 275 280 285Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 290 295 300Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln305 310 315 320Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 325 330 335Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 340 345 350Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 355 360 365Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 370 375 380Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser385 390 395 400Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 405 410 415Pro Arg801281DNAArtificial SequenceNKG2D-28BBZ nucleotide 80atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atttttgggt cctggtggtc gtgggagggg tgctggcatg ttactcactg 660ctggtcaccg tggccttcat catcttctgg gtgcggagca agaggtcccg cctgctgcac 720agcgactata tgaacatgac cccacggaga cccggcccta cacggaaaca ttaccagccc 780tatgctccac cccgggactt cgcagcttac agaagtaagc ggggaagaaa gaaactgctg 840tacatcttca aacagccctt tatgaggcct gtgcagacca cacaggagga agacggctgc 900tcctgccggt tccccgagga agaggaaggc gggtgcgagc tgcgagtgaa gttcagcagg 960tccgccgacg ctcctgcata ccagcaggga cagaaccagc tgtataacga gctgaatctg 1020ggccggagag aggaatacga cgtgctggac aaaaggcggg gccgggaccc cgaaatggga 1080gggaagccac gacggaaaaa cccccaggag ggcctgtaca atgagctgca aaaggacaaa 1140atggccgagg cttattctga aatcgggatg aagggagaga gaaggcgcgg aaaaggccac 1200gatggcctgt accaggggct gagcaccgct acaaaggaca cctatgatgc actgcacatg 1260caggccctgc cccctcggtg a 128181426PRTArtificial SequenceNKG2D-28BBZ amino acid 81Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu 195 200 205Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 210 215 220Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His225 230 235 240Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 245 250 255His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 260 265 270Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 275 280 285Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 290 295 300Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg305 310 315 320Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 325 330 335Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 340 345 350Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 355 360 365Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 370 375 380Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His385 390 395 400Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 405 410 415Ala Leu His Met Gln Ala Leu Pro Pro Arg 420 425821263DNAArtificial SequenceNKG2D-28OX40Z nucleotide 82atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc 480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atttttgggt cctggtggtc gtgggagggg tgctggcatg ttactcactg 660ctggtcaccg tggccttcat catcttctgg gtgcggagca agaggtcccg cctgctgcac 720agcgactata tgaacatgac cccacggaga cccggcccta cacggaaaca ttaccagccc 780tatgctccac cccgggactt cgcagcttac agaagtcggg accagagact gccacctgat 840gcacacaagc caccaggagg aggcagcttc aggaccccca tccaggagga acaggccgac 900gctcattcca cactggccaa aattcgagtg aagttcagca ggtccgccga cgctcctgca 960taccagcagg gacagaacca gctgtataac gagctgaatc tgggccggag agaggaatac 1020gacgtgctgg acaaaaggcg gggccgggac cccgaaatgg gagggaagcc acgacggaaa 1080aacccccagg agggcctgta caatgagctg caaaaggaca aaatggccga ggcttattct 1140gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggcct gtaccagggg 1200ctgagcaccg ctacaaagga cacctatgat gcactgcaca tgcaggccct gccccctcgg 1260tga 126383420PRTArtificial SequenceNKG2D-28OX40Z amino acid 83Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu 195 200 205Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 210 215 220Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His225 230 235 240Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 245 250 255His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 260 265 270Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly 275 280 285Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr 290 295 300Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala305 310 315 320Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 325 330 335Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 340 345 350Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 355 360 365Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 370 375 380Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly385 390 395 400Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 405 410 415Leu Pro Pro Arg 420841221DNAArtificial SequenceNKG2D-28OX40LZ nucleotide 84atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc aaaacctacc

480acaactcctg caccacgccc ccctactcca gcacctacca tcgcatctca gccactgagt 540ctgcgaccag aggcctgccg gcccgccgcc ggcggggccg tccataccag agggctggac 600tttgcctgcg atttttgggt cctggtggtc gtgggagggg tgctggcatg ttactcactg 660ctggtcaccg tggccttcat catcttctgg gtgcggagca agaggtcccg cctgctgcac 720agcgactata tgaacatgac cccacggaga cccggcccta cacggaaaca ttaccagccc 780tatgctccac cccgggactt cgcagcttac agaagtgaaa gagtgcagcc cctggaagag 840aatgtcggga atgccgctcg cccaagattt gaaaggaaca aacgagtgaa gttcagcagg 900tccgccgacg ctcctgcata ccagcaggga cagaaccagc tgtataacga gctgaatctg 960ggccggagag aggaatacga cgtgctggac aaaaggcggg gccgggaccc cgaaatggga 1020gggaagccac gacggaaaaa cccccaggag ggcctgtaca atgagctgca aaaggacaaa 1080atggccgagg cttattctga aatcgggatg aagggagaga gaaggcgcgg aaaaggccac 1140gatggcctgt accaggggct gagcaccgct acaaaggaca cctatgatgc actgcacatg 1200caggccctgc cccctcggtg a 122185406PRTArtificial SequenceNKG2D-28OX40LZ amino acid 85Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Lys Pro Thr145 150 155 160Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 165 170 175Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 180 185 190Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu 195 200 205Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 210 215 220Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His225 230 235 240Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 245 250 255His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 260 265 270Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg Pro 275 280 285Arg Phe Glu Arg Asn Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 290 295 300Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu305 310 315 320Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 325 330 335Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 340 345 350Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 355 360 365Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 370 375 380Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met385 390 395 400Gln Ala Leu Pro Pro Arg 405861254DNAArtificial SequenceNKG2D-28(H)BBZ nucleotide 86atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtgat cgaggtcatg 480tacccccctc catatctgga caacgaaaag tccaatggaa ctatcattca cgtgaagggc 540aaacatctgt gcccatctcc cctgttccct gggccaagta aacctttttg ggtcctggtg 600gtcgtgggag gggtgctggc atgttactca ctgctggtca ccgtggcctt catcatcttc 660tgggtgcgga gcaagaggtc ccgcctgctg cacagcgact atatgaacat gaccccacgg 720agacccggcc ctacacggaa acattaccag ccctatgctc caccccggga cttcgcagct 780tacagaagta agcggggaag aaagaaactg ctgtacatct tcaaacagcc ctttatgagg 840cctgtgcaga ccacacagga ggaagacggc tgctcctgcc ggttccccga ggaagaggaa 900ggcgggtgcg agctgcgagt gaagttcagc aggtccgccg acgctcctgc ataccagcag 960ggacagaacc agctgtataa cgagctgaat ctgggccgga gagaggaata cgacgtgctg 1020gacaaaaggc ggggccggga ccccgaaatg ggagggaagc cacgacggaa aaacccccag 1080gagggcctgt acaatgagct gcaaaaggac aaaatggccg aggcttattc tgaaatcggg 1140atgaagggag agagaaggcg cggaaaaggc cacgatggcc tgtaccaggg gctgagcacc 1200gctacaaagg acacctatga tgcactgcac atgcaggccc tgccccctcg gtga 125487417PRTArtificial SequenceNKG2D-28(H)BBZ amino acid 87Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ile Glu Val Met145 150 155 160Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile 165 170 175His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro 180 185 190Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 195 200 205Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 210 215 220Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg225 230 235 240Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 245 250 255Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr 260 265 270Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 275 280 285Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 290 295 300Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln305 310 315 320Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 325 330 335Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 340 345 350Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 355 360 365Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 370 375 380Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr385 390 395 400Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 405 410 415Arg881236DNAArtificial SequenceNKG2D-28(H)OX40Z nucleotide 88atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtgat cgaggtcatg 480tacccccctc catatctgga caacgaaaag tccaatggaa ctatcattca cgtgaagggc 540aaacatctgt gcccatctcc cctgttccct gggccaagta aacctttttg ggtcctggtg 600gtcgtgggag gggtgctggc atgttactca ctgctggtca ccgtggcctt catcatcttc 660tgggtgcgga gcaagaggtc ccgcctgctg cacagcgact atatgaacat gaccccacgg 720agacccggcc ctacacggaa acattaccag ccctatgctc caccccggga cttcgcagct 780tacagaagtc gggaccagag actgccacct gatgcacaca agccaccagg aggaggcagc 840ttcaggaccc ccatccagga ggaacaggcc gacgctcatt ccacactggc caaaattcga 900gtgaagttca gcaggtccgc cgacgctcct gcataccagc agggacagaa ccagctgtat 960aacgagctga atctgggccg gagagaggaa tacgacgtgc tggacaaaag gcggggccgg 1020gaccccgaaa tgggagggaa gccacgacgg aaaaaccccc aggagggcct gtacaatgag 1080ctgcaaaagg acaaaatggc cgaggcttat tctgaaatcg ggatgaaggg agagagaagg 1140cgcggaaaag gccacgatgg cctgtaccag gggctgagca ccgctacaaa ggacacctat 1200gatgcactgc acatgcaggc cctgccccct cggtga 123689411PRTArtificial SequenceNKG2D-28(H)OX40Z amino acid 89Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ile Glu Val Met145 150 155 160Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile 165 170 175His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro 180 185 190Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 195 200 205Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 210 215 220Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg225 230 235 240Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 245 250 255Asp Phe Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp Ala 260 265 270His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu 275 280 285Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser 290 295 300Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr305 310 315 320Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 325 330 335Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 340 345 350Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 355 360 365Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 370 375 380His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr385 390 395 400Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 405 410901194DNAArtificial SequenceNKG2D-28(H)OX40LZ nucleotide 90atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa 240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtgat cgaggtcatg 480tacccccctc catatctgga caacgaaaag tccaatggaa ctatcattca cgtgaagggc 540aaacatctgt gcccatctcc cctgttccct gggccaagta aacctttttg ggtcctggtg 600gtcgtgggag gggtgctggc atgttactca ctgctggtca ccgtggcctt catcatcttc 660tgggtgcgga gcaagaggtc ccgcctgctg cacagcgact atatgaacat gaccccacgg 720agacccggcc ctacacggaa acattaccag ccctatgctc caccccggga cttcgcagct 780tacagaagtg aaagagtgca gcccctggaa gagaatgtcg ggaatgccgc tcgcccaaga 840tttgaaagga acaaacgagt gaagttcagc aggtccgccg acgctcctgc ataccagcag 900ggacagaacc agctgtataa cgagctgaat ctgggccgga gagaggaata cgacgtgctg 960gacaaaaggc ggggccggga ccccgaaatg ggagggaagc cacgacggaa aaacccccag 1020gagggcctgt acaatgagct gcaaaaggac aaaatggccg aggcttattc tgaaatcggg 1080atgaagggag agagaaggcg cggaaaaggc cacgatggcc tgtaccaggg gctgagcacc 1140gctacaaagg acacctatga tgcactgcac atgcaggccc tgccccctcg gtga 119491397PRTArtificial SequenceNKG2D-28(H)OX40LZ amino acid 91Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ile Glu Val Met145 150 155 160Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile 165 170 175His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro 180 185 190Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 195 200 205Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 210 215 220Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg225 230 235 240Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 245 250 255Asp Phe Ala Ala Tyr Arg Ser Glu Arg Val Gln Pro Leu Glu Glu Asn 260 265 270Val Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys Arg Val Lys 275 280 285Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 290 295 300Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu305 310 315 320Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 325 330 335Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 340 345 350Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 355 360 365Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 370 375 380Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg385 390 395921203DNAArtificial SequenceNKG2D-AAA-28(H)OX40LZ nucleotide 92atggctctgc cagtgactgc actgctgctg ccactggccc tgctgctgca cgcagctcga 60cctctgttca accaggaggt ccagatccca ctgaccgaaa gttactgcgg accatgtccc 120aagaactgga tctgctacaa gaacaactgt taccagttct ttgacgagtc taagaactgg 180tatgaatctc aggccagttg catgtcacag aatgcttcac tgctgaaggt gtacagcaaa

240gaggaccagg atctgctgaa gctggtgaaa tcctatcact ggatgggcct ggtccatatc 300ccaaccaacg ggtcttggca gtgggaggac ggaagcattc tgtcccccaa tctgctgaca 360atcattgaaa tgcagaaggg cgattgtgct ctgtacgcaa gctccttcaa agggtatatc 420gagaactgct ccacccccaa tacatacatt tgtatgcaga ggacagtggc cgccgctatc 480gaggtcatgt acccccctcc atatctggac aacgaaaagt ccaatggaac tatcattcac 540gtgaagggca aacatctgtg cccatctccc ctgttccctg ggccaagtaa acctttttgg 600gtcctggtgg tcgtgggagg ggtgctggca tgttactcac tgctggtcac cgtggccttc 660atcatcttct gggtgcggag caagaggtcc cgcctgctgc acagcgacta tatgaacatg 720accccacgga gacccggccc tacacggaaa cattaccagc cctatgctcc accccgggac 780ttcgcagctt acagaagtga aagagtgcag cccctggaag agaatgtcgg gaatgccgct 840cgcccaagat ttgaaaggaa caaacgagtg aagttcagca ggtccgccga cgctcctgca 900taccagcagg gacagaacca gctgtataac gagctgaatc tgggccggag agaggaatac 960gacgtgctgg acaaaaggcg gggccgggac cccgaaatgg gagggaagcc acgacggaaa 1020aacccccagg agggcctgta caatgagctg caaaaggaca aaatggccga ggcttattct 1080gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggcct gtaccagggg 1140ctgagcaccg ctacaaagga cacctatgat gcactgcaca tgcaggccct gccccctcgg 1200tga 120393400PRTArtificial SequenceNKG2D-AAA-28(H)OX40LZ amino acid 93Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr 20 25 30Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 35 40 45Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys65 70 75 80Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 85 90 95Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 100 105 110Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 115 120 125Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 130 135 140Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Ala Ala Ile145 150 155 160Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly 165 170 175Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe 180 185 190Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val 195 200 205Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp 210 215 220Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met225 230 235 240Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 245 250 255Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Glu Arg Val Gln Pro Leu 260 265 270Glu Glu Asn Val Gly Asn Ala Ala Arg Pro Arg Phe Glu Arg Asn Lys 275 280 285Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 290 295 300Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr305 310 315 320Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 325 330 335Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 340 345 350Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 355 360 365Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 370 375 380Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg385 390 395 400

Claims

1-30. (canceled)

31. A chimeric antigen receptor, comprising: an intracellular signaling domain which includes the whole or a portion of OX40 ligand (CD252).

32. The chimeric antigen receptor according to claim 31, further comprising: a transmembrane domain linked to the intracellular signaling domain; a spacer domain linked to the transmembrane domain; and an extracellular domain linked to the spacer domain.

33. The chimeric antigen receptor according to claim 32, further comprising a signal sequence linked to the extracellular domain.

34. The chimeric antigen receptor according to claim 33, wherein the signal sequence includes the whole or a portion of CD16 and the signal sequence includes the whole or a portion of CD8.alpha., respectively.

35. The chimeric antigen receptor according to claim 32, wherein the extracellular domain includes the whole or a portion of any one selected from the group consisting of an antigen-binding fragment of an antibody, a Fc receptor, a natural cytotoxicity receptor, NKG2D, 2B4 and DNAM-1.

36. The chimeric antigen receptor according to claim 35, wherein the antigen-binding fragment is Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment.

37. The chimeric antigen receptor according to claim 36, wherein the Fv fragment is a single-chain variable fragment (ScFv).

38. The chimeric antigen receptor according to claim 35, wherein the Fc receptor is selected from the group consisting of CD16, CD32, CD64, CD23, CD89 and variants thereof.

39. The chimeric antigen receptor according to claim 38, wherein the Fc receptor is CD16 or variants thereof.

40. The chimeric antigen receptor according to claim 35, wherein the natural cytotoxicity receptor is selected from the group consisting of NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.

41. The chimeric antigen receptor according to claim 32, wherein the spacer domain includes the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

42. The chimeric antigen receptor according to claim 32, wherein the transmembrane domain includes the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

43. The chimeric antigen receptor according to claim 31, wherein the intracellular signaling domain further includes the whole or a portion of CD3-zeta.

44. The chimeric antigen receptor according to claim 43, wherein the whole or a portion of CD3-zeta and the whole or a portion of OX40 ligand are arranged in order from a cell membrane toward an inside of the cell.

45. The chimeric antigen receptor according to claim 32, further comprising an AAA linker between the extracellular domain and the spacer domain.

46. A chimeric antigen receptor, comprising intracellular signaling domains which comprise: a first intracellular signaling domain including the whole or a portion of any one selected from the group consisting of CD28 and 4-1BB; a second intracellular signaling domain including the whole or a portion of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB; and a third intracellular signaling domain including the whole or a portion of CD3-zeta, wherein the first, second and third intracellular signaling domains are arranged in order from a cell membrane toward an inside of the cell.

47. The chimeric antigen receptor according to claim 46, further comprising: a transmembrane domain linked to the intracellular signaling domain; a spacer domain linked to the transmembrane domain; and an extracellular domain linked to the spacer domain.

48. The chimeric antigen receptor according to claim 47, further comprising a signal sequence linked to the extracellular domain.

49. The chimeric antigen receptor according to claim 48, wherein the signal sequence includes the whole or a portion of CD16 and the signal sequence includes the whole or a portion of CD8.alpha., respectively.

50. The chimeric antigen receptor according to claim 47, wherein the extracellular domain includes the whole or a portion of any one selected from the group consisting of an antigen-binding fragment of an antibody, a Fc receptor, a natural cytotoxicity receptor, NKG2D, 2B4 and DNAM-1.

51. The chimeric antigen receptor according to claim 47, wherein the spacer domain includes the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

52. The chimeric antigen receptor according to claim 47, wherein the transmembrane domain includes the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.

53. The chimeric antigen receptor according to claim 46, wherein the first intracellular signaling domain includes the whole or a portion of CD28; the second intracellular signaling domain includes the whole or a portion of the OX40 ligand; and the third intracellular signaling domain includes the whole or a portion of CD3-zeta.

54. The chimeric antigen receptor according to claim 47, further comprising an AAA linker between the extracellular domain and the spacer domain.

55. An immune cell expressing the chimeric antigen receptor according to claim 31.

56. The immune cell according to claim 55, wherein the immune cell is a natural killer cell (NK cell).

57. A pharmaceutical composition for treatment of tumor, comprising the immune cell according to claim 55 as an active ingredient.

58. The pharmaceutical composition according to claim 57, further comprising an antibody as an active ingredient when the extracellular domain is a Fc receptor.

59. A nucleic acid sequence encoding the chimeric antigen receptor according to claim 31.

60. The nucleic acid sequence according to claim 59, wherein the nucleic acid sequence encodes one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 33, 41, 43, 45, 47, 49, 51, 53, 55, 69, 71, 77, 81, 83, 85, 87, 89, 91 and 93, or variants thereof having a sequence identity of 80% or more.

61. The nucleic acid sequence according to claim 60, wherein the nucleic acid sequence includes one or more nucleic acid sequence selected from the group consisting of SEQ ID NOs: 32, 40, 42, 44, 46, 48, 50, 52, 54, 68, 70, 76, 80, 82, 84, 86, 88, 90 and 92, or variants thereof having a sequence identity of 80% or more.

62. A method for treatment of tumor, comprising administering the immune cell according to claim 55 to a subject.