ENHANCED CHIMERIC ANTIGEN RECEPTORS AND USE THEREOF

Abstract

Provided herein are chimeric antigen receptors (CARs) comprising a truncated EGFRvIII (Ev3) in the hinge region of the CAR and/or a humanized scFv. Further provided herein are immune cells expressing the CARs as well as methods of their use in the treatment of immune disorders.

Description

[0001] The present application is a continuation of U.S. patent application Ser. No. 16/632,762 filed Jan. 21, 2020, which is a national phase application under 35 U.S.C. .sctn. 371 that claims priority to International Application No. PCT/US2018/043779 filed Jul. 25, 2018, which claims the priority benefit of U.S. Provisional Application Ser. No. 62/536,934, filed Jul. 25, 2017, all of which are hereby incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] The sequence listing that is contained in the file named "MDAC_P1331USC1_1001160638_SL.txt", which is 289 KB (as measured in Microsoft Windows) and was created on Apr. 16, 2021, is filed herewith by electronic submission and is incorporated by reference herein.

BACKGROUND

1. Field

[0003] The present invention relates generally to the fields of immunology and molecular biology. More particularly, it concerns enhanced chimeric antigen receptors (CARs), such as for immune cells.

2. Description of Related Art

[0004] Despite technological advancements in the diagnosis and treatment options available to patients diagnosed with cancer, the prognosis still often remains poor and many patients cannot be cured. Immunotherapy holds the promise of offering a potent, yet targeted, treatment to patients diagnosed with various tumors with the potential to eradicate the malignant tumor cells without damaging normal tissues. In theory, the T cells of the immune system are capable of recognizing protein patterns specific for tumor cells and to mediate their destruction through a variety of effector mechanisms. The administration of immune cells expressing chimeric antigen receptors (CARs), such as adoptive T cell therapy or NK cell therapy, is an attempt to harness and amplify the tumor-eradicating capacity of a patient's own immune cells and then return these effectors to the patient in such a state that they effectively eliminate residual tumor, however without damaging healthy tissue. Commonly, CARs comprise a single chain variable fragment (scFv) of an antibody specific for a tumor associated antigen (TAA) coupled via hinge and transmembrane regions to cytoplasmic domains of T-cell signaling molecules. However, many drawbacks in the clinical use of immune cells therapy impair the full use of this approach in cancer treatments. Thus, there is an unmet need for improved CARs for use in immune cell therapy.

SUMMARY

[0005] In certain embodiments, the present disclosure provides enhanced chimeric antigen receptors and methods of their use for the treatment of diseases and disorders, including cancer and autoimmune disorders. In one embodiment, there is provided a truncated EGFRvIII, referred to as Ev3 (or tEv3), which does not comprise an endodomain or functional EGF binding domain. In particular aspects, Ev3 comprises truncated domain 1 (L1), truncated domain 2 (CR1), domain 3 (L2), and domain 4 (CR2) of EGFR (as depicted in FIG. 2B). In some aspects, the Ev3 may be located in the hinge region of a CAR and can be used for CAR detection and/or ablation of CAR-expressing cells. In some embodiments, the CAR comprises both a humanized scFv and an Ev3 hinge. In another embodiment, there are provided humanized antigen-binding domains for the CARs, such as humanized single chain variable fragments (scFvs). Further embodiments provide methods of treating immune-associated disorders by administering immune cells, such as T cells or NK cells, which express the CAR(s) of the embodiments to a subject.

[0006] In another embodiment, there is provided a chimeric antigen receptor (CAR) comprising a Ev3 in the hinge of said CAR, wherein said Ev3 hinge links an extracellular domain and at least one intracellular immune signaling domain. In some aspects, the Ev3 hinge comprises a transmembrane domain and a non-functional ectodomain of EGFRvIII. In particular aspects, the Ev3 hinge does not comprise EGFRvIII endodomain. In some aspects, the non-functional ectodomain of EGFRvIII has essentially no binding capability to epidermal growth factor (EGF).

[0007] In some aspects, the Ev3 hinge comprises an amino acid sequence with at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:2. In some aspects, the Ev3 hinge comprises an amino acid sequence of SEQ ID NO:2.

[0008] The Ev3 may comprise or have at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2 LEEKK-GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRK-VCNGIGIGEFKDSLSINATNIKHFKNC- TSISGDLHILPVAFRGDSFTHTPPLDPQELDIL KTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLK EISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQ-VCHALCSPEGCWGPEPRDCVSC- RNVSRGRECVDKCNLLEGEPREFVENSECIQCHPE CLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHV CHLCHPNCTYGCTGPGLEGCPTNGPKIPS (comprising partial Domain 1-partial Domain 2-Domain 3-Domain 4). In certain aspects, the Ev3 may have one, two, three, or more modifications.

[0009] In some aspects, the Ev3 hinge is encoded by a nucleotide sequence with at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:1. In certain aspects, the Ev3 hinge is encoded by a nucleotide sequence of SEQ ID NO:1.

[0010] In certain aspects, the extracellular domain of the CAR comprises an antigen-binding domain selected from the group consisting of F(ab')2, Fab', Fab, Fv, and scFv. In some aspects, the antigen-binding domain comprises a scFv. In specific aspects, the scFv is further defined as a humanized scFv. In some aspects, the humanized scFv is a scFv according to the embodiments.

[0011] In some aspects, the antigen binding region of the CAR binds one or more tumor associated antigens. In certain aspects, the one or more tumor associated antigens are selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL/DR4, VEGFR2, CD33, CLL-1 and CD99. In particular aspects, the one or more tumor-associated antigens are CD19, CD319, CD123, CD5, ROR1, mesothelin, CD33, CLL-1, and/or CD99. In some aspects, the scFv does not comprise an EGFR binding domain.

[0012] In certain aspects, the at least one signaling domain comprises CD3, CD28, OX40/CD134, 4-1BB/CD137, Fc.epsilon.RI.gamma., ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP12, CD70, CD40, or a combination thereof. In particular aspects, the at least one signaling domain comprises DAP12. In some aspects, the CAR comprises IL-15. In one specific aspects, the CAR comprises CD3.zeta., CD28, DAP12, and IL-15.

[0013] In some aspects, the CAR further comprises a suicide gene. In particular aspects, the suicide gene is inducible caspase 9.

[0014] In one embodiment, there are provided isolated antigen-specific humanized single chain variable fragments (scFvs) comprising a light chain variable region (V.sub.L) and a heavy chain variable region (V.sub.H), wherein the scFv is:

[0015] (a) CD19-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:7 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:8;

[0016] (b) CD123-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:13 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:14;

[0017] (c) mesothelin-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:19 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:20;

[0018] (d) ROR1-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:25 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:26;

[0019] (e) CD5-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:31 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:32;

[0020] (f) CLL-1-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:59 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:60;

[0021] (g) CD99-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:63 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:64 or

[0022] (h) a sequence with 90% sequence identity to framework regions of any one of (a)-(g).

[0023] In some aspects, the scFv sequences comprise 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the framework regions of SEQ ID NOs:6, 12, 18, 24, or 30 and 100% sequence identity to the CDRs of said sequences. In certain aspects, the CDRs of said sequences may have one, two, or three modifications.

[0024] In some aspects, the scFv is CD19-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:7 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:8. In one specific aspect, the CD19-specific scFv comprises an amino acid sequence of SEQ ID NO:6. In certain aspects, the scFv is CD123-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:13 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:14. In particular aspects, the CD123-specific scFv comprises an amino acid sequence of SEQ ID NO:12. In some aspects, the scFv is mesothelin-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:19 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:20. In specific aspects, the mesothelin-specific scFv comprises an amino acid sequence of SEQ ID NO:18. In some aspects, the scFv is ROR1-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:25 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:26. In specific aspects, the ROR1-specific comprises an amino acid sequence of SEQ ID NO:24. In some aspects, the scFv is CD5-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:31 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:32. In certain aspects, the CD5-specific comprises an amino acid sequence of SEQ ID NO:30.

[0025] In some aspects, the scFv comprises an amino acid sequence with at least 95%, such as at least 96, 97, 98, or 99% sequence identity to framework regions an amino acid sequence of SEQ ID NO:6, 12, 18, 24, or 30.

[0026] Further provided herein are isolated polynucleotides encoding the isolated scFvs of the above embodiments. In some aspects, the polynucleotide comprises SEQ ID NO:3, 9, 15, 21, or 27. In other aspects, the polynucleotide comprises a sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:3, 9, 15, 21, or 27.

[0027] Also provided herein are expression vectors comprising isolated nucleotides of the embodiments. In some aspects, the vector is further defined as a viral vector.

[0028] Further provided herein are isolated nucleic acids comprising a nucleotide sequence encoding the CAR according to any one of the present embodiments.

[0029] Also provided herein are host cells engineered to express a CAR comprising a humanized scFv of the present embodiments and/or a truncated EGFRvIII (Ev3) in the hinge region of said CAR. In some aspects, the CAR is according to any one of the present embodiments. In some aspects, DNA encoding the CAR is integrated into the genome of the cell.

[0030] In some aspects, the host cell is further defined as a CAR immune cell. In particular aspects, the immune cell is a T cell, peripheral blood lymphocyte, NK cell, invariant NK cell, NKT cell, or stem cell. In specific aspects, the immune cell is a T cell or a NK cell. In some aspects, the stem cell is a mesenchymal stem cell (MSC) or an induced pluripotent stem (iPS) cell. In certain aspects, the immune cell is derived from an iPS cell. In some aspects, the T cell is a CD8.sup.+ T cell, CD4.sup.+ T cell, or gamma-delta T cell. In certain aspects, the T cell is a cytotoxic T lymphocyte (CTL). In some aspects, the immune cell is allogeneic or autologous. In some aspects, the immune cell is isolated from peripheral blood, cord blood, or bone marrow. In certain aspects, the immune cell is isolated from cord blood. In particular aspects, the cord blood is pooled from 2 or more individual cord blood units.

[0031] Further provided herein are pharmaceutical compositions comprising a population of immune cells according to any of the present embodiments. Also provided herein are compositions comprising a population of immune cells of any one of present embodiments for use in the treatment of an immune-related disorder.

[0032] In another embodiment, there is provided a method of treating an immune-related disorder in a subject comprising administering an effective amount of CAR immune cells of any one of the present embodiments, such as comprising a humanized scFv and/or a Ev3 hinge.

[0033] In some aspects of the above embodiments, the immune-related disorder is a cancer, autoimmune disorder, graft versus host disease, allograft rejection, or inflammatory condition. In certain aspects, the immune-related disorder is an inflammatory condition and the immune cells have essentially no expression of glucocorticoid receptor. In particular aspects, the immune-related disorder is a cancer. In some aspects, the immune cells are autologous or allogeneic. In some aspects, the cancer is a solid cancer or a hematologic malignancy.

[0034] In additional aspects, the method further comprises administering at least a second therapeutic agent. In some aspects, the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, or biotherapy. In certain aspects, the immune cells and/or the at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.

[0035] In some aspects, the method further comprises administering an anti-EGFR antibody. In some aspects, the anti-EGFR antibody is a monoclonal antibody. In certain aspects, the anti-EGFR antibody is cetuximab or panitumumab. In some aspects, the anti-EGFR antibody selectively ablates Ev3-CAR immune cells through antibody-dependent cell-mediated cytotoxicity (ADCC). In certain aspects, the anti-EGFR antibody is fused to a detectable tag and/or a cytotoxic agent. In some aspects, the method further comprises imaging the detectable tag, thereby detecting the Ev3-CAR immune cells. In certain aspects, the anti-EGFR antibody is fused to a cytotoxic agent. In some aspects, the cytotoxic agent induces activation of the suicide gene in the Ev3-CAR immune cells. In some aspects, the cytotoxic agent results in the ablation of the Ev3-CAR immune cells.

[0036] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0038] FIGS. 1A-1B: Comparisons between current CARs and Ev3-CAR designs. (FIG. 1A) The Ev3 stalk serves as an integrative structure to transduce signals from antigen binding, such as by scFv, as well as an "off" switch, such that it can be recognized by clinically accessible antibodies such as cetuximab to ablate CAR-positive immune cells. (FIG. 1B) As an enhancement over CARs, such as T cell use, DAP12 may be utilized as the signal booster in Ev3-DAP12 CARs to increase the efficacies of CAR functions in NK cells.

[0039] FIGS. 2A-2C: (FIG. 2A) Schematic depicting Ev3 as compared to EGFR which comprise the endodomain and the EGF binding domain. (FIG. 2B) Schematic depicting EGFR variants and fragments. (FIG. 2C) Sequence of Ev3 comprising only extracellular domains.

[0040] FIGS. 3A-3F: Diagrams for retroviral vectors for the antigens CD19 (FIG. 3A), hCD19 (FIG. 3B), CD123 (FIG. 3C), hCD123 (FIG. 3D), and CD99 (FIG. 3E). (FIG. 3F) Construct map for CAR 2.0 using humanized scFv against CD19 which can be replaced with other scFvs.

[0041] FIGS. 4A-4B: (FIG. 4A) Ev3 antibody recognizes CAR19.Ev3.CD28.CD3.zeta., CAR19.Ev3.DAP12.CD3.zeta. or humanized CAR19.Ev3.CD3.zeta. transduced NK cells. (FIG. 4B) In chromium release assays to assess the killing activity of CAR.19 transduced NK cells against CD19.sup.+ Raji tumor targets, it was shown that CAR19.Ev3.CD28.DAP12 NK cells are as effective as CAR19.IgG1.CD28.CD3z transduced NK cells in killing CD19.sup.+ Raji cells at multiple effector:target ratios.

[0042] FIGS. 5A-5B: (FIG. 5A) Schematic depicting platform for humanization of CARs. (FIG. 5B) Monoclonal antibody sequence analysis.

[0043] FIG. 6: Schematic depicting isotypes, allotypes, and idiotypes of antibodies. Structural determinants of antibodies are pictured to define the elements isolated and used for the analysis and subsequent CDR/framework graftings. 5 major isotypes for human immunoglobulins are further subclassified as indicated (IgG, 2, 3, 4, IgA1, 2).

[0044] FIGS. 7A-7C: (FIG. 7A) Amino acid sequences for hCD19 scFv and mCD19 scFv including V.sub.L and V.sub.H. The CDR regions are underlined. (FIG. 7B) BLAST analysis of hCD19 scFv and mCD19 scFv to verify their respective species origins by sequence identities or homologies. (FIG. 3C) Summary of analysis comparing mCD19 scFv vs humanized CD19 scFv.

[0045] FIGS. 8A-8D: (FIG. 8A) Schematic depicting 3D structure modeling. (FIGS. 8B-D) 3D Structure modeling of human CD19 variable light chain. Using SWISS-MODEL*, a fully automated protein structure homology-modeler, 3-dimensional models of humanized V.sub.L and V.sub.H domains were generated (anti-CD19 as specific example shown). Structure files were extracted from SAbDab, a structural antibody database, as templates for homology modeling, as well as using PDB structure files which are automatically accessed within SWISS-MODEL.

[0046] FIGS. 9A-9C: 3D structure modeling of human CD19 variable heavy chain. For the specific example of hCD19V.sub.L domain, (FIG. 9A) a list was generated in SWISS-MODEL for the best matching (structurally homologous) structure with their scores (GMQE) and matching methods (BLAST or HHblits). (FIGS. 9B-C) The best scoring structure (2fgw) was then aligned with the hCD19V.sub.L domain ("target") as shown in the bottom panel.

[0047] FIGS. 10A-10G: (FIG. 10A) Schematic depicting human framework grafting showing identification and isolation of the CDR regions from mouse antibodies (left top), distillation of human framework regions (right panel), and human framework/murine CDR grafting (left bottom). The CDRs and framework regions are gleaned from sequence analysis as described above. (FIG. 10B) Baseline humanization scoring of monoclonal antibody using T20 scoring system (Gao et al., 2013) to enumerate the `humanness` of particular Ig domains. As controls, V.sub.L and V.sub.H domains were extracted from antibodies which are currently in the market place for scoring and comparisons. The humanness scores for the indicated antibodies (right most columns) are listed for V.sub.L and V.sub.H domains. (FIG. 10C) Early therapeutic antibodies approved for the commercial marketplace are either murine or chimeric antibodies (variable mouse regions+human constant regions), while later approvals tend to be humanized or human antibodies. The humanness scores tend to cluster for antibodies of the same type (murine, chimeric, humanized, or human), with increasing humanness scores correlative to human contents. (FIG. 10D) Humanization scoring of variable heavy chains by year of FDA approval of antibody. The humanness scores tend to cluster for antibodies of the same type (murine, chimeric, humanized, or human), with increasing humanness scores correlative to human contents. Due to the larger sizes of the V.sub.H domains, the humanness scores tend to be lower (compared to V.sub.L). (FIG. 10E) Humanization scoring for indicated antibodies. The frequency of anti-therapeutic antibody responses is shown as % and the size of the patient group evaluated is given in brackets. In general, the more human or humanized an antibody, the less immunogenicity is reported in clinical use. (FIG. 10F) Humanization scoring of variable light chain of hCD19-CAR (SEQ ID NO:1) and mCD19-CAR (SEQ ID NO:3). (FIG. 10G) Humanization scoring of variable heavy chain of hCD19-CAR (SEQ ID NO:2) and mCD19-CAR (SEQ ID NO:4)

[0048] FIGS. 11A-11E: (FIG. 11A) Schematic of CD19-CAR and hCD19-CAR constructs. (FIG. 11B) Transduction efficiency of CAR. (FIG. 11C) Cytotoxicity of different CAR constructs against K562 cells. (FIG. 11D) Cytotoxicity of different CAR constructs against Raji cells. (FIG. 11E) Cytotoxicity assay for cells with control CD3.zeta./IgG1 CAR or humanized CD3.zeta./IgG1 CAR against K562 or Raji cells at indicated effector:target ratios.

[0049] FIGS. 12A-12I: (FIG. 12A) CD319-CAR NK transduction. (FIG. 12B) Growth curve of NK cells. (FIG. 12C) CD319 expression in cell lines. (FIGS. 12D-F) CD319-CAR NK cytotoxicity assay. (FIG. 12G) Cytotoxicity assay with CD319-CAR NK cells against indicated cell types including K562, MM.1S, and OPM2. (FIGS. 12H-I) CD319-CAR NK cells have an effector phenotype.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0050] Chimeric antigen receptor (CAR) based cellular immunotherapy has shown significant efficacies in cancer treatment, in particular for treatment of leukemia and lymphoma. Nevertheless, currently available CARs can comprise various design defects. Recent clinical results using CARs to reprogram immune cells for targeted cancer immunotherapy have shown potent anti-tumor cytotoxicity. Since most CAR single chain variable fragment (scFv) modules are derivatives of murine antibodies, human anti-mouse immune-reactivity compromises the efficacy of CARs thus derived.

[0051] Thus, in some embodiments, the present disclosure provides a platform technology to engineer humanized CARs (hCARs) against antigens. The antigens may include CD19 (hCD19CAR), CD123 (hCD123CAR), mesothelin (hMesoCAR), ROR1 (hROR1CAR), CD5 (hCD5CAR), CLL-1 (hCLL-1CAR), and/or CD99 (hCD99CAR) as well as other antigens that may be targeted for treatment of a disease, such as cancer or autoimmune disorders. Accordingly, in certain embodiments, the present disclosure further provides humanized CARs and methods of their use in the treatment of cancer.

[0052] In some embodiments, the present disclosure provides a platform for the humanization of CARs, specifically the scFv of the CAR. The process follows 5 modular components as described in Example 1. These can include sequence analysis, 3D structure modeling, human framework grafting, humanization scoring, and functional analysis. Next, IgG is focused on for the humanization process, making use of extensive sequence variations in the allotypes (i.e., versions of the same isotype) to fine tune for human framework selection (FIG. 2).

[0053] In the first step, the antibody sequences for human heavy and light chains can be curated in a local database from sources such as GenBank. The variable domains may be aligned using IgBLAST (a version of BLAST) to delineate allotypes and sort the sequences according to homologies. CDR regions can then be identified and framework sequences are cataloged (FIG. 1A). 3D structure modeling may then be performed, such as using a protein structure homology-modeler (e.g., SWISS-MODEL).

[0054] In some embodiments, immune cells are engineered to express CARs with the humanized scFvs provided herein. The immune cells may be NK cells and/or T cells, such as derived from peripheral blood, umbilical cord blood, or marrow-derived infiltrating lymphocytes. The immune cells with a scFv derived from the present humanization platform can mitigate the possibility of human anti-mouse antibody (HAMA) production and associated immune-mediated rejection of genetically modified immune cell therapy products.

[0055] Thus, further embodiments provide methods of treating diseases or disorders by administering immune cells which express CARs with humanized scFvs. These methods can be used to lower immune rejection as well as longer persistence of infused cell therapy products. Exemplary subjects that may be treated include cancer patients with CD19.sup.+ CLL, ALL and NHL; CD123-positive malignancies including AML, CML. MDS and BPDCN; Mesothelin-positive cancers including cancers of the cervix, endometrium, and the head and neck; ROR1-positive malignancies including CLL (e.g., without prior CAR therapy or those who relapse following CD19-CAR with CD19-negative disease), breast, ovary and pancreatic cancers; CD5-positive malignancies including ALL and NHL as well as thymic cancer.

[0056] In further embodiments, the present disclosure provides CARs comprising truncated EGFRvIII wherein the receptor does not comprise the cytoplasmic endodomain or a functional EGF binding domain, referred to herein as Ev3. EGFRvIII is an EGFR variant that is found only in tumor cells, such as glioblastoma multiforme and lung cancer, and not in normal EGFR expressing cells such as epithelial cells. EGFRvIII has a somatic mutation resulting in deletion of exons 2-7 (801 base pair in-frame deletion, 267 amino acid missing compared to wild-type). The deletion of EGFRvIII also covers most of domains 1 and 2 resulting in a truncated domain 1 and truncated domain 2. Further, EGFRvIII has a specific epitope as a result of the fusion between exon 1 and exon 8 creating an extra glycine residue (amino acids 6-273 are replaced by a glycine residue; i.e., LEEKKGNYVVTD), which forms a tumor-specific antigen, such as for glioblastoma multiforme.

[0057] The truncated EGFRvIII provided herein, referred to as Ev3 (or tEv3), does not comprise an endodomain or functional EGF binding domain. Specifically, the Ev3 extracellular domain may be used as the CAR stalk, excluding the transmembrane and intracellular signaling domain, for a) signal transduction, b) cellular marker for assigning CAR positivity and, c) antigenic marker for cell ablation via antibody binding (e.g., with Cetuximab). In particular aspects, Ev3 comprises truncated domain 1 (L1), truncated domain 2 (CR1), domain 3 (L2), and domain 4 (CR2) of EGFR (as depicted in FIG. 2B). An exemplary sequence of Ev3 is depicted in FIG. 2C. The present Ev3 may comprise a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence in FIG. 2C. The cetuximab binding site (in domain 3) is preserved in both Ev3 and wild-type EGFR extracellular domains. In addition to the cetuximab (and/or panitumumab) binding site, the Ev3 epitope presents additional binding sites for other antibodies (e.g., MR1, mAb 806, and DH8.3)

[0058] The CARs provided herein can use Ev3 as an integral signal transduction hinge which can double as an antigenic safety switch to covalently link the scFv (i.e., the antigen recognition domain) with the signaling domains (i.e., CD28 and CD3.zeta.). The Ev3 also allows for detection of CAR immune cells in vitro and in vivo. In addition, the Ev3 hinge ensures retention of genetic loads (CARs).

[0059] A further aspect of the Ev3 hinge that is provided herein is its ability to be targeted by antibodies, such as MR1, which as specific to EGFRvIII and will not cross-react with domain III and IV of EGFR which is present in other tissues and cells, such as epithelial cells. Cetuximab and panitumumab are clinically available antibodies and FDA-approved which obviates the need to acquire regulatory approval for idiotypic antibodies for each CAR with different antigenic specificities, such as CD19, CD123, CD5, ROR1, CD33, CD99, and mesothelin. The ability to target Ev3-CARs can be used to detect CAR-positive cells and/or controlled cellular ablation. Cellular ablation may be desired in the event of adverse events, such as cytokine release syndrome, neurotoxicity, and/or on-target or off tumor activity. As Ev3 is not normally expressed in immune cells, such as T or NK cells, enforced expression of Ev3 will specifically mark genetically-modified immune cells. Finally, Ev3 does not comprise a signaling endodomain as the cytoplasmic domain is removed) or an EGF binding domain, CARs comprising Ev3 will not respond or crosstalk with EGF signaling pathways (e.g., AR, ARF4, CAV1, CAV3, CBL, CBLB, CBLC, CD44, CDC25A, CRK, CTNNB1, DCN, EGF, GRB14, Grb2, JAK2, MUC1, NCK1, NCK2, PKC.alpha., PLCG1, PLSCR1, PTPN1, PTPN11, PTPN6, PTPRK, SH2D3A, SH3KBP1, SHC1, SOS1, Src, STAT1, STAT3, STAT3, STAT5A, UBC, WAS) either outside or inside the cell. Accordingly, in particular aspects, none of these gene products are expected to interfere with the Ev3 hinge of Ev3-CARS. In certain aspects, the Ev3 may have one, two, three, or more modifications.

[0060] In additional aspects, the function, efficacy, and cellular persistence of Ev3-CAR may be further enhanced by the incorporation of the DNAX-activation protein 12 (DAP12) signaling module, an important element in NK cells activation, in Ev3-DAP12-CARs.

[0061] Further embodiments provide methods of introducing the CARs provided herein to immune cells, such as T cell and NK cells. The CAR-T or CAR-NK cells may be administered to a subject, such as for the treatment of cancer. In particular, the cancers that may be treated by the CAR-T or CAR-NK cells include leukemia and lymphoma, such as chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), and non-hodgkin lymphoma (NHL).

I. Definitions

[0062] As used herein, "essentially free," in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0063] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0064] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more. The terms "about", "substantially" and "approximately" mean, in general, the stated value plus or minus 5%.

[0065] The term "exogenous," when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or in relation to a cell, the term refers to a cell that was isolated and subsequently introduced to other cells or to an organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid that occurs naturally within the organism or cell. An exogenous cell may be from a different organism, or it may be from the same organism. By way of a non-limiting example, an exogenous nucleic acid is one that is in a chromosomal location different from where it would be in natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.

[0066] By "expression construct" or "expression cassette" is meant a nucleic acid molecule that is capable of directing transcription. An expression construct includes, at a minimum, one or more transcriptional control elements (such as promoters, enhancers or a structure functionally equivalent thereof) that direct gene expression in one or more desired cell types, tissues or organs. Additional elements, such as a transcription termination signal, may also be included.

[0067] A "vector" or "construct" (sometimes referred to as a gene delivery system or gene transfer "vehicle") refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo.

[0068] A "plasmid," a common type of a vector, is an extra-chromosomal DNA molecule separate from the chromosomal DNA that is capable of replicating independently of the chromosomal DNA. In certain cases, it is circular and double-stranded.

[0069] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.

[0070] An "immune disorder," "immune-related disorder," or "immune-mediated disorder" refers to a disorder in which the immune response plays a key role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allograft rejection, graft versus host disease and inflammatory and allergic conditions.

[0071] An "immune response" is a response of a cell of the immune system, such as a B cell, or a T cell, or innate immune cell to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response").

[0072] The terms "tumor-associated antigen," "tumor antigen" and "cancer cell antigen" are used interchangeably herein. In each case, the terms refer to proteins, glycoproteins or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0073] An "epitope" is the site on an antigen recognized by an antibody as determined by the specificity of the amino acid sequence. Two antibodies are said to bind to the same epitope if each competitively inhibits (blocks) binding of the other to the antigen as measured in a competitive binding assay. Alternatively, two antibodies have the same epitope if most amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are said to have overlapping epitopes if each partially inhibits binding of the other to the antigen, and/or if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0074] "Treating" or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.

[0075] Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, "treating" or "treatment" may include "preventing" or "prevention" of disease or undesirable condition. In addition, "treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0076] The term "effective," as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. "Effective amount," "Therapeutically effective amount" or "pharmaceutically effective amount" when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease.

[0077] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0078] "Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0079] As used herein, the term "framework region(s)" refers to regions of the variable region of an antibody which act as a scaffold for the CDRs. Thus, the framework regions may comprise the non-CDR sequences of the variable light chain and variable heavy chain. The CDRs of a variable region may be determined by methods known in the art, such as by using the Kabat numbering system as described in Sela-Culang et al., 2013; incorporated herein by reference in its entirety. The system described by Kabat (CITE) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs.

[0080] As generally used herein "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[0081] "Pharmaceutically acceptable salts" means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0082] A "pharmaceutically acceptable carrier," "drug carrier," or simply "carrier" is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and/or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and/or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.

[0083] The term "chimeric antigen receptors (CARs)," as used herein, may refer to artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a tumor associated antigen, for example. In some embodiments, CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor associated antigen binding region. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death. In certain cases, CARs comprise domains for additional co-stimulatory signaling, such as CD3.zeta., FcR, CD27, CD28, CD137, DAP10, and/or OX40. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0084] The term "antigen presenting cells (APCs)" refers to a class of cells capable of presenting one or more antigens in the form of peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. APCs can be intact whole cells such as macrophages, B-cells, endothelial cells, activated T-cells, and dendritic cells; or other molecules, naturally occurring or synthetic, such as purified MHC Class I molecules complexed to .beta.2-microglobulin. While many types of cells may be capable of presenting antigens on their cell surface for T-cell recognition, only dendritic cells have the capacity to present antigens in an efficient amount to activate naive T-cells for cytotoxic T-lymphocyte (CTL) responses.

[0085] The term "culturing" refers to the in vitro maintenance, differentiation, and/or propagation of cells in suitable media. By "enriched" is meant a composition comprising cells present in a greater percentage of total cells than is found in the tissues where they are present in an organism.

[0086] An "anti-cancer" agent is capable of negatively affecting a cancer cell/tumor in a subject, for example, by promoting killing of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[0087] The term "suicide gene" as used herein is defined as a gene which may be used to selectively target cells for killing.

[0088] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" or "homology" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.

[0089] M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.

[0090] The term "truncated" as used herein refers to a fragment of a protein or domain of a protein which is shorter than the full length protein or domain of a protein. The truncation may be by removal of 1 or more amino acids, such as 5, 10, 15, 20, 25, 30 or more amino acids of the full length sequence.

II. Immune Cells

[0091] Certain embodiments of the present disclosure concern immune cells which express a CAR, such as with Ev3 as the hinge of the CAR and/or a humanized scFv as the antigen binding domain of the cAR. The immune cells may be T cells (e.g., regulatory T cells, CD4.sup.+ T cells, CD8.sup.+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, NKT cells, stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotent stem (iPSC) cells). In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils. Also provided herein are methods of producing and engineering the immune cells as well as methods of using and administering the cells for adoptive cell therapy, in which case the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, such as to target cancer cells.

[0092] The immune cells may be isolated from subjects, particularly human subjects. The immune cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject who is undergoing therapy for a particular disease or condition. Immune cells can be collected from any location in which they reside in the subject including, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells may be used directly, or they can be stored for a period of time, such as by freezing.

[0093] The immune cells may be enriched/purified from any tissue where they reside including, but not limited to, blood (including blood collected by blood banks or cord blood banks), spleen, bone marrow, tissues removed and/or exposed during surgical procedures, and tissues obtained via biopsy procedures. Tissues/organs from which the immune cells are enriched, isolated, and/or purified may be isolated from both living and non-living subjects, wherein the non-living subjects are organ donors. In particular embodiments, the immune cells are isolated from blood, such as peripheral blood or cord blood. In some aspects, immune cells isolated from cord blood have enhanced immunomodulation capacity, such as measured by CD4- or CD8-positive T cell suppression. In specific aspects, the immune cells are isolated from pooled blood, particularly pooled cord blood, for enhanced immunomodulation capacity. The pooled blood may be from 2 or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0094] The population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject in need of therapy. Alternatively, the population of immune cells can be obtained from a donor, preferably a histocompatibility matched donor. The immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ/tissue in which immune cells reside in said subject or donor. The immune cells can be isolated from a pool of subjects and/or donors, such as from pooled cord blood.

[0095] When the population of immune cells is obtained from a donor distinct from the subject, the donor is preferably allogeneic, provided the cells obtained are subject-compatible in that they can be introduced into the subject. Allogeneic donor cells are may or may not be human-leukocyte-antigen (HLA)-compatible.

[0096] A. T Cells

[0097] In some embodiments, the immune cells are T cells. Several basic approaches for the derivation, activation and expansion of functional anti-tumor effector cells have been described in the last two decades. These include: autologous cells, such as tumor-infiltrating lymphocytes (TILs); T cells activated ex-vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs) or beads coated with T cell ligands and activating antibodies, or cells isolated by virtue of capturing target cell membrane; allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR); and non-tumor-specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express tumor-reactive TCR or chimeric TCR molecules displaying antibody-like tumor recognition capacity known as "T-bodies". These approaches have given rise to numerous protocols for T cell preparation and immunization which can be used in the methods described herein.

[0098] In some embodiments, the T cells are derived from the blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some aspects, the cells are human cells. The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4.sup.+ cells, CD8.sup.+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.

[0099] Among the sub-types and subpopulations of T cells (e.g., CD4.sup.+ and/or CD8.sup.+ T cells) are naive T (T.sub.N) cells, effector T cells (T.sub.EFF), memory T cells and sub-types thereof, such as stem cell memory T (TSC.sub.M), central memory T (T.sub.CM), effector memory T (T.sub.EM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

[0100] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for a specific marker, such as surface markers, or that are negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells).

[0101] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4.sup.+ or CD8.sup.+ selection step is used to separate CD4.sup.+ helper and CD8.sup.+ cytotoxic T cells. Such CD4.sup.+ and CD8.sup.+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.

[0102] In some embodiments, CD8.sup.+ T cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (T.sub.CM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations.

[0103] In some embodiments, the T cells are autologous T cells. In this method, tumor samples are obtained from patients and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS.TM. Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL-2).

[0104] The cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T-cells of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days.

[0105] Expansion can be accomplished by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. The non-specific T-cell receptor stimulus can include around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil, Raritan, N.J.). Alternatively, T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growth factor, such as 300 IU/ml IL-2 or IL-15, with IL-2 being preferred. The in vitro-induced T-cells are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2.sup.+ allogeneic lymphocytes and IL-2, for example.

[0106] The autologous T cells can be modified to express a T cell growth factor that promotes the growth and activation of the autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable methods of modification are known in the art. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3.sup.rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, N Y, 1994. In particular aspects, modified autologous T cells express the T cell growth factor at high levels. T cell growth factor coding sequences, such as that of IL-12, are readily available in the art, as are promoters, the operable linkage of which to a T cell growth factor coding sequence promote high-level expression.

[0107] B. NK Cells

[0108] In some embodiments, the immune cells are natural killer (NK) cells. NK cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans. NK cells do not express T cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors.

[0109] In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art. Particularly, umbilical CB is used to derive NK cells. In certain aspects, the NK cells are isolated and expanded by the previously described method of ex vivo expansion of NK cells (Spanholtz et al., 2011; Shah et al., 2013). In this method, CB mononuclear cells are isolated by ficoll density gradient centrifugation and cultured in a bioreactor with IL-2 and artificial antigen presenting cells (aAPCs). After 7 days, the cell culture is depleted of any cells expressing CD3 and re-cultured for an additional 7 days. The cells are again CD3-depleted and characterized to determine the percentage of CD56.sup.+/CD3.sup.- cells or NK cells. In other methods, umbilical CB is used to derive NK cells by the isolation of CD34.sup.+ cells and differentiation into CD56.sup.+/CD3.sup.- cells by culturing in medium contain SCF, IL-7, IL-15, and IL-2.

[0110] C. Stem Cells

[0111] In some embodiments, the immune cells of the present disclosure may be stem cells, such as induced pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs).

[0112] The pluripotent stem cells used herein may be induced pluripotent stem (iPS) cells, commonly abbreviated iPS cells or iPSCs. With the exception of germ cells, any cell can be used as a starting point for iPSCs. For example, cell types could be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or stomach cells. There is no limitation on the degree of cell differentiation or the age of an animal from which cells are collected; even undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be used as sources of somatic cells in the methods disclosed herein.

[0113] Somatic cells can be reprogrammed to produce iPS cells using methods known to one of skill in the art. Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from a somatic cell. In some embodiments, at least three, or at least four, of Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3/4, Sox2, c-Myc and Klf4 are utilized or Oct3/4, Sox2, Nanog, and Lin28.

[0114] Once derived, iPSCs can be cultured in a medium sufficient to maintain pluripotency. In certain embodiments, undefined conditions may be used; for example, pluripotent cells may be cultured on fibroblast feeder cells or a medium that has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state. In some embodiments, the cell is cultured in the co-presence of mouse embryonic fibroblasts treated with radiation or an antibiotic to terminate the cell division, as feeder cells. Alternately, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using a defined, feeder-independent culture system, such as a TESR.TM. medium or E8.TM./Essential 8.TM. medium.

III. Enhanced Chimeric Antigen Receptors

[0115] A. Truncated EGFRvIII (Ev3)

[0116] In certain embodiments, the present disclosure provides enhanced CARs which comprise Ev3 as the hinge or stalk region of the CAR by covalently linking the antigen binding region to the intracellular signaling domain(s). The antigen binding region may comprise a single-chain variable fragment (scFv) derived from an anti-antigen binding domain derived from an antibody, such as a mouse anti-human antibody. The scFv can target any potential antigen. The scFv may be humanized, such as in the framework region of the scFv. In particular aspects, the accessibility to the detection or ablation antibodies (e.g., via ADCC) is maintained.

[0117] The Ev3 may comprise or have at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2 LEEKK-GNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRK-VCNGIGIGEFKDSLSINATNIKHFKNC- TSISGDLHILPVAFRGDSFTHTPPLDPQELDIL KTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLK EISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQ-VCHALCSPEGCWGPEPRDCVSC- RNVSRGRECVDKCNLLEGEPREFVENSECIQCHPE CLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHV CHLCHPNCTYGCTGPGLEGCPTNGPKIPS (comprising partial Domain 1-partial Domain 2-Domain 3-Domain 4). In certain aspects, the Ev3 may have one, two, three, or more modifications.

[0118] In one embodiment, there is provided a CD19Ev3-CAR comprising from N-terminal to C-terminal a scFv derived from a mouse anti-human CD19 monoclonal antibody, a Ev3 hinge including the transmembrane domain, a CD28 endodomain, and a CD3.zeta. endodomain. In another embodiment, the CD19Ev3-CAR comprises a humanized CD19 (hCD19) referred to as hCD19Ev3-CAR. The CAR may further comprise DAP12 endodomain, such as between the Ev3 transmembrane domain and the CD3.zeta. endodomain, such a CD19Ev3DAP12-CAR or hCD19Ev3DAP12-CAR.

[0119] In one embodiment, there is provided a CD123Ev3-CAR comprising from N-terminal to C-terminal a scFv derived from a mouse anti-human CD123 monoclonal antibody, a Ev3 hinge including the transmembrane domain, a CD28 endodomain, and a CD3.zeta. endodomain. In another embodiment, the CD123Ev3-CAR comprises a humanized CD123 (hCD123) referred to as hCD123Ev3-CAR. The CAR may further comprise DAP12 endodomain, such as between the Ev3 transmembrane domain and the CD3.zeta. endodomain, such a CD123Ev3DAP12-CAR or hCD123Ev3DAP12-CAR.

[0120] In one embodiment, there is provided a MesoEv3-CAR comprising from N-terminal to C-terminal a scFv derived from a mouse anti-human mesothelin monoclonal antibody, a Ev3 hinge including the transmembrane domain, a CD28 endodomain, and a CD3.zeta. endodomain. In another embodiment, the MesoEv3-CAR comprises a humanized Meso (hMeso) referred to as hMesoEv3-CAR. The CAR may further comprise DAP12 endodomain, such as between the Ev3 transmembrane domain and the CD3.zeta. endodomain, such a MesoEv3DAP12-CAR or hMesoEv3DAP12-CAR.

[0121] In one embodiment, there is provided a ROR1-Ev3-CAR comprising from N-terminal to C-terminal a scFv derived from a mouse anti-human ROR1 monoclonal antibody, a Ev3 hinge including the transmembrane domain, a CD28 endodomain, and a CD3.zeta. endodomain. In another embodiment, the ROR1-Ev3-CAR comprises a humanized ROR1 (hROR1) referred to as hROR1Ev3-CAR. The CAR may further comprise DAP12 endodomain, such as between the Ev3 transmembrane domain and the CD3.zeta. endodomain, such a ROR1-Ev3DAP12-CAR or hROR1-Ev3DAP12-CAR.

[0122] In one embodiment, there is provided a CD5Ev3-CAR comprising from N-terminal to C-terminal a scFv derived from a mouse anti-human CD5 monoclonal antibody, a Ev3 hinge including the transmembrane domain, a CD28 endodomain, and a CD3.zeta. endodomain. In another embodiment, the CD5Ev3-CAR comprises a humanized CD5 (hCD5) referred to as hCD5Ev3-CAR. The CAR may further comprise DAP12 endodomain, such as between the Ev3 transmembrane domain and the CD3.zeta. endodomain, such a CD5Ev3DAP12-CAR or hCD5Ev3DAP12-CAR.

[0123] In one embodiment, there is provided a CD99Ev3-CAR comprising from N-terminal to C-terminal a scFv derived from a mouse anti-human CD99 monoclonal antibody, a Ev3 hinge including the transmembrane domain, a CD28 endodomain, and a CD3.zeta. endodomain. In another embodiment, the CD5Ev3-CAR comprises a humanized CD99 (hCD99) referred to as hCD99Ev3-CAR. The CAR may further comprise DAP12 endodomain, such as between the Ev3 transmembrane domain and the CD3.zeta. endodomain, such a CD99Ev3DAP12-CAR or hCD99Ev3DAP12-CAR.

[0124] EGFRvIII is the result of a mutation of exons 2-7 of the EGFR gene which renders the mutant receptor incapable of binding any known ligand as the receptor does not have a complete ligand binding domain. The truncated EGFRvIII, Ev3, of the present disclosure further comprises a deletion of the intracellular domain or endodomain of EGFRvIII. Thus, Ev3 does not comprise a functional EGF binding domain or the intracellular signaling domain of EGFRvIII.

[0125] The Ev3 hinge may comprise a nucleotide sequence of SEQ ID NO:1 and/or an amino acid sequence of SEQ ID NO:2. In particular embodiments, the Ev3 hinge has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:1 or SEQ ID NO:2. The Ev3 hinge region may comprise a fragment of SEQ ID NO:2 or may comprise additional amino acids on the N-terminal or C-terminal end of the receptor fragment. An exemplary schematic of Ev3 is depicted in FIG. 2.

[0126] The inclusion of the truncated EGFRvIII in the hinge region of a CAR allows for retention of genetic cargo load. The Ev3 can also be used a safety switch and/or for detection of the CAR in vitro or in vivo. The integral Ev4 hinge can be recognized by antibodies, such as clinically accessible monoclonal antibodies (e.g., cetuximab), for detection or cellular ablation. In addition, the Ev3 does not comprise a ligand binding domain; thus, there is no endogenous function or crosstalk with other pathways. The Ev3 is also plays a role in the CAR signal transduction and is tumor specific which can minimize side-effects of CAR therapies.

[0127] B. Antigens

[0128] The CARs provided herein may have any antigenic specificity useful in the treatment of a disease or disorder. The CARs provided herein may comprise more than one antigen, such as two antigens. Exemplary antigens include, but are not limited to, CD19, CD123, mesothelin, CD5, CD47, CLL-1, CD33, CD99, CLL-1, U5snRNP200, CD200, CS1, BAFF-R, ROR-1, CD99, Mesothelin, or BCMA.

[0129] The humanization platform provided herein may be used for the development of a humanized CAR for any given antigen. Exemplary antigens with humanized CARs include, but are not limited to, CD19, CD123, mesothelin, CD5, CD47, CLL-1, CD33, CD99, U5snRNP200, CD200, CS1, BAFF-R, ROR-1, or BCMA.

[0130] In one embodiment, the encoded light chain variable region comprises one, two, three or all four framework regions of the human PH0879 germline sequence. The encoded heavy chain variable region of hCD19 may comprise one, two, three or all four framework regions of the human ACD43442 germline sequence.

[0131] In one embodiment, the encoded antigen binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence of SEQ ID NO:6, 12, 18, 24, 30, 58 or 61. In one embodiment, the antigen binding domain (e.g., an scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 10, 20 or 30 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 90-99% identity with an amino acid sequence of SEQ ID NOs:7, 13, 19, 25, 31, 59 or 63; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 10, 20 or 30 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region of SEQ ID NOs:8, 14, 20, 26, 32, 60 or 64; or a sequence with 90-99% identity to an amino acid sequence of SEQ ID NOs:6, 12, 18, 24, 30, 58 or 61.

[0132] In some embodiments, a humanized scFv for CD19 is provided herein. The humanized CD19 (hCD19) scFv may have a variable light chain sequence of SEQ ID NO:7 (DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKVPKLLIYHTSRLHS GVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPYTFGQGTKVEIKR) and a variable heavy chain of SEQ ID NO:8 (EVQLVESGGGLVQPGGSLRLSCAASGVSLPDYGVSWVRQAPGKGLEWIGVIWGSE TTYYNSALKSKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARHYYYGGSYAMDY WGQGTLVTVSS). In other aspects, the hCD19 scFv may comprise a V.sub.L with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7 and/or a V.sub.H with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8. The hCD19 scFv may comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6. The hCD19 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3. The V.sub.L of the hCD19 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4. The V.sub.H of the hCD19 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5.

[0133] In some embodiments, a humanized scFv for CD123 is provided herein. The humanized CD123 (hCD123) scFv may have a variable light chain sequence of SEQ ID NO:13 and a variable heavy chain of SEQ ID NO:14. In other aspects, the hCD123 scFv may comprise a V.sub.L with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:13 and/or a V.sub.H with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:14. The hCD123 scFv may comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:12. The hCD123 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9. The V.sub.L of the hCD123 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:10. The V.sub.H of the hCD123 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:11.

[0134] In some embodiments, a humanized scFv for mesothelin is provided herein. The humanized mesothelin (hMeso) scFv may have a variable light chain sequence of SEQ ID NO:19 and a variable heavy chain of SEQ ID NO:20. In other aspects, the hMeso scFv may comprise a V.sub.L with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:19 and/or a V.sub.H with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20. The hMeso scFv may comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:18. The hMeso scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:15. The V.sub.L of the hMeso scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:16. The V.sub.H of the hMeso scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:17.

[0135] In some embodiments, a humanized scFv for ROR1 is provided herein. The humanized ROR1 (hROR1) scFv may have a variable light chain sequence of SEQ ID NO:25 and a variable heavy chain of SEQ ID NO:26. In other aspects, the hROR1 scFv may comprise a V.sub.L with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25 and/or a V.sub.H with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:26. The hROR1 scFv may comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:24. The hROR1 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21. The V.sub.L of the hROR1 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22. The V.sub.H of the hROR1 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:23.

[0136] In some embodiments, a humanized scFv for CD5 is provided herein. The humanized CD5 (hCD5) scFv may have a variable light chain sequence of SEQ ID NO:31 and a variable heavy chain of SEQ ID NO:32. In other aspects, the hCD5 scFv may comprise a V.sub.L with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:31 and/or a V.sub.H with at least 80%, 85%,90%, 91%, 92%,93%, 94%, 95%,96%,97%, 98%, or 99% sequence identity to SEQ ID NO:32. The hCD5 scFv may comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30. The hCD5 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:27. The V.sub.L of the hCD5 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:28. The V.sub.H of the hCD5 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:29.

[0137] In some embodiments, a humanized scFv for CD99 is provided herein. The humanized CD99 (hCD99) scFv may have a variable light chain sequence of SEQ ID NO:63 and a variable heavy chain of SEQ ID NO:64. In other aspects, the hCD99 scFv may comprise a V.sub.L with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:63 and/or a V.sub.H with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:64. The hCD99 scFv may comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:61. The hCD99 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to SEQ ID NO:61.

[0138] In some embodiments, a humanized scFv for CD99 is provided herein. The humanized CD99 (hCD99) scFv may have a variable light chain sequence of SEQ ID NO:59 and a variable heavy chain of SEQ ID NO:60. In other aspects, the hCD99 scFv may comprise a V.sub.L with at least 80%, 85%, 90%, 91%, 921%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:59 and/or a V.sub.H with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:60. The hCD99 scFv may comprise an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:58. The hCD99 scFv may be encoded by a polynucleotide with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:57.

[0139] Among the antigens targeted by the genetically engineered antigen receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues, antigens associated with autoimmune or alloimmune disorders, or pathogen-specific antigens. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.

[0140] Any suitable antigen may find use in the present method. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, auto-/self-antigens, tumor-/cancer-associated antigens, and tumor neoantigens. In particular aspects, the antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL/DR4, and CEA.

[0141] Tumor-associated antigens may be derived from prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, or melanoma cancers. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE 1, 3, and MAGE 4; PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types such as melanoma, lung carcinoma, sarcoma, and bladder carcinoma. Prostate cancer tumor-associated antigens include, for example, prostate specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphates, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

[0142] Other tumor associated antigens include Plu-1, HASH-1, HasH-2, Cripto and Criptin. Additionally, a tumor antigen may be a self peptide hormone, such as whole length gonadotrophin hormone releasing hormone, a short 10 amino acid long peptide, useful in the treatment of many cancers.

[0143] Tumor antigens include tumor antigens derived from cancers that are characterized by tumor-associated antigen expression, such as HER-2/neu expression. Tumor-associated antigens of interest include lineage-specific tumor antigens such as the melanocyte-melanoma lineage antigens MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-related protein. Illustrative tumor-associated antigens include, but are not limited to, tumor antigens derived from or comprising any one or more of, p53, Ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms' tumor antigen (WT), AFP, -catenin/m, Caspase-8/m, CEA, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notch1-4), c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1 and idiotype.

[0144] Antigens may include epitopic regions or epitopic peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutated ras, bcr/abl rearrangement, Her2/neu, mutated or wild-type p53, cytochrome P450 1B1, and abnormally expressed intron sequences such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes generating unique idiotypes in myeloma and B-cell lymphomas; tumor antigens that include epitopic regions or epitopic peptides derived from oncoviral processes, such as human papilloma virus proteins E6 and E7; Epstein bar virus protein LMP2; nonmutated oncofetal proteins with a tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0145] In other embodiments, an antigen is obtained or derived from a pathogenic microorganism or from an opportunistic pathogenic microorganism (also called herein an infectious disease microorganism), such as a virus, fungus, parasite, and bacterium. In certain embodiments, antigens derived from such a microorganism include full-length proteins.

[0146] Illustrative pathogenic organisms whose antigens are contemplated for use in the method described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Influenza A, B, and C, vesicular stomatitis virus (VSV), vesicular stomatitis virus (VSV), polyomavirus (e.g., BK virus and JC virus), adenovirus, Staphylococcus species including Methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species including Streptococcus pneumoniae. As would be understood by the skilled person, proteins derived from these and other pathogenic microorganisms for use as antigen as described herein and nucleotide sequences encoding the proteins may be identified in publications and in public databases such as GENBANK.RTM., Swiss-Prot.RTM., and TrEMBL.RTM..

[0147] Antigens derived from human immunodeficiency virus (HIV) include any of the HIV virion structural proteins (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or HIV proteins encoded by tat, rev, nef, vif, vpr and vpu.

[0148] Antigens derived from herpes simplex virus (e.g., HSV 1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late group of genes predominantly encodes proteins that form the virion particle. Such proteins include the five proteins from (UL) which form the viral capsid: UL6, UL18, UL35, UL38 and the major capsid protein UL19, UL45, and UL27, each of which may be used as an antigen as described herein. Other illustrative HSV proteins contemplated for use as antigens herein include the ICP27 (H1, H2), glycoprotein B (gB) and glycoprotein D (gD) proteins. The HSV genome comprises at least 74 genes, each encoding a protein that could potentially be used as an antigen.

[0149] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed during the immediate early and early phases of virus replication, glycoproteins I and III, capsid protein, coat protein, lower matrix protein pp65 (ppUL83), p52 (ppUL44), 1E1 and 1E2 (UL123 and UL122), protein products from the cluster of genes from UL128-UL150 (Rykman, et al., 2006), envelope glycoprotein B (gB), gH, gN, and pp150. As would be understood by the skilled person, CMV proteins for use as antigens described herein may be identified in public databases such as GenBank.RTM., Swiss-Prot.RTM., and TrEMBL.RTM..

[0150] Antigens derived from Epstein-Ban virus (EBV) that are contemplated for use in certain embodiments include EBV lytic proteins gp350 and gp110, EBV proteins produced during latent cycle infection including Epstein-Ban nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP) and latent membrane proteins (LMP)-1, LMP-2A and LMP-2B.

[0151] Antigens derived from respiratory syncytial virus (RSV) that are contemplated for use herein include any of the eleven proteins encoded by the RSV genome, or antigenic fragments thereof: NS 1, NS2, N (nucleocapsid protein), M (Matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcription regulation), RNA polymerase, and phosphoprotein P.

[0152] Antigens derived from Vesicular stomatitis virus (VSV) that are contemplated for use include any one of the five major proteins encoded by the VSV genome, and antigenic fragments thereof: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M).

[0153] Antigens derived from an influenza virus that are contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0154] Exemplary viral antigens also include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., a calicivirus capsid antigen), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (a hepatitis B core or surface antigen, a hepatitis C virus E1 or E2 glycoproteins, core, or non-structural proteins), herpesvirus polypeptides (including a herpes simplex virus or varicella zoster virus glycoprotein), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides, papilloma virus polypeptides, parainfluenza virus polypeptides (e.g., the hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picorna virus polypeptides (e.g., a poliovirus capsid polypeptide), pox virus polypeptides (e.g., a vaccinia virus polypeptide), rabies virus polypeptides (e.g., a rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

[0155] In certain embodiments, the antigen may be bacterial antigens. In certain embodiments, a bacterial antigen of interest may be a secreted polypeptide. In other certain embodiments, bacterial antigens include antigens that have a portion or portions of the polypeptide exposed on the outer cell surface of the bacteria.

[0156] Antigens derived from Staphylococcus species including Methicillin-resistant Staphylococcus aureus (MRSA) that are contemplated for use include virulence regulators, such as the Agr system, Sar and Sae, the Arl system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), the Srr system and TRAP. Other Staphylococcus proteins that may serve as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes for two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As would be understood by the skilled person, Staphylococcus proteins for use as antigens may also be identified in other public databases such as GenBank.RTM., Swiss-Prot.RTM., and TrEMBL.RTM..

[0157] Antigens derived from Streptococcus pneumoniae that are contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as an antigen in some embodiments. The complete genome sequence of a virulent strain of Streptococcus pneumoniae has been sequenced and, as would be understood by the skilled person, S. pneumoniae proteins for use herein may also be identified in other public databases such as GenBank.RTM., Swiss-Prot.RTM., and TrEMBL.RTM.. Proteins of particular interest for antigens according to the present disclosure include virulence factors and proteins predicted to be exposed at the surface of the pneumococci.

[0158] Examples of bacterial antigens that may be used as antigens include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae type b outer membrane protein), Helicobacter polypeptides, Klebsiella polypeptides, L-form bacteria polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., S. pneumoniae polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, group A streptococcus polypeptides (e.g., S. pyogenes M proteins), group B streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y. pestis F1 and V antigens).

[0159] Examples of fungal antigens include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

[0160] Examples of protozoan parasite antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides. Examples of helminth parasite antigens include, but are not limited to, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necator polypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, Physaloptera polypeptides, Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides. (e.g., P. falciparum circumsporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSA1 c-term), and exported protein 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

[0161] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens as well as allergens) from fleas; ticks, including hard ticks and soft ticks; flies, such as midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs.

[0162] C. Chimeric Antigen Receptors

[0163] In some embodiments, the CAR contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

[0164] In some embodiments, the chimeric antigen receptor comprises: a) an intracellular signaling domain, b) a hinge and transmembrane domain, such as a Ev3 hinge, and c) an extracellular domain comprising an antigen binding region.

[0165] In some embodiments, the engineered antigen receptors include chimeric antigen receptors (CARs), including activating or stimulatory CARs, costimulatory CARs (see WO2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., 2013). The CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.

[0166] Certain embodiments of the present disclosure concern the use of nucleic acids, including nucleic acids encoding an antigen-specific CAR polypeptide, including a CAR that has been humanized to reduce immunogenicity (hCAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope comprising the shared space between one or more antigens. In certain embodiments, the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen binding fragments thereof. In another embodiment, that specificity is derived from a peptide (e.g., cytokine) that binds to a receptor.

[0167] It is contemplated that the human CAR nucleic acids may be human genes used to enhance cellular immunotherapy for human patients. In a specific embodiment, the invention includes a full-length CAR cDNA or coding region. The antigen binding regions or domain can comprise a fragment of the V.sub.H and V.sub.L chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Pat. No. 7,109,304, incorporated herein by reference. The fragment can also be any number of different antigen binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.

[0168] The arrangement could be multimeric, such as a diabody or multimers. The multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into a diabody. The hinge portion of the construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine. The Fc portion can be deleted. Any protein that is stable and/or dimerizes can serve this purpose. One could use just one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin. One could also use the hinge, CH2 and CH3 region of a human immunoglobulin that has been modified to improve dimerization. One could also use just the hinge portion of an immunoglobulin. One could also use portions of CD8alpha.

[0169] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137). In addition to a primary signal initiated by CD3.zeta., an additional signal provided by a human costimulatory receptor inserted in a human CAR is important for full activation of NK cells and could help improve in vivo persistence and the therapeutic success of the adoptive immunotherapy. The CAR may express one or more cytokines, such as IL-15. The inclusion of one or more cytokines may promote in vivo persistence. In some aspects, the CAR comprises the antigen binding domain, such as humanized scFv, CD3.zeta., DAP12, and IL-15, optionally in combination with the Ev3 hinge.

[0170] In some embodiments, CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and/or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (V.sub.H) and variable light (V.sub.L) chains of a monoclonal antibody (mAb).

[0171] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as an extracellular domain comprising an antigen binding region) comprises a tumor associated antigen or a pathogen-specific antigen binding domain. Antigens include carbohydrate antigens recognized by pattern-recognition receptors, such as Dectin-1. A tumor associated antigen may be of any kind so long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor associated antigens include CD19, CD319 (CS1), CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, and so forth. In certain embodiments, the CAR may be co-expressed with a cytokine to improve persistence when there is a low amount of tumor-associated antigen. For example, CAR may be co-expressed with IL-15.

[0172] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0173] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.

[0174] Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use in accordance with the method of the present disclosure are non-replicating in the immune cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[0175] In some aspects, the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0176] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D, and DAP molecules. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0177] In certain embodiments, the platform technologies disclosed herein to genetically modify immune cells, such as NK cells, comprise (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector), (ii) CARs that signal through endodomains (e.g., CD28/CD3-.zeta., CD137/CD3-.zeta., or other combinations), (iii) CARs with variable lengths of extracellular domains connecting the antigen-recognition domain to the cell surface, and, in some cases, (iv) artificial antigen presenting cells (aAPC) derived from K562 to be able to robustly and numerically expand CAR.sup.+ immune cells (Singh et al., 2008; Singh et al., 2011).

[0178] In some embodiments, the CAR may be retrovirally tranduced in the cells. The retroviral vector may be the pSFG4 vector. The retroviral transfer vector pSFG4 comprises a backbone based on pUC19 plasmid (large fragment [2.63 kb] in between HindIII and EcoRI restriction enzyme sites) carrying viral components from Moloney Murine Leukemia Virus (MoMLV) including 5' LTR, psi packaging sequence, and 3' LTR. LTRs are long terminal repeats found on either side of a retroviral provirus, and in the case of transfer vector, brackets the genetic cargo of interest (in our case mainly CARs and associated genetic components). The psi packaging sequence, which is a target site for packaging by nucleocapsid, is also incorporated in cis, sandwiched between the 5' LTR and the CAR coding sequence. Thus, the basic structure of the pSFG4 transfer vector can be outlined as such: pUC19 sequence--5' LTR--psi packaging sequence--genetic cargo of interest--3' LTR--pUC19 sequence.

[0179] D. Antigen-Presenting Cells

[0180] Antigen-presenting cells, which include macrophages, B lymphocytes, and dendritic cells, are distinguished by their expression of a particular MHC molecule. APCs internalize antigen and re-express a part of that antigen, together with the MHC molecule on their outer cell membrane. The major histocompatibility complex (MHC) is a large genetic complex with multiple loci. The MHC loci encode two major classes of MHC membrane molecules, referred to as class I and class II MHCs. T helper lymphocytes generally recognize antigen associated with MHC class II molecules, and T cytotoxic lymphocytes recognize antigen associated with MHC class I molecules. In humans the MHC is referred to as the HLA complex and in mice the H-2 complex.

[0181] aAPC systems may comprise at least one exogenous assisting molecule. Any suitable number and combination of assisting molecules may be employed. The assisting molecule may be selected from assisting molecules such as co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD86, CD64 (Fc.gamma.RI), 41BB ligand, and IL-21. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which promote, for example, cell-to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selection, cloning, preparation, and expression of exemplary assisting molecules, including co-stimulatory molecules and adhesion molecules.

IV. Methods of Use

[0182] In some embodiments, the present disclosure provides methods for immunotherapy comprising administering an effective amount of the immune cells expressing CARs (e.g., CARs with Ev3 hinge and/or humanized scFv) of the present disclosure. In one embodiments, a medical disease or disorder is treated by transfer of an immune cell population that elicits an immune response. In certain embodiments of the present disclosure, cancer or infection is treated by transfer of an immune cell population that elicits an immune response. Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount an antigen-specific cell therapy. The present methods may be applied for the treatment of immune disorders, solid cancers, hematologic cancers, and viral infections.

[0183] In particular embodiments, methods are provided for treating cancer patients by administering immune cells, such as NK cells and/or T cells, expressing CARs provided herein with an antigen-binding domain specific for the antigen expressed by said cancer. For example, a subject with chronic lymphocytic leukemia (CLL), B cell acute lymphoblastic leukemia (ALL), or non-Hodgkin lymphoma (NHL) may be treated with CD19-Ev3-CAR immune cells to ablate CD19-positive cells such as cancer stem cells. In other aspects, the subject may have acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), or plasmacytoid dendritic cell leukemia (PDCL) and be treated with CD123-Ev3-CAR immune cells. In additional aspects, the subject may have T-cell leukemia and lymphoma and be treated with CD5-Ev3-CAR immune cells. Subjects with CLL, breast cancer, pancreatic cancer or lung cancer may be treated with ROR1-Ev3-CAR immune cells. Patients with cervical, endometrial or ovarian cancer may be treated with Meso-Ev3-CAR immune cells.

[0184] Subjects with multiple myeloma (MM) or other CD319-expressing cancers may be treated with CD319-CAR immune cells. In some aspects, patient with amyloidosis are treated by ablating CD319-positive cells, such as cancer stem cells. In some aspects, patients with monoclonal gammopathy of unknown significance (MGUS) or smoldering myeloma are treated with CD319-CAR immune cells. Subjects with CD319-positive cells in autoimmune disorders, such as rheumatoid arthritis, SLE, or autoimmune hemolytic anemia may be treated by immune cells of the present embodiments. In particular aspects, a CD319-expressing cancer is treated by administering immune cells expressing Ev3-CAR with CD319 scFv, CD3.zeta., DAP12, and IL-15.

[0185] In some embodiments, cells comprising the Ev3 CARs provided herein may be ablated and/or detected by anti-EGFR antibodies. Exemplary anti-EGFR antibodies include, but are not limited to, cetuximab (Erbitux) and panitumumab (Vectibix) as well as biosimilars. Both of these antibodies have been approved by the FDA for treatment of colorectal cancer. In some embodiments, the detection of cells expressing Ev3-CARs comprises the administration of an anti-EGFR antibody with a detectable tag. In other aspects, the ablation of cells expressing Ev3-CARs comprises the administration of an anti-EGFR antibody which induces ADCC. In some aspects, the anti-EGFR antibody may be fused to a pro-drug or cytotoxic agent, such as a toxin.

[0186] Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0187] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0188] Particular embodiments concern methods of treatment of leukemia. Leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is part of the broad group of diseases called hematological neoplasms. Leukemia is a broad term covering a spectrum of diseases. Leukemia is clinically and pathologically split into its acute and chronic forms.

[0189] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof, such as an individual that has cancer or an infection. The cells then enhance the individual's immune system to attack the respective cancer or pathogenic cells. In some cases, the individual is provided with one or more doses of the immune cells. In cases where the individual is provided with two or more doses of the immune cells, the duration between the administrations should be sufficient to allow time for propagation in the individual, and in specific embodiments the duration between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0190] Certain embodiments of the present disclosure provide methods for treating or preventing an immune-mediated disorder. In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac spate-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (such as minimal change disease, focal glomerulosclerosis, or mebranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitides (such as polyarteritis nodosa, takayasu arteritis, temporal arteritis/giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Thus, some examples of an autoimmune disease that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosis, type I diabetes mellitus, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subject can also have an allergic disorder such as Asthma.

[0191] In yet another embodiment, the subject is the recipient of a transplanted organ or stem cells and immune cells are used to prevent and/or treat rejection. In particular embodiments, the subject has or is at risk of developing graft versus host disease. GVHD is a possible complication of any transplant that uses or contains stem cells from either a related or an unrelated donor. There are two kinds of GVHD, acute and chronic. Acute GVHD appears within the first three months following transplantation. Signs of acute GVHD include a reddish skin rash on the hands and feet that may spread and become more severe, with peeling or blistering skin. Acute GVHD can also affect the stomach and intestines, in which case cramping, nausea, and diarrhea are present. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD has affected the liver. Chronic GVHD is ranked based on its severity: stage/grade 1 is mild; stage/grade 4 is severe. Chronic GVHD develops three months or later following transplantation. The symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD may also affect the mucous glands in the eyes, salivary glands in the mouth, and glands that lubricate the stomach lining and intestines. Any of the populations of immune cells disclosed herein can be utilized. Examples of a transplanted organ include a solid organ transplant, such as kidney, liver, skin, pancreas, lung and/or heart, or a cellular transplant such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The transplant can be a composite transplant, such as tissues of the face. Immune cells can be administered prior to transplantation, concurrently with transplantation, or following transplantation. In some embodiments, the immune cells are administered prior to the transplant, such as at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to the transplant. In one specific, non-limiting example, administration of the therapeutically effective amount of immune cells occurs 3-5 days prior to transplantation.

[0192] In some embodiments, the subject can be administered nonmyeloablative lymphodepleting chemotherapy prior to the immune cell therapy. The nonmyeloablative lymphodepleting chemotherapy can be any suitable such therapy, which can be administered by any suitable route. The nonmyeloablative lymphodepleting chemotherapy can comprise, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which can be metastatic. An exemplary route of administering cyclophosphamide and fludarabine is intravenously. Likewise, any suitable dose of cyclophosphamide and fludarabine can be administered. In particular aspects, around 60 mg/kg of cyclophosphamide is administered for two days after which around 25 mg/m.sup.2 fludarabine is administered for five days.

[0193] In certain embodiments, a growth factor that promotes the growth and activation of the immune cells is administered to the subject either concomitantly with the immune cells or subsequently to the immune cells. The immune cell growth factor can be any suitable growth factor that promotes the growth and activation of the immune cells. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.

[0194] Therapeutically effective amounts of immune cells can be administered by a number of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection, or infusion.

[0195] The therapeutically effective amount of immune cells for use in adoptive cell therapy is that amount that achieves a desired effect in a subject being treated. For instance, this can be the amount of immune cells necessary to inhibit advancement, or to cause regression of an autoimmune or alloimmune disease, or which is capable of relieving symptoms caused by an autoimmune disease, such as pain and inflammation. It can be the amount necessary to relieve symptoms associated with inflammation, such as pain, edema and elevated temperature. It can also be the amount necessary to diminish or prevent rejection of a transplanted organ.

[0196] The immune cell population can be administered in treatment regimens consistent with the disease, for example a single or a few doses over one to several days to ameliorate a disease state or periodic doses over an extended time to inhibit disease progression and prevent disease recurrence. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. The therapeutically effective amount of immune cells will be dependent on the subject being treated, the severity and type of the affliction, and the manner of administration. In some embodiments, doses that could be used in the treatment of human subjects range from at least 3.8.times.10.sup.4, at least 3.8.times.10.sup.5, at least 3.8.times.10.sup.6, at least 3.8.times.10.sup.7, at least 3.8.times.10.sup.8, at least 3.8.times.10.sup.9, or at least 3.8.times.10.sup.10 immune cells/m.sup.2. In a certain embodiment, the dose used in the treatment of human subjects ranges from about 3.8.times.10.sup.9 to about 3.8.times.10.sup.10 immune cells/m.sup.2. In additional embodiments, a therapeutically effective amount of immune cells can vary from about 5.times.10.sup.6 cells per kg body weight to about 7.5.times.10.sup.8 cells per kg body weight, such as about 2.times.10.sup.7 cells to about 5.times.10.sup.8 cells per kg body weight, or about 5.times.10.sup.7 cells to about 2.times.10.sup.8 cells per kg body weight. The exact amount of immune cells is readily determined by one of skill in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0197] The immune cells may be administered in combination with one or more other therapeutic agents for the treatment of the immune-mediated disorder. Combination therapies can include, but are not limited to, one or more anti-microbial agents (for example, antibiotics, anti-viral agents and anti-fungal agents), anti-tumor agents (for example, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune-depleting agents (for example, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (for example, azathioprine, or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (for example, glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non-steroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen or naproxen sodium), cytokines (for example, interleukin-10 or transforming growth factor-beta), hormones (for example, estrogen), or a vaccine. In addition, immunosuppressive or tolerogenic agents including but not limited to calcineurin inhibitors (e.g., cyclosporin and tacrolimus); mTOR inhibitors (e.g., Rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., Methotrexate, Treosulfan, Busulfan); irradiation; or chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) can be administered. Such additional pharmaceutical agents can be administered before, during, or after administration of the immune cells, depending on the desired effect. This administration of the cells and the agent can be by the same route or by different routes, and either at the same site or at a different site.

[0198] B. Pharmaceutical Compositions

[0199] Also provided herein are pharmaceutical compositions and formulations comprising immune cells (e.g., T cells or NK cells) and a pharmaceutically acceptable carrier.

[0200] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22.sup.nd edition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[0201] C. Combination Therapies

[0202] In certain embodiments, the compositions and methods of the present embodiments involve an immune cell population in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0203] In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent. The additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0204] An immune cell therapy may be administered before, during, after, or in various combinations relative to an additional cancer therapy, such as immune checkpoint therapy. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the immune cell therapy is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0205] Various combinations may be employed. For the example below a CAR immune cell therapy is "A" and an anti-cancer therapy is "B":

TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

[0206] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.

[0207] 1. Chemotherapy

[0208] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0209] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and calicheamicin omegaI1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0210] 2. Radiotherapy

[0211] Other factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation, and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0212] 3. Immunotherapy

[0213] The skilled artisan will understand that additional immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells

[0214] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs and may be used in combination therapies. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. Exemplary ADC drugs include ADCETRIS.RTM. (brentuximab vedotin) and KADCYLA.RTM. (trastuzumab emtansine or T-DM1).

[0215] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0216] Examples of immunotherapies include immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds); cytokine therapy, e.g., interferons .alpha., .beta., and .gamma., IL-1, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-1, IL-2, and p53; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.

[0217] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.

[0218] The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies. Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present disclosure. For example it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

[0219] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0220] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO.RTM., is an anti-PD-1 antibody that may be used. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA.RTM., and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

[0221] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0222] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0223] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy.RTM.) or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0224] 4. Surgery

[0225] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).

[0226] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

[0227] 5. Other Agents

[0228] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.

V. Articles of Manufacture or Kits

[0229] An article of manufacture or a kit is provided comprising immune cells is also provided herein. The article of manufacture or kit can further comprise a package insert comprising instructions for using the immune cells to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer. Any of the antigen-specific immune cells described herein may be included in the article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.

VI. Examples

[0230] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1--Development of Truncated EGFRvIII (Ev3) CAR

[0231] An Ev3-CAR was designed in which the Ev3 stalk serves as an integrative structure to transduce signals from antigen binding, such as by scFv, as well as an "off" switch, meaning it can be recognized by clinically accessible antibodies such as cetuximab to ablate CAR-positive immune cells (FIG. 1A). The Ev3-CAR may further comprise DAP12 signaling domain and is designated Ev3-DAP12-CAR. A schematic depicting Ev3 as compared to EGFR which comprise the endodomain and the EGF binding domain is shown in FIG. 2.

[0232] Retroviral vectors encoding the Ev3-CAR or Ev3-DAP12-CAR with various antigen binding domains were generated as depicted in FIGS. 3A-3D. The antigen-binding domain was both the scFv derived from the mouse anti-human antibody as well as a humanized scFv. The vectors comprise CD28 and CD3.zeta. signaling domains and, optionally, DAP12. The vectors also comprise a suicide gene, inducible caspase 9.

[0233] The CD19-specific CARs including CAR19.Ev3.CD28.CD3.zeta., CAR19.Ev3.DAP12.CD3.zeta. or humanized CAR19.Ev3.CD3.zeta. were transduced into NK cells (FIG. 4A). The CAR-NK cells were tested for their efficacy at killing CD19.sup.+ Raji or K562 cells (FIG. 4B). The CAR19-Ev3-CD28-DAP12-CAR NK cells were observed to have the highest cytoxicity. Thus, the use of Ev3 in the hinge region of the CAR in combination with DAP12 endodomain produces CARs that are highly cytotoxic against target cells.

Example 2--Development of Humanized CARs

[0234] A platform was developed to generate humanized scFvs for use in producing humanized CARs. Due to structural constraints, some residues contribute more to antigen binding, thus 3-D modeling was used to identify potentially critical interactions to generate the humanized CARs. The stepwise process detailed below may be repeated iteratively to improve on previous cycles.

[0235] The CAR humanization workflow provided herein comprises 5 basic process modules as detailed below and depicted in FIG. 5A.

[0236] Module I comprises monoclonal antibody (mAb) sequence analysis. The BLAST program was harnessed in search of immunoglobulin (IG) sequences for the best match against the starting queries, such as the murine mAb sequences (FIG. 5B). BLAST can analyze nucleotide and amino acid sequences, either in series (one-by-one) or in parallel (in batches), as well as search against germline gene databases including curated sequence databases simultaneously thus maximizing the chance of getting the best matching variable (V gene. For reference sequences, databases of germline variable (V), diversity (D) and joining (J) genes were used to delineate IG V domain framework regions (FR1-4s) and complementarity determining regions (CDRs). For each antibody, the variable domains were analyzed for the light and heavy chains, each are encoded by multiple genes, including the gene V, D and J genes. The focus of this study was mainly on the V genes to compare variations in the context of defined regions, finding the boundaries for each FR and CDR regions and comparing with other IG sequences in a database.

[0237] Due to individual variations within the genomic sequences (i.e., estimated at 1 single nucleotide polymorphism per 1,000 bp DNA sequence), the germline IG sequences comprise many thousands of allotypes (i.e., alleles or versions of a gene) (FIG. 6). Databases of human allotypes were generated and exploited for CAR humanization.

[0238] As an example, a humanized domain was derived using a particular human allotype by CDR-grafting to transform an anti-human CD19-binding domain VL and VH sequences from a mouse antibody (clone FMC63) (FIG. 7A). The CDR regions are underlined in FIG. 7A.

[0239] The sources of the framework sequences were confirmed by BLAST (Altshul et al., 1997) analyses shown in FIG. 7B. Additionally, the sequence homologies and identities were readily quantified and are summarized in FIG. 7C. As a comparison, the sequences that were created de novo were compared to previously available sequences and substantial and readily distinguishable differences were detected at the amino acid and nucleotide levels.

[0240] Module II comprised 3D structure modeling. To identify critical residues for antigen binding, 3D structures were built to evaluate the constructs. SWISS-MODEL is a protein structure homology-modeler that was used to make 3-dimensional models of humanized mAbs. Structure files were extracted from SAbDab, a structural antibody database, as templates for homology modeling. As an example of characterizing the 3D conformation of CDR regions of VL and VH domains, homology modeling was employed using SWISS-MODEL (Arnold et al., 2006; Benkert et al., 2011) algorithms with the following primary amino acid sequences:

TABLE-US-00002 hCD 19 V.sub.L (SEQ ID NO: 9): DIQMTQSPSSLSASVGDRVTITCRASQDIS KYLNWYQQKPGKVPKLLIYHTSRLHSGVPD RFSGSGSGTDFTLTISSLQPEDVATYYCQQ GNTLPYTFGQGTKVEIKR hCD19 V.sub.H (SEQ ID NO: 8): EVQLVESGGGLVQPGGSLRLSCAASGVSLP DYGVSWVRQAPGKGLEWIGVIWGSETTYYN SALKSKFIISRDNAKNSLYLQMNSLRAEDT AVYYCARHYYYGGSYAMDYWGQGTLVTVSS

[0241] The SWISS-MODEL template library (SMTL version 2017-06-28, PDB release 2017-06-23) was searched with BLAST (Altschul et al., 1997) and HHBlits (Remmert et al., 2012) for evolutionary related structures matching the target sequences hCD19 V.sub.L and hCD19 V.sub.H.

[0242] Overall, 13,535 templates were found for hCD19 V.sub.L, with 2fgw.1.A showing the best fit (FIG. 8A) and used for further analysis. The target sequence (hCD19 V.sub.L) was searched with BLAST against the primary amino acid sequence contained in the SMTL.

[0243] A total of 761 templates were found. An initial HHblits profile had been built using the procedure outlined by Remmert et al., followed by 1 iteration of HHblits against NR20. The profile thus obtained was then searched against all profiles of the SMTL. A total of 12,946 templates were found.

[0244] In all, 14,631 templates were found for hCD19 V.sub.H, with 4uv7.1.B showing the best fit (FIG. 8A) and used for further analysis. The target sequence (hCD19 V.sub.L) was searched with BLAST against the primary amino acid sequence contained in the SMTL. A total of 755 templates were found. An initial HHblits profile had been built using the procedure outlined by Remmert et al., followed by 1 iteration of HHblits against NR20. The profile thus obtained was then searched against all profiles of the SMTL. A total of 13,964 templates were found.

[0245] Template Selection: For each identified template, the template's quality had been predicted from features of the target-template alignment. The templates with the highest quality were then been selected for model building.

[0246] Model Building: Models were built based on the target-template alignment using ProMod3. Coordinates which were conserved between the target and the template were copied from the template to the model. Insertions and deletions were remodeled using a fragment library. Side chains were then rebuilt. Finally, the geometry of the resulting model was regularized by using a force field. In case loop modeling with ProMod3 failed, an alternative model was built with PROMOD-II (Geux et al., 1997).

[0247] Model Quality Estimation: The global and per-residue model quality had been assessed using the QMEAN scoring function (Benkert et al., 2011). For improved performance, weights of the individual QMEAN terms had been trained specifically for SWISS-MODEL.

[0248] The 3-dimensional models of hCD19 V.sub.L and hCD19 V.sub.H showed that the CDR residues exposed the water-phase and were able to form paratopic surfaces consistently with their antigen binding properties.

[0249] Module III comprised human framework grafting. As part of the synthetic CAR building process, a robust and reliable CDR grafting technique was included in the platform technology. The antigen binding sequences (CDRs) were identified and spliced using custom analysis algorithms (a combination of traditional Kabat/IMGT and novel methods) from antibody variable domains into selective human framework sequences (carefully curated custom databases based homology and position of key residues) to create a panel of full length humanized scFvs for robust expression.

[0250] Fully humanized scFvs are an essential step in the process of incorporating the antigen-binding domains of mAbs into CARs for clinical application while reducing the possibility of human anti-mouse reactivity ensuring persistence and viability of CAR-modified therapeutic cells. The present CAR humanization platform's efficacy was shown by the de novo creation of a panel of at least 5 high quality, full-length, humanized CARs for expression characterization, functional verification, and pre-clinical testing.

[0251] Module IV comprises humanization scoring. Scoring metrics were used to quantify and objectively evaluate the synthetic constructs as part of the CAR humanization process. The T20.sup.1 scoring algorithm was applied to a series of commercially available and FDA-approved monoclonal antibodies (mAbs) as listed in FIG. 10B to test its utility. As expected, the V.sub.L and V.sub.H (FIG. 10C) sequences score higher with increasing degrees of humanization. Interestingly, V.sub.L sequences trend higher in T20 scores as compared to paired V.sub.H sequences.

[0252] As a correlative analysis, information regarding FDA approved antibody therapeutics was assessed and the level of reported immunogenicity observed in patients from prescribing information in the public domain. As summarized in FIG. 10E, higher levels of humanization tend to be correlated with decreased instances of reported immunotoxicity in patients.

[0253] As an example of humanization scoring, T20 scores for the V.sub.L and V.sub.H sequences were evaluated before and after the humanization process for mCD19CAR vs hCD19CAR (FIGS. 10F-G). Indeed, higher T20 scores were observed for both processed fragments (humanized V.sub.L and V.sub.H). Since the workflow can be iterative, the CAR humanization process can be used to produce CARs with lower immunogenicity, better persistence, and robust function.

[0254] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

[0255] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0256] Altschul, S. F., et al., Nucleic Acids Res, 25, 3389-3402, 1997.

[0257] Arnold, K., et al., Bioinformatics, 22, 195-201, 2006.

[0258] Benkert, P., et al., Bioinformatics, 27, 343-350, 2011.

[0259] Fedorov et al., Sci. Transl. Medicine, 5(215), 2013.

[0260] Gao S H, et al., BMC Biotechnology. 13:55, 2013.

[0261] Guex, N. et al., Electrophoresis, 18, 2714-2723, 1997.

[0262] Marco Biasini et al., Nucleic Acids Research, 42 (W1): W252-W258, 2014.

[0263] Remmert, M., et al., Nat Methods, 9, 173-175, 2012.

[0264] Sela-Culang et al., Frontiers in Immunology, 4:302, 2013.

[0265] Shah et al., PLoS One, 8:e776781, 2013.

[0266] Singh et al., Cancer Research, 68:2961-2971, 2008.

[0267] Singh et al., Cancer Research, 71:3516-3527, 2011.

[0268] U.S. Pat. No. 7,109,304

Sequence CWU 1

1

6411062DNAHomo sapiens 1ctggaagaga agaaaggcaa ttacgtcgtg accgaccacg gcagctgtgt gcgggcttgt 60ggcgccgata gctacgagat ggaagaggac ggcgtgcgga agtgcaagaa gtgcgagggc 120ccctgcagaa aagtgtgcaa cggcatcggc atcggagagt tcaaggatag cctgagcatc 180aacgccacca acatcaagca cttcaagaac tgcaccagca tcagcggcga cctgcacatc 240ctgcccgtgg cctttagagg cgacagcttc acccacaccc ccccactgga tccccaggaa 300ctggacatcc tgaaaaccgt gaaagagatc acaggctttc tgctgattca ggcctggccc 360gagaaccgga cagacctgca cgccttcgag aacctggaaa tcatcagagg ccggaccaag 420cagcacggcc agttttctct ggccgtggtg tccctgaaca tcaccagcct gggcctgcgg 480agcctgaaag aaatcagcga cggcgacgtg atcatctccg gcaacaagaa cctgtgctac 540gccaacacca tcaattggaa gaagctgttc ggcacctccg gccagaaaac aaagatcatc 600tctaaccggg gcgagaacag ctgcaaagcc accggacaag tgtgccacgc cctgtgtagc 660cctgagggct gttggggacc cgagcccaga gattgcgtgt cctgccggaa tgtgtccaga 720ggccgcgagt gcgtggacaa gtgcaacctg ctggaaggcg agccccgcga gtttgtggaa 780aacagcgagt gcatccagtg ccaccccgag tgtctgcccc aggccatgaa cattacctgc 840accggcagag gccccgacaa ctgtatccag tgcgcccact acatcgacgg cccccactgc 900gtgaaaacct gtccagctgg cgtgatggga gagaacaaca ccctcgtgtg gaagtacgcc 960gacgccggcc atgtgtgcca cctgtgtcac cccaattgca cctacggctg taccggccct 1020ggcctggaag gctgtcctac caacggcccc aagatccctt ct 10622354PRTHomo sapiens 2Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys1 5 10 15Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val 20 25 30Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly 35 40 45Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn 50 55 60Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile65 70 75 80Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu 85 90 95Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly 100 105 110Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala 115 120 125Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln 130 135 140Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg145 150 155 160Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys 165 170 175Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr 180 185 190Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys 195 200 205Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys 210 215 220Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg225 230 235 240Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg 245 250 255Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu 260 265 270Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys 275 280 285Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys 290 295 300Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala305 310 315 320Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly 325 330 335Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile 340 345 350Pro Ser3801DNAArtificial SequencehCD19-scFv 3atggagttcg gtttgagctg gctctttctt gttgctatcc tcaagggggt gcagtgttca 60agggatatcc agatgactca gagccctagc tcccttagcg cgtccgtcgg cgaccgggtt 120acaattactt gtcgcgcatc acaagacata agtaaatact tgaactggta tcaacaaaag 180ccaggaaagg ttccaaaact cctcatctat cacacgagta gactccattc cggggtccct 240gatcggttct caggctctgg ttctggaact gacttcactc tcacgatctc tagtctgcaa 300cctgaggatg tcgctaccta ttattgccaa cagggaaaca ccctccctta cacatttggc 360caaggtacca aagttgagat caagaggggc tccacatctg ggagcgggaa accgggaagt 420ggagaaggct caaccaaagg tgaggtgcaa cttgtagaga gtggtggagg cctggttcag 480ccgggagggt ccctcagact tagttgcgcg gcttctggcg tttcactgcc ggactacggg 540gtcagttggg ttaggcaggc gcctgggaaa ggacttgagt ggatcggcgt gatatggggt 600agtgaaacaa cctactacaa tagtgctctt aaatcaaaat tcattatcag ccgagacaat 660gctaagaact ccctctatct ccaaatgaat agtcttagag cagaagacac agccgtttat 720tactgtgccc gccattatta ctatgggggc agctacgcaa tggattattg gggccaaggg 780accctggtca cggtatcaag c 8014324DNAArtificial SequencehCD19-scFv VL 4gatatccaga tgactcagag ccctagctcc cttagcgcgt ccgtcggcga ccgggttaca 60attacttgtc gcgcatcaca agacataagt aaatacttga actggtatca acaaaagcca 120ggaaaggttc caaaactcct catctatcac acgagtagac tccattccgg ggtccctgat 180cggttctcag gctctggttc tggaactgac ttcactctca cgatctctag tctgcaacct 240gaggatgtcg ctacctatta ttgccaacag ggaaacaccc tcccttacac atttggccaa 300ggtaccaaag ttgagatcaa gagg 3245360DNAArtificial SequencehCD19-scFv VH 5gaggtgcaac ttgtagagag tggtggaggc ctggttcagc cgggagggtc cctcagactt 60agttgcgcgg cttctggcgt ttcactgccg gactacgggg tcagttgggt taggcaggcg 120cctgggaaag gacttgagtg gatcggcgtg atatggggta gtgaaacaac ctactacaat 180agtgctctta aatcaaaatt cattatcagc cgagacaatg ctaagaactc cctctatctc 240caaatgaata gtcttagagc agaagacaca gccgtttatt actgtgcccg ccattattac 300tatgggggca gctacgcaat ggattattgg ggccaaggga ccctggtcac ggtatcaagc 3606267PRTArtificial SequencehCD19-scFv 6Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val 50 55 60Pro Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro65 70 75 80Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln145 150 155 160Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu 165 170 175Pro Asp Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 180 185 190Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205Ala Leu Lys Ser Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser 210 215 220Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr225 230 235 240Tyr Cys Ala Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 2657108PRTArtificial SequencehCD19-scFv VL 7Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 1058120PRTArtificial SequencehCD19-scFv VH 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60Ser Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 1209786DNAArtificial SequencehCD123-scFv 9atggaattcg ggctttcatg gttgttcctc gttgcgatac ttaaaggcgt tcagtgcagt 60agagacattc agatgaccca aagccctagt agcctctccg ccagtgtggg tgaccgcgtg 120actattacgt gccgagcaag caagagtatt agcaaagatt tggcatggta ccagcagaaa 180ccaggcaagg tccctaagtt gcttatatat agcgggtcta cactccaatc tggagtgccg 240gatcgcttct caggttccgg atcaggaacc gatttcactc ttacaatttc ctctctgcaa 300ccggaggacg tggcaacata ctattgccag cagcataaca aatatcccta tactttcggt 360caaggtacaa aagtagaaat aaaacgaggt tccacctccg ggagtgggaa gcccggctcc 420ggcgaaggat ccaccaaagg cgaggtacag ctcgttgaaa gcgggggcgg tctcgtccaa 480ccaggagggt ctcttcgact ttcttgtgct gcaagtgggt acacgtttac ttcctattgg 540atgaattggg ttcggcaagc ccccggtaaa gggcttgaat ggatagggag gatagacccc 600tacgattctg agacgcatta taatcagaaa ttcaaagata agttcataat aagcagagac 660aatgcaaaaa atagtttgta cctccagatg aatagtcttc gggcagaaga cacagcggta 720tattactgtg ctcggggtaa ctgggatgat tactggggtc agggaactct ggttactgtg 780tctagc 78610324DNAArtificial SequencehCD123-scFv VL 10gacattcaga tgacccaaag ccctagtagc ctctccgcca gtgtgggtga ccgcgtgact 60attacgtgcc gagcaagcaa gagtattagc aaagatttgg catggtacca gcagaaacca 120ggcaaggtcc ctaagttgct tatatatagc gggtctacac tccaatctgg agtgccggat 180cgcttctcag gttccggatc aggaaccgat ttcactctta caatttcctc tctgcaaccg 240gaggacgtgg caacatacta ttgccagcag cataacaaat atccctatac tttcggtcaa 300ggtacaaaag tagaaataaa acga 32411345DNAArtificial SequencehCD123-scFv VH 11gaggtacagc tcgttgaaag cgggggcggt ctcgtccaac caggagggtc tcttcgactt 60tcttgtgctg caagtgggta cacgtttact tcctattgga tgaattgggt tcggcaagcc 120cccggtaaag ggcttgaatg gatagggagg atagacccct acgattctga gacgcattat 180aatcagaaat tcaaagataa gttcataata agcagagaca atgcaaaaaa tagtttgtac 240ctccagatga atagtcttcg ggcagaagac acagcggtat attactgtgc tcggggtaac 300tgggatgatt actggggtca gggaactctg gttactgtgt ctagc 34512262PRTArtificial SequencehCD123-scFv 12Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys 35 40 45Ser Ile Ser Lys Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val 50 55 60Pro Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro65 70 75 80Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln His 100 105 110Asn Lys Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln145 150 155 160Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe 165 170 175Thr Ser Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 180 185 190Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn 195 200 205Gln Lys Phe Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn 210 215 220Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val225 230 235 240Tyr Tyr Cys Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr 245 250 255Leu Val Thr Val Ser Ser 26013108PRTArtificial SequencehCD123-scFv VL 13Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 10514115PRTArtificial SequencehCD123-scFv VH 14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11515777DNAArtificial SequencehMeso-scFv 15atggagttcg gcctttcttg gctgtttttg gtcgcaatat tgaagggcgt tcagtgttca 60agggacatac agatgacgca gtcacctagc tctctttcag cctctgtggg agaccgcgtt 120acaattacgt gttctgcttc tagttcagtg tcttacatgc attggtacca gcaaaaaccc 180ggcaaggttc caaagctcct tatatacgat acgtccaaac tggcttcagg cgtgccagac 240aggtttagtg gatctgggag cggcactgac ttcacactta caatatccag tctgcaaccc 300gaagatgttg cgacatacta ttgtcagcaa tggagcggct atccgctcac cggacagggg 360acaaaagtgg agataaagcg gggctccacc agtggttccg ggaagcctgg tagtggtgag 420ggttctacga aaggcgaagt gcagttggtg gagtctggtg gcgggcttgt tcaacccggt 480ggttctctca ggcttagctg tgcagcctct gggtacacaa tgaattgggt aaggcaagcg 540cctggaaagg gccttgagtg gatcggtttg ataaccccat acaacggagc ttcaagttac 600aaccaaaagt tccgcgggaa gtttattata agcagggata acgcaaagaa cagcctctat 660cttcaaatga actcactccg ggctgaagac acagccgtct attattgtgc ccgggggggc 720tacgacggac gagggttcga ttactggggt cagggcacac tcgtgaccgt gtcaagc 77716318DNAArtificial SequencehMeso-scFv VL 16gacatacaga tgacgcagtc acctagctct ctttcagcct ctgtgggaga ccgcgttaca 60attacgtgtt ctgcttctag ttcagtgtct tacatgcatt ggtaccagca aaaacccggc 120aaggttccaa agctccttat atacgatacg tccaaactgg cttcaggcgt gccagacagg 180tttagtggat ctgggagcgg cactgacttc acacttacaa tatccagtct gcaacccgaa 240gatgttgcga catactattg tcagcaatgg agcggctatc cgctcaccgg acaggggaca 300aaagtggaga taaagcgg 31817342DNAArtificial SequencehMeso-scFv VH 17gaagtgcagt tggtggagtc tggtggcggg cttgttcaac ccggtggttc tctcaggctt 60agctgtgcag cctctgggta cacaatgaat tgggtaaggc aagcgcctgg aaagggcctt 120gagtggatcg gtttgataac cccatacaac ggagcttcaa gttacaacca aaagttccgc 180gggaagttta ttataagcag ggataacgca aagaacagcc tctatcttca aatgaactca 240ctccgggctg aagacacagc cgtctattat tgtgcccggg ggggctacga cggacgaggg 300ttcgattact ggggtcaggg cacactcgtg accgtgtcaa gc 34218259PRTArtificial SequencehMeso-scFv 18Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser 35 40 45Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro 50 55 60Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Asp65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys

Gln Gln Trp Ser 100 105 110Gly Tyr Pro Leu Thr Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly 115 120 125Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 130 135 140Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly145 150 155 160Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Met Asn Trp 165 170 175Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile Thr 180 185 190Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Phe 195 200 205Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn 210 215 220Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly225 230 235 240Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255Val Ser Ser19106PRTArtificial SequencehMeso-scFv VL 19Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Val Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Leu Thr 85 90 95Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 10520114PRTArtificial SequencehMeso-scFv VH 20Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Met Asn Trp Val 20 25 30Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile Thr Pro 35 40 45Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Phe Ile 50 55 60Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser65 70 75 80Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr 85 90 95Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110Ser Ser21798DNAArtificial SequencehROR1-scFv 21atggagttcg gacttagttg gcttttcctc gtcgccatac tcaaaggtgt acaatgctca 60cgcgatatac aaatgacgca gagtccatcc tcactgtctg cctcagtagg cgacagggtc 120acaataacct gtaaagcaag cccggatatc aactcttacc tcagttggtt ccaacagaaa 180ccaggaaagg tgccgaagtt gttgatttac cgcgcaaacc gcctcgttga cggcgtgcca 240gataggtttt ctggcagcgg gagcggcact gactttacgc tcactatatc aagcctgcag 300ccggaggacg tagcaactta ctattgcctc caatacgacg aattccctta cacgttcggg 360caaggaacta aagttgaaat aaaaagagga agcacaagtg gttctggaaa acccggttcc 420ggtgagggca gtaccaaagg agaggtgcag ttggtagagt ctggtggggg tttggtgcaa 480cccggagggt ctctgaggtt gtcttgcgcg gcatccggct ttactttttc ctcatatgcg 540atgtcatggg tacgacaggc cccaggcaag ggactggagt ggatcgcatc tatttcacga 600ggagggacga catattaccc ggattcagtg aagggcaaat tcataatttc acgcgataac 660gccaagaact ccctctacct gcaaatgaac agtcttagag cggaagatac ggctgtgtat 720tactgtgcgc gatatgacta cgatggatat tacgcgatgg attattgggg acaaggtaca 780ctggtaaccg tttccagc 79822324DNAArtificial SequencehROR1-scFv VL 22gatatacaaa tgacgcagag tccatcctca ctgtctgcct cagtaggcga cagggtcaca 60ataacctgta aagcaagccc ggatatcaac tcttacctca gttggttcca acagaaacca 120ggaaaggtgc cgaagttgtt gatttaccgc gcaaaccgcc tcgttgacgg cgtgccagat 180aggttttctg gcagcgggag cggcactgac tttacgctca ctatatcaag cctgcagccg 240gaggacgtag caacttacta ttgcctccaa tacgacgaat tcccttacac gttcgggcaa 300ggaactaaag ttgaaataaa aaga 32423357DNAArtificial SequencehROR1-scFv VH 23gaggtgcagt tggtagagtc tggtgggggt ttggtgcaac ccggagggtc tctgaggttg 60tcttgcgcgg catccggctt tactttttcc tcatatgcga tgtcatgggt acgacaggcc 120ccaggcaagg gactggagtg gatcgcatct atttcacgag gagggacgac atattacccg 180gattcagtga agggcaaatt cataatttca cgcgataacg ccaagaactc cctctacctg 240caaatgaaca gtcttagagc ggaagatacg gctgtgtatt actgtgcgcg atatgactac 300gatggatatt acgcgatgga ttattgggga caaggtacac tggtaaccgt ttccagc 35724266PRTArtificial SequencehROR1-scFv 24Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Pro 35 40 45Asp Ile Asn Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Val 50 55 60Pro Lys Leu Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro65 70 75 80Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr 100 105 110Asp Glu Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln145 150 155 160Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 165 170 175Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 180 185 190Glu Trp Ile Ala Ser Ile Ser Arg Gly Gly Thr Thr Tyr Tyr Pro Asp 195 200 205Ser Val Lys Gly Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser 210 215 220Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr225 230 235 240Tyr Cys Ala Arg Tyr Asp Tyr Asp Gly Tyr Tyr Ala Met Asp Tyr Trp 245 250 255Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 26525108PRTArtificial SequencehROR1-scFv VL 25Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Pro Asp Ile Asn Ser Tyr 20 25 30Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 10526119PRTArtificial SequencehROR1-scFv VH 26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ala Ser Ile Ser Arg Gly Gly Thr Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60Gly Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Asp Tyr Asp Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11527792DNAArtificial SequencehCD5-scFv 27atggagttcg gtctcagttg gctgtttttg gttgctatct tgaagggcgt ccaatgcagc 60cgggacatcc agatgaccca gtctccctct agcatgtcag cgagtcttgg tgatcgagtg 120acgattacct gcagagcctc tcaagatata aacagctatc tttcatggtt ccaacagaag 180ccggggaagt ccccaaaaac tctcatatac agggcgaatc gactcgtaga cggtgtgcct 240tcaaggtttt ccgggagtgg tagtggcaca gattacacac ttacaatctc ttcattgcag 300tatgaggatt tcgggatcta ctactgtcaa cagtacgacg aatccccatg gacgtttggg 360ggcgggacca aacttgagat aaaagggagc acatctggaa gtggtaaacc tgggtcaggg 420gagggttcca caaaaggaca aattcaactt gtccaaagcg gtcctggtct taagaagcct 480ggagggtctg tcaggataag ttgtgcggca tccggctaca ccttcaccaa ctatgggatg 540aactgggtga aacaagcgcc tgggaaaggt cttcgatgga tgggctggat taatacccac 600actggagagc ccacttacgc tgatgatttc aaaggacgat ttaccttctc cttggatact 660tccaagagta ccgcgtactt gcaaatcaac agtctccggg ctgaagacac ggccacatac 720ttctgtacgc ggagagggta tgactggtat tttgatgtgt ggggtcaggg aacaaccgtg 780actgtttcaa gc 79228321DNAArtificial SequencehCD5-scFv VL 28gacatccaga tgacccagtc tccctctagc atgtcagcga gtcttggtga tcgagtgacg 60attacctgca gagcctctca agatataaac agctatcttt catggttcca acagaagccg 120gggaagtccc caaaaactct catatacagg gcgaatcgac tcgtagacgg tgtgccttca 180aggttttccg ggagtggtag tggcacagat tacacactta caatctcttc attgcagtat 240gaggatttcg ggatctacta ctgtcaacag tacgacgaat ccccatggac gtttgggggc 300gggaccaaac ttgagataaa a 32129354DNAArtificial SequencehCD5-scFv VH 29caaattcaac ttgtccaaag cggtcctggt cttaagaagc ctggagggtc tgtcaggata 60agttgtgcgg catccggcta caccttcacc aactatggga tgaactgggt gaaacaagcg 120cctgggaaag gtcttcgatg gatgggctgg attaataccc acactggaga gcccacttac 180gctgatgatt tcaaaggacg atttaccttc tccttggata cttccaagag taccgcgtac 240ttgcaaatca acagtctccg ggctgaagac acggccacat acttctgtac gcggagaggg 300tatgactggt attttgatgt gtggggtcag ggaacaaccg tgactgtttc aagc 35430264PRTArtificial SequencehCD5-scFv 30Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 35 40 45Asp Ile Asn Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser 50 55 60Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95Ser Ser Leu Gln Tyr Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr 100 105 110Asp Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 130 135 140Lys Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Gly Leu Lys Lys Pro145 150 155 160Gly Gly Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 165 170 175Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg 180 185 190Trp Met Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp 195 200 205Asp Phe Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr 210 215 220Ala Tyr Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr225 230 235 240Phe Cys Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln 245 250 255Gly Thr Thr Val Thr Val Ser Ser 26031107PRTArtificial SequencehCD5-scFv VL 31Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr65 70 75 80Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10532118PRTArtificial SequencehCD5-scFv VH 32Gln Ile Gln Leu Val Gln Ser Gly Pro Gly Leu Lys Lys Pro Gly Gly1 5 10 15Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met 35 40 45Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser 1153310253DNAArtificial SequencehCD19-CAR 33aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggtt

tgagctggct ctttcttgtt 3180gctatcctca agggggtgca gtgttcaagg gatatccaga tgactcagag ccctagctcc 3240cttagcgcgt ccgtcggcga ccgggttaca attacttgtc gcgcatcaca agacataagt 3300aaatacttga actggtatca acaaaagcca ggaaaggttc caaaactcct catctatcac 3360acgagtagac tccattccgg ggtccctgat cggttctcag gctctggttc tggaactgac 3420ttcactctca cgatctctag tctgcaacct gaggatgtcg ctacctatta ttgccaacag 3480ggaaacaccc tcccttacac atttggccaa ggtaccaaag ttgagatcaa gaggggctcc 3540acatctggga gcgggaaacc gggaagtgga gaaggctcaa ccaaaggtga ggtgcaactt 3600gtagagagtg gtggaggcct ggttcagccg ggagggtccc tcagacttag ttgcgcggct 3660tctggcgttt cactgccgga ctacggggtc agttgggtta ggcaggcgcc tgggaaagga 3720cttgagtgga tcggcgtgat atggggtagt gaaacaacct actacaatag tgctcttaaa 3780tcaaaattca ttatcagccg agacaatgct aagaactccc tctatctcca aatgaatagt 3840cttagagcag aagacacagc cgtttattac tgtgcccgcc attattacta tgggggcagc 3900tacgcaatgg attattgggg ccaagggacc ctggtcacgg tatcaagcta cgtcaccgtc 3960tcttcacagg atcccgccga gcccaaatct cctgacaaaa ctcacacatg cccaccgtgc 4020ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 4080accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 4140gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 4200aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 4260caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 4320gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 4380accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 4440aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca accggagaac 4500aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 4560ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 4620gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaaaaagat 4680cccaaatttt gggtgctggt ggtggttggt ggagtcctgg cttgctatag cttgctagta 4740acagtggcct ttattatttt ctgggtgagg agtaagagga gcaggctcct gcacagtgac 4800tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 4860ccaccacgcg acttcgcagc ctatcgctcc agagtgaagt tcagcaggag cgcagacgcc 4920cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 4980gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 5040aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 5100tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 5160cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 5220cctcgcggac cgcagtgtac taattatgct ctcttgaaat tggctggaga tgttgagagc 5280aatcccgggc ccatgcggat cagcaagccc cacctgcgga gcatcagcat ccagtgctac 5340ctgtgcctgc tgctgaacag ccacttcctg accgaggccg gcatccacgt gttcatcctg 5400ggctgcttca gcgccggact gcccaagacc gaggccaact gggtgaacgt gatcagcgac 5460ctgaagaaga tcgaggacct gatccagagc atgcacatcg acgccaccct gtacaccgag 5520agcgacgtgc accccagctg caaggtgacc gccatgaagt gctttctgct ggaactgcag 5580gtgatcagcc tggaaagcgg cgacgccagc atccacgaca ccgtggagaa cctgatcatc 5640ctggccaaca acagcctgag cagcaacggc aacgtgaccg agagcggctg caaagagtgc 5700gaggaactgg aagagaagaa catcaaagag tttctgcaga gcttcgtgca catcgtgcag 5760atgttcatca acaccagctg acgcgtcatc atcgatccgg attagtccaa tttgttaaag 5820acaggatatc agtggtccag gctctagttt tgactcaaca atatcaccag ctgaagccta 5880tagagtacga gccatagata aaataaaaga ttttatttag tctccagaaa aaggggggaa 5940tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 6000ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag 6060ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 6120gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc 6180ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag 6240agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg 6300aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat 6360aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg 6420tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct 6480tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca cacatgcagc 6540atgtatcaaa attaatttgg ttttttttct taagtattta cattaaatgg ccatagtact 6600taaagttaca ttggcttcct tgaaataaac atggagtatt cagaatgtgt cataaatatt 6660tctaatttta agatagtatc tccattggct ttctactttt tcttttattt ttttttgtcc 6720tctgtcttcc atttgttgtt gttgttgttt gtttgtttgt ttgttggttg gttggttaat 6780ttttttttaa agatcctaca ctatagttca agctagacta ttagctactc tgtaacccag 6840ggtgaccttg aagtcatggg tagcctgctg ttttagcctt cccacatcta agattacagg 6900tatgagctat catttttggt atattgattg attgattgat tgatgtgtgt gtgtgtgatt 6960gtgtttgtgt gtgtgactgt gaaaatgtgt gtatgggtgt gtgtgaatgt gtgtatgtat 7020gtgtgtgtgt gagtgtgtgt gtgtgtgtgt gcatgtgtgt gtgtgtgact gtgtctatgt 7080gtatgactgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttgtgaa 7140aaaatattct atggtagtga gagccaacgc tccggctcag gtgtcaggtt ggtttttgag 7200acagagtctt tcacttagct tggaattcac tggccgtcgt tttacaacgt cgtgactggg 7260aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 7320gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 7380aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 7440ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 7500caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 7560ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 7620cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 7680tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 7740ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 7800aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 7860tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 7920atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 7980agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 8040tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 8100tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 8160atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 8220ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 8280tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 8340acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 8400ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 8460aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 8520ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 8580cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 8640gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 8700actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 8760agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 8820cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 8880tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 8940agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 9000ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 9060acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 9120ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 9180gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 9240gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 9300gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 9360tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 9420caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 9480tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 9540gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 9600agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 9660ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 9720gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 9780ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 9840atgaccatga ttacgccaag ctttgctctt aggagtttcc taatacatcc caaactcaaa 9900tatataaagc atttgacttg ttctatgccc tagggggcgg ggggaagcta agccagcttt 9960ttttaacatt taaaatgtta attccatttt aaatgcacag atgtttttat ttcataaggg 10020tttcaatgtg catgaatgct gcaatattcc tgttaccaaa gctagtataa ataaaaatag 10080ataaacgtgg aaattactta gagtttctgt cattaacgtt tccttcctca gttgacaaca 10140taaatgcgct gctgagcaag ccagtttgca tctgtcagga tcaatttccc attatgccag 10200tcatattaat tactagtcaa ttagttgatt tttatttttg acatatacat gtg 102533410238DNAArtificial SequencehCD123-CAR 34aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gaattcgggc tttcatggtt gttcctcgtt 3180gcgatactta aaggcgttca gtgcagtaga gacattcaga tgacccaaag ccctagtagc 3240ctctccgcca gtgtgggtga ccgcgtgact attacgtgcc gagcaagcaa gagtattagc 3300aaagatttgg catggtacca gcagaaacca ggcaaggtcc ctaagttgct tatatatagc 3360gggtctacac tccaatctgg agtgccggat cgcttctcag gttccggatc aggaaccgat 3420ttcactctta caatttcctc tctgcaaccg gaggacgtgg caacatacta ttgccagcag 3480cataacaaat atccctatac tttcggtcaa ggtacaaaag tagaaataaa acgaggttcc 3540acctccggga gtgggaagcc cggctccggc gaaggatcca ccaaaggcga ggtacagctc 3600gttgaaagcg ggggcggtct cgtccaacca ggagggtctc ttcgactttc ttgtgctgca 3660agtgggtaca cgtttacttc ctattggatg aattgggttc ggcaagcccc cggtaaaggg 3720cttgaatgga tagggaggat agacccctac gattctgaga cgcattataa tcagaaattc 3780aaagataagt tcataataag cagagacaat gcaaaaaata gtttgtacct ccagatgaat 3840agtcttcggg cagaagacac agcggtatat tactgtgctc ggggtaactg ggatgattac 3900tggggtcagg gaactctggt tactgtgtct agctacgtca ccgtctcttc acaggatccc 3960gccgagccca aatctcctga caaaactcac acatgcccac cgtgcccagc acctgaactc 4020ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 4080cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 4140ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 4200cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 4260aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 4320accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 4380cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 4440agcgacatcg ccgtggagtg ggagagcaat gggcaaccgg agaacaacta caagaccacg 4500cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 4560agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 4620cactacacgc agaagagcct ctccctgtct ccgggtaaaa aagatcccaa attttgggtg 4680ctggtggtgg ttggtggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt 4740attttctggg tgaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact 4800ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc 4860gcagcctatc gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 4920ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 4980gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 5040gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 5100atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 5160gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg cggaccgcag 5220tgtactaatt atgctctctt gaaattggct ggagatgttg agagcaatcc cgggcccatg 5280cggatcagca agccccacct gcggagcatc agcatccagt gctacctgtg cctgctgctg 5340aacagccact tcctgaccga ggccggcatc cacgtgttca tcctgggctg cttcagcgcc 5400ggactgccca agaccgaggc caactgggtg aacgtgatca gcgacctgaa gaagatcgag 5460gacctgatcc agagcatgca catcgacgcc accctgtaca ccgagagcga cgtgcacccc 5520agctgcaagg tgaccgccat gaagtgcttt ctgctggaac tgcaggtgat cagcctggaa 5580agcggcgacg ccagcatcca cgacaccgtg gagaacctga tcatcctggc caacaacagc 5640ctgagcagca acggcaacgt gaccgagagc ggctgcaaag agtgcgagga actggaagag 5700aagaacatca aagagtttct gcagagcttc gtgcacatcg tgcagatgtt catcaacacc 5760agctgacgcg tcatcatcga tccggattag tccaatttgt taaagacagg atatcagtgg 5820tccaggctct agttttgact caacaatatc accagctgaa gcctatagag tacgagccat 5880agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct 5940gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac 6000tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa tatgggccaa 6060acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag 6120ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 6180gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt 6240ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt 6300tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac 6360ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa 6420taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc 6480tgagtgattg actacccgtc agcgggggtc tttcacacat gcagcatgta tcaaaattaa 6540tttggttttt tttcttaagt atttacatta aatggccata gtacttaaag ttacattggc 6600ttccttgaaa taaacatgga gtattcagaa tgtgtcataa atatttctaa ttttaagata 6660gtatctccat tggctttcta ctttttcttt tatttttttt tgtcctctgt cttccatttg 6720ttgttgttgt tgtttgtttg tttgtttgtt ggttggttgg ttaatttttt tttaaagatc 6780ctacactata gttcaagcta gactattagc tactctgtaa cccagggtga ccttgaagtc 6840atgggtagcc tgctgtttta gccttcccac atctaagatt acaggtatga gctatcattt 6900ttggtatatt gattgattga ttgattgatg tgtgtgtgtg tgattgtgtt tgtgtgtgtg 6960actgtgaaaa tgtgtgtatg ggtgtgtgtg aatgtgtgta tgtatgtgtg tgtgtgagtg 7020tgtgtgtgtg tgtgtgcatg tgtgtgtgtg tgactgtgtc tatgtgtatg actgtgtgtg 7080tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtt gtgaaaaaat attctatggt 7140agtgagagcc aacgctccgg ctcaggtgtc aggttggttt ttgagacaga gtctttcact 7200tagcttggaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 7260cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 7320cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc 7380ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta 7440caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 7500cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 7560ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc 7620tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag 7680gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 7740caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 7800ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 7860gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt

7920tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 7980ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 8040tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 8100atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 8160gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 8220caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 8280ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 8340ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 8400ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 8460ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 8520gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 8580ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 8640taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 8700agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 8760atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 8820aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 8880caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 8940ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 9000cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 9060tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 9120gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 9180ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 9240gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 9300caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 9360ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 9420tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 9480ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 9540agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 9600aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 9660gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 9720tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 9780tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 9840ccaagctttg ctcttaggag tttcctaata catcccaaac tcaaatatat aaagcatttg 9900acttgttcta tgccctaggg ggcgggggga agctaagcca gcttttttta acatttaaaa 9960tgttaattcc attttaaatg cacagatgtt tttatttcat aagggtttca atgtgcatga 10020atgctgcaat attcctgtta ccaaagctag tataaataaa aatagataaa cgtggaaatt 10080acttagagtt tctgtcatta acgtttcctt cctcagttga caacataaat gcgctgctga 10140gcaagccagt ttgcatctgt caggatcaat ttcccattat gccagtcata ttaattacta 10200gtcaattagt tgatttttat ttttgacata tacatgtg 102383510229DNAArtificial SequencehMeso-CAR 35aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggcc tttcttggct gtttttggtc 3180gcaatattga agggcgttca gtgttcaagg gacatacaga tgacgcagtc acctagctct 3240ctttcagcct ctgtgggaga ccgcgttaca attacgtgtt ctgcttctag ttcagtgtct 3300tacatgcatt ggtaccagca aaaacccggc aaggttccaa agctccttat atacgatacg 3360tccaaactgg cttcaggcgt gccagacagg tttagtggat ctgggagcgg cactgacttc 3420acacttacaa tatccagtct gcaacccgaa gatgttgcga catactattg tcagcaatgg 3480agcggctatc cgctcaccgg acaggggaca aaagtggaga taaagcgggg ctccaccagt 3540ggttccggga agcctggtag tggtgagggt tctacgaaag gcgaagtgca gttggtggag 3600tctggtggcg ggcttgttca acccggtggt tctctcaggc ttagctgtgc agcctctggg 3660tacacaatga attgggtaag gcaagcgcct ggaaagggcc ttgagtggat cggtttgata 3720accccataca acggagcttc aagttacaac caaaagttcc gcgggaagtt tattataagc 3780agggataacg caaagaacag cctctatctt caaatgaact cactccgggc tgaagacaca 3840gccgtctatt attgtgcccg ggggggctac gacggacgag ggttcgatta ctggggtcag 3900ggcacactcg tgaccgtgtc aagctacgtc accgtctctt cacaggatcc cgccgagccc 3960aaatctcctg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 4020ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 4080gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 4140tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 4200agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 4260gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 4320aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 4380ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 4440gccgtggagt gggagagcaa tgggcaaccg gagaacaact acaagaccac gcctcccgtg 4500ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 4560cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 4620cagaagagcc tctccctgtc tccgggtaaa aaagatccca aattttgggt gctggtggtg 4680gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat tattttctgg 4740gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 4800cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 4860cgctccagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 4920cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 4980cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 5040tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 5100gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 5160gacacctacg acgcccttca catgcaggcc ctgccccctc gcggaccgca gtgtactaat 5220tatgctctct tgaaattggc tggagatgtt gagagcaatc ccgggcccat gcggatcagc 5280aagccccacc tgcggagcat cagcatccag tgctacctgt gcctgctgct gaacagccac 5340ttcctgaccg aggccggcat ccacgtgttc atcctgggct gcttcagcgc cggactgccc 5400aagaccgagg ccaactgggt gaacgtgatc agcgacctga agaagatcga ggacctgatc 5460cagagcatgc acatcgacgc caccctgtac accgagagcg acgtgcaccc cagctgcaag 5520gtgaccgcca tgaagtgctt tctgctggaa ctgcaggtga tcagcctgga aagcggcgac 5580gccagcatcc acgacaccgt ggagaacctg atcatcctgg ccaacaacag cctgagcagc 5640aacggcaacg tgaccgagag cggctgcaaa gagtgcgagg aactggaaga gaagaacatc 5700aaagagtttc tgcagagctt cgtgcacatc gtgcagatgt tcatcaacac cagctgacgc 5760gtcatcatcg atccggatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc 5820tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca tagataaaat 5880aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg 5940gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag 6000agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat 6060ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg 6120ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 6180gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt 6240gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc 6300gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc 6360ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc 6420ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt 6480gactacccgt cagcgggggt ctttcacaca tgcagcatgt atcaaaatta atttggtttt 6540ttttcttaag tatttacatt aaatggccat agtacttaaa gttacattgg cttccttgaa 6600ataaacatgg agtattcaga atgtgtcata aatatttcta attttaagat agtatctcca 6660ttggctttct actttttctt ttattttttt ttgtcctctg tcttccattt gttgttgttg 6720ttgtttgttt gtttgtttgt tggttggttg gttaattttt ttttaaagat cctacactat 6780agttcaagct agactattag ctactctgta acccagggtg accttgaagt catgggtagc 6840ctgctgtttt agccttccca catctaagat tacaggtatg agctatcatt tttggtatat 6900tgattgattg attgattgat gtgtgtgtgt gtgattgtgt ttgtgtgtgt gactgtgaaa 6960atgtgtgtat gggtgtgtgt gaatgtgtgt atgtatgtgt gtgtgtgagt gtgtgtgtgt 7020gtgtgtgcat gtgtgtgtgt gtgactgtgt ctatgtgtat gactgtgtgt gtgtgtgtgt 7080gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt tgtgaaaaaa tattctatgg tagtgagagc 7140caacgctccg gctcaggtgt caggttggtt tttgagacag agtctttcac ttagcttgga 7200attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 7260atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 7320atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc 7380tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 7440ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 7500gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 7560tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 7620gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 7680ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 7740atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 7800tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 7860tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 7920gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 7980aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 8040gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 8100ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 8160gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 8220gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 8280atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 8340ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 8400cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 8460cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 8520gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 8580cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 8640cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 8700taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 8760ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 8820aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 8880caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 8940taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 9000gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 9060cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 9120taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 9180agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 9240ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 9300gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 9360acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 9420acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 9480tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 9540ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 9600agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 9660acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 9720tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 9780ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttt 9840gctcttagga gtttcctaat acatcccaaa ctcaaatata taaagcattt gacttgttct 9900atgccctagg gggcgggggg aagctaagcc agcttttttt aacatttaaa atgttaattc 9960cattttaaat gcacagatgt ttttatttca taagggtttc aatgtgcatg aatgctgcaa 10020tattcctgtt accaaagcta gtataaataa aaatagataa acgtggaaat tacttagagt 10080ttctgtcatt aacgtttcct tcctcagttg acaacataaa tgcgctgctg agcaagccag 10140tttgcatctg tcaggatcaa tttcccatta tgccagtcat attaattact agtcaattag 10200ttgattttta tttttgacat atacatgtg 102293610250DNAArtificial SequencehROR1-CAR 36aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct

ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggac ttagttggct tttcctcgtc 3180gccatactca aaggtgtaca atgctcacgc gatatacaaa tgacgcagag tccatcctca 3240ctgtctgcct cagtaggcga cagggtcaca ataacctgta aagcaagccc ggatatcaac 3300tcttacctca gttggttcca acagaaacca ggaaaggtgc cgaagttgtt gatttaccgc 3360gcaaaccgcc tcgttgacgg cgtgccagat aggttttctg gcagcgggag cggcactgac 3420tttacgctca ctatatcaag cctgcagccg gaggacgtag caacttacta ttgcctccaa 3480tacgacgaat tcccttacac gttcgggcaa ggaactaaag ttgaaataaa aagaggaagc 3540acaagtggtt ctggaaaacc cggttccggt gagggcagta ccaaaggaga ggtgcagttg 3600gtagagtctg gtgggggttt ggtgcaaccc ggagggtctc tgaggttgtc ttgcgcggca 3660tccggcttta ctttttcctc atatgcgatg tcatgggtac gacaggcccc aggcaaggga 3720ctggagtgga tcgcatctat ttcacgagga gggacgacat attacccgga ttcagtgaag 3780ggcaaattca taatttcacg cgataacgcc aagaactccc tctacctgca aatgaacagt 3840cttagagcgg aagatacggc tgtgtattac tgtgcgcgat atgactacga tggatattac 3900gcgatggatt attggggaca aggtacactg gtaaccgttt ccagctacgt caccgtctct 3960tcacaggatc ccgccgagcc caaatctcct gacaaaactc acacatgccc accgtgccca 4020gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 4080ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 4140cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 4200ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 4260caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 4320cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 4380ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 4440ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcaacc ggagaacaac 4500tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 4560accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 4620gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa aaaagatccc 4680aaattttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 4740gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 4800atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 4860ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 4920gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 4980tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 5040aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 5100agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 5160ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 5220cgcggaccgc agtgtactaa ttatgctctc ttgaaattgg ctggagatgt tgagagcaat 5280cccgggccca tgcggatcag caagccccac ctgcggagca tcagcatcca gtgctacctg 5340tgcctgctgc tgaacagcca cttcctgacc gaggccggca tccacgtgtt catcctgggc 5400tgcttcagcg ccggactgcc caagaccgag gccaactggg tgaacgtgat cagcgacctg 5460aagaagatcg aggacctgat ccagagcatg cacatcgacg ccaccctgta caccgagagc 5520gacgtgcacc ccagctgcaa ggtgaccgcc atgaagtgct ttctgctgga actgcaggtg 5580atcagcctgg aaagcggcga cgccagcatc cacgacaccg tggagaacct gatcatcctg 5640gccaacaaca gcctgagcag caacggcaac gtgaccgaga gcggctgcaa agagtgcgag 5700gaactggaag agaagaacat caaagagttt ctgcagagct tcgtgcacat cgtgcagatg 5760ttcatcaaca ccagctgacg cgtcatcatc gatccggatt agtccaattt gttaaagaca 5820ggatatcagt ggtccaggct ctagttttga ctcaacaata tcaccagctg aagcctatag 5880agtacgagcc atagataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga 5940aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga 6000aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagctg 6060aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 6120cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg 6180gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga 6240accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac 6300taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa 6360agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac 6420ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg 6480gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcacac atgcagcatg 6540tatcaaaatt aatttggttt tttttcttaa gtatttacat taaatggcca tagtacttaa 6600agttacattg gcttccttga aataaacatg gagtattcag aatgtgtcat aaatatttct 6660aattttaaga tagtatctcc attggctttc tactttttct tttatttttt tttgtcctct 6720gtcttccatt tgttgttgtt gttgtttgtt tgtttgtttg ttggttggtt ggttaatttt 6780tttttaaaga tcctacacta tagttcaagc tagactatta gctactctgt aacccagggt 6840gaccttgaag tcatgggtag cctgctgttt tagccttccc acatctaaga ttacaggtat 6900gagctatcat ttttggtata ttgattgatt gattgattga tgtgtgtgtg tgtgattgtg 6960tttgtgtgtg tgactgtgaa aatgtgtgta tgggtgtgtg tgaatgtgtg tatgtatgtg 7020tgtgtgtgag tgtgtgtgtg tgtgtgtgca tgtgtgtgtg tgtgactgtg tctatgtgta 7080tgactgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg ttgtgaaaaa 7140atattctatg gtagtgagag ccaacgctcc ggctcaggtg tcaggttggt ttttgagaca 7200gagtctttca cttagcttgg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 7260accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 7320atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 7380ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 7440gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 7500cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 7560tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 7620gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 7680cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 7740tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 7800aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 7860ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 7920ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 7980tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 8040tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 8100actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 8160gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 8220acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 8280gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 8340acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 8400gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 8460ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 8520gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 8580cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 8640agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 8700catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 8760tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 8820cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 8880gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 8940taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 9000ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 9060tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 9120ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 9180cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 9240agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 9300gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 9360atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 9420gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 9480gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 9540ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 9600cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 9660cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 9720acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 9780cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 9840accatgatta cgccaagctt tgctcttagg agtttcctaa tacatcccaa actcaaatat 9900ataaagcatt tgacttgttc tatgccctag ggggcggggg gaagctaagc cagctttttt 9960taacatttaa aatgttaatt ccattttaaa tgcacagatg tttttatttc ataagggttt 10020caatgtgcat gaatgctgca atattcctgt taccaaagct agtataaata aaaatagata 10080aacgtggaaa ttacttagag tttctgtcat taacgtttcc ttcctcagtt gacaacataa 10140atgcgctgct gagcaagcca gtttgcatct gtcaggatca atttcccatt atgccagtca 10200tattaattac tagtcaatta gttgattttt atttttgaca tatacatgtg 102503710244DNAArtificial SequencehCD5-CAR 37aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggtc tcagttggct gtttttggtt 3180gctatcttga agggcgtcca atgcagccgg gacatccaga tgacccagtc tccctctagc 3240atgtcagcga gtcttggtga tcgagtgacg attacctgca gagcctctca agatataaac 3300agctatcttt catggttcca acagaagccg gggaagtccc caaaaactct catatacagg 3360gcgaatcgac tcgtagacgg tgtgccttca aggttttccg ggagtggtag tggcacagat 3420tacacactta caatctcttc attgcagtat gaggatttcg ggatctacta ctgtcaacag 3480tacgacgaat ccccatggac gtttgggggc gggaccaaac ttgagataaa agggagcaca 3540tctggaagtg gtaaacctgg gtcaggggag ggttccacaa aaggacaaat tcaacttgtc 3600caaagcggtc ctggtcttaa gaagcctgga gggtctgtca ggataagttg tgcggcatcc 3660ggctacacct tcaccaacta tgggatgaac tgggtgaaac aagcgcctgg gaaaggtctt 3720cgatggatgg gctggattaa tacccacact ggagagccca cttacgctga tgatttcaaa 3780ggacgattta ccttctcctt ggatacttcc aagagtaccg cgtacttgca aatcaacagt 3840ctccgggctg aagacacggc cacatacttc tgtacgcgga gagggtatga ctggtatttt 3900gatgtgtggg gtcagggaac aaccgtgact gtttcaagct acgtcaccgt ctcttcacag 3960gatcccgccg agcccaaatc tcctgacaaa actcacacat gcccaccgtg cccagcacct 4020gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 4080atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 4140gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 4200gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 4260tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 4320gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 4380ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 4440tatcccagcg acatcgccgt ggagtgggag agcaatgggc aaccggagaa caactacaag 4500accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 4560gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 4620cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaaaaaga tcccaaattt 4680tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 4740tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 4800atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 4860gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 4920cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 4980gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 5040cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 5100attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 5160agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcgga 5220ccgcagtgta ctaattatgc tctcttgaaa ttggctggag atgttgagag caatcccggg 5280cccatgcgga tcagcaagcc ccacctgcgg agcatcagca tccagtgcta cctgtgcctg 5340ctgctgaaca gccacttcct gaccgaggcc ggcatccacg tgttcatcct gggctgcttc 5400agcgccggac tgcccaagac cgaggccaac tgggtgaacg tgatcagcga cctgaagaag 5460atcgaggacc tgatccagag catgcacatc gacgccaccc tgtacaccga gagcgacgtg 5520caccccagct gcaaggtgac cgccatgaag tgctttctgc tggaactgca ggtgatcagc 5580ctggaaagcg gcgacgccag catccacgac accgtggaga acctgatcat cctggccaac 5640aacagcctga gcagcaacgg caacgtgacc gagagcggct gcaaagagtg cgaggaactg 5700gaagagaaga acatcaaaga gtttctgcag agcttcgtgc acatcgtgca gatgttcatc 5760aacaccagct gacgcgtcat catcgatccg gattagtcca atttgttaaa gacaggatat 5820cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg 5880agccatagat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc 5940ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata 6000cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg 6060ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 6120gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 6180ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc 6240agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 6300atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 6360cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt 6420atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt 6480ctcctctgag tgattgacta cccgtcagcg ggggtctttc acacatgcag catgtatcaa 6540aattaatttg gttttttttc ttaagtattt acattaaatg gccatagtac ttaaagttac 6600attggcttcc ttgaaataaa catggagtat tcagaatgtg tcataaatat ttctaatttt 6660aagatagtat ctccattggc tttctacttt ttcttttatt tttttttgtc ctctgtcttc 6720catttgttgt tgttgttgtt tgtttgtttg tttgttggtt ggttggttaa ttttttttta 6780aagatcctac actatagttc aagctagact attagctact ctgtaaccca gggtgacctt 6840gaagtcatgg gtagcctgct gttttagcct tcccacatct aagattacag gtatgagcta 6900tcatttttgg tatattgatt gattgattga ttgatgtgtg tgtgtgtgat tgtgtttgtg 6960tgtgtgactg tgaaaatgtg tgtatgggtg tgtgtgaatg tgtgtatgta tgtgtgtgtg 7020tgagtgtgtg tgtgtgtgtg tgcatgtgtg tgtgtgtgac tgtgtctatg tgtatgactg 7080tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttgtga aaaaatattc

7140tatggtagtg agagccaacg ctccggctca ggtgtcaggt tggtttttga gacagagtct 7200ttcacttagc ttggaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 7260gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 7320aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 7380tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 7440tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 7500ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 7560tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 7620agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 7680cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 7740tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 7800gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 7860cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 7920atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 7980agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 8040gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 8100ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 8160cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 8220ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 8280atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 8340gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 8400tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 8460gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 8520gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 8580tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 8640ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 8700tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 8760ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 8820ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 8880tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 8940ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 9000tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 9060tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 9120actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 9180cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 9240gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 9300tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 9360ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 9420ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 9480cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 9540cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 9600gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 9660attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 9720ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 9780gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 9840attacgccaa gctttgctct taggagtttc ctaatacatc ccaaactcaa atatataaag 9900catttgactt gttctatgcc ctagggggcg gggggaagct aagccagctt tttttaacat 9960ttaaaatgtt aattccattt taaatgcaca gatgttttta tttcataagg gtttcaatgt 10020gcatgaatgc tgcaatattc ctgttaccaa agctagtata aataaaaata gataaacgtg 10080gaaattactt agagtttctg tcattaacgt ttccttcctc agttgacaac ataaatgcgc 10140tgctgagcaa gccagtttgc atctgtcagg atcaatttcc cattatgcca gtcatattaa 10200ttactagtca attagttgat ttttattttt gacatataca tgtg 102443810625DNAArtificial SequencepSFG4-CD19CAR-Ev3 38aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagtttgggc tgagctggct ttttcttgtg 3180gctattttaa aaggtgtcca gtgctctaga gacatccaga tgacacagac tacatcctcc 3240ctgtctgcct ctctgggaga cagagtcacc atcagttgca gggcaagtca ggacattagt 3300aaatatttaa attggtatca gcagaaacca gatggaactg ttaaactcct gatctaccat 3360acatcaagat tacactcagg agtcccatca aggttcagtg gcagtgggtc tggaacagat 3420tattctctca ccattagcaa cctggagcaa gaagatattg ccacttactt ttgccaacag 3480ggtaatacgc ttccgtacac gttcggaggg gggaccaagc tggagctgaa acgtggtggt 3540ggtggttctg gtggtggtgg ttctggcggc ggcggctccg gtggtggtgg atccgaggtg 3600cagctgcagc agtctggacc tggcctggtg gcgccctcac agagcctgtc cgtcacatgc 3660actgtctcag gggtctcatt acccgactat ggtgtaagct ggattcgcca gcctccacga 3720aagggtctgg agtggctggg agtaatatgg ggtagtgaaa ccacatacta taattcagct 3780ctcaaatcca gactgaccat catcaaggac aactccaaga gccaagtttt cttaaaaatg 3840aacagtctgc aaactgatga cacagccatt tactactgtg ccaaacatta ttactacggt 3900ggtagctatg ctatggacta ctggggccaa gggaccacgg tcaccgtctc ctcgtacgtc 3960accgtctctt cacaggatcc cgccctggaa gagaagaaag gcaattacgt cgtgaccgac 4020cacggcagct gtgtgcgggc ttgtggcgcc gatagctacg agatggaaga ggacggcgtg 4080cggaagtgca agaagtgcga gggcccctgc agaaaagtgt gcaacggcat cggcatcgga 4140gagttcaagg atagcctgag catcaacgcc accaacatca agcacttcaa gaactgcacc 4200agcatcagcg gcgacctgca catcctgccc gtggccttta gaggcgacag cttcacccac 4260acccccccac tggatcccca ggaactggac atcctgaaaa ccgtgaaaga gatcacaggc 4320tttctgctga ttcaggcctg gcccgagaac cggacagacc tgcacgcctt cgagaacctg 4380gaaatcatca gaggccggac caagcagcac ggccagtttt ctctggccgt ggtgtccctg 4440aacatcacca gcctgggcct gcggagcctg aaagaaatca gcgacggcga cgtgatcatc 4500tccggcaaca agaacctgtg ctacgccaac accatcaatt ggaagaagct gttcggcacc 4560tccggccaga aaacaaagat catctctaac cggggcgaga acagctgcaa agccaccgga 4620caagtgtgcc acgccctgtg tagccctgag ggctgttggg gacccgagcc cagagattgc 4680gtgtcctgcc ggaatgtgtc cagaggccgc gagtgcgtgg acaagtgcaa cctgctggaa 4740ggcgagcccc gcgagtttgt ggaaaacagc gagtgcatcc agtgccaccc cgagtgtctg 4800ccccaggcca tgaacattac ctgcaccggc agaggccccg acaactgtat ccagtgcgcc 4860cactacatcg acggccccca ctgcgtgaaa acctgtccag ctggcgtgat gggagagaac 4920aacaccctcg tgtggaagta cgccgacgcc ggccatgtgt gccacctgtg tcaccccaat 4980tgcacctacg gctgtaccgg ccctggcctg gaaggctgtc ctaccaacgg ccccaagatc 5040ccttctaaag atcccaaatt ttgggtgctg gtggtggttg gtggagtcct ggcttgctat 5100agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 5160ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 5220cagccctatg ccccaccacg cgacttcgca gcctatcgct ccagagtgaa gttcagcagg 5280agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 5340ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 5400ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 5460atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 5520gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 5580caggccctgc cccctcgcgg accgcagtgt actaattatg ctctcttgaa attggctgga 5640gatgttgaga gcaatcccgg gcccatgcgg atcagcaagc cccacctgcg gagcatcagc 5700atccagtgct acctgtgcct gctgctgaac agccacttcc tgaccgaggc cggcatccac 5760gtgttcatcc tgggctgctt cagcgccgga ctgcccaaga ccgaggccaa ctgggtgaac 5820gtgatcagcg acctgaagaa gatcgaggac ctgatccaga gcatgcacat cgacgccacc 5880ctgtacaccg agagcgacgt gcaccccagc tgcaaggtga ccgccatgaa gtgctttctg 5940ctggaactgc aggtgatcag cctggaaagc ggcgacgcca gcatccacga caccgtggag 6000aacctgatca tcctggccaa caacagcctg agcagcaacg gcaacgtgac cgagagcggc 6060tgcaaagagt gcgaggaact ggaagagaag aacatcaaag agtttctgca gagcttcgtg 6120cacatcgtgc agatgttcat caacaccagc tgacgcgtca tcatcgatcc ggattagtcc 6180aatttgttaa agacaggata tcagtggtcc aggctctagt tttgactcaa caatatcacc 6240agctgaagcc tatagagtac gagccataga taaaataaaa gattttattt agtctccaga 6300aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat 6360tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa 6420cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg 6480gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa 6540gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc 6600agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct 6660gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc 6720cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact 6780gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt 6840ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt 6900cacacatgca gcatgtatca aaattaattt ggtttttttt cttaagtatt tacattaaat 6960ggccatagta cttaaagtta cattggcttc cttgaaataa acatggagta ttcagaatgt 7020gtcataaata tttctaattt taagatagta tctccattgg ctttctactt tttcttttat 7080ttttttttgt cctctgtctt ccatttgttg ttgttgttgt ttgtttgttt gtttgttggt 7140tggttggtta attttttttt aaagatccta cactatagtt caagctagac tattagctac 7200tctgtaaccc agggtgacct tgaagtcatg ggtagcctgc tgttttagcc ttcccacatc 7260taagattaca ggtatgagct atcatttttg gtatattgat tgattgattg attgatgtgt 7320gtgtgtgtga ttgtgtttgt gtgtgtgact gtgaaaatgt gtgtatgggt gtgtgtgaat 7380gtgtgtatgt atgtgtgtgt gtgagtgtgt gtgtgtgtgt gtgcatgtgt gtgtgtgtga 7440ctgtgtctat gtgtatgact gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 7500gtgtgttgtg aaaaaatatt ctatggtagt gagagccaac gctccggctc aggtgtcagg 7560ttggtttttg agacagagtc tttcacttag cttggaattc actggccgtc gttttacaac 7620gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 7680tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 7740gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 7800cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 7860cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 7920cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 7980caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 8040tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 8100ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 8160gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 8220cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 8280tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 8340tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 8400cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 8460tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 8520agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 8580ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 8640ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 8700aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 8760gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 8820tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 8880ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 8940cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 9000atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 9060cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 9120ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 9180cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 9240ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 9300tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 9360taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 9420caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 9480agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 9540gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 9600gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 9660ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 9720acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 9780tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 9840ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 9900ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 9960ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc 10020tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag 10080cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt 10140tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca 10200caggaaacag ctatgaccat gattacgcca agctttgctc ttaggagttt cctaatacat 10260cccaaactca aatatataaa gcatttgact tgttctatgc cctagggggc ggggggaagc 10320taagccagct ttttttaaca tttaaaatgt taattccatt ttaaatgcac agatgttttt 10380atttcataag ggtttcaatg tgcatgaatg ctgcaatatt cctgttacca aagctagtat 10440aaataaaaat agataaacgt ggaaattact tagagtttct gtcattaacg tttccttcct 10500cagttgacaa cataaatgcg ctgctgagca agccagtttg catctgtcag gatcaatttc 10560ccattatgcc agtcatatta attactagtc aattagttga tttttatttt tgacatatac 10620atgtg 106253910619DNAArtificial SequencepSFG4-hCD19-Ev3-CAR 39aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac

cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggtt tgagctggct ctttcttgtt 3180gctatcctca agggggtgca gtgttcaagg gatatccaga tgactcagag ccctagctcc 3240cttagcgcgt ccgtcggcga ccgggttaca attacttgtc gcgcatcaca agacataagt 3300aaatacttga actggtatca acaaaagcca ggaaaggttc caaaactcct catctatcac 3360acgagtagac tccattccgg ggtccctgat cggttctcag gctctggttc tggaactgac 3420ttcactctca cgatctctag tctgcaacct gaggatgtcg ctacctatta ttgccaacag 3480ggaaacaccc tcccttacac atttggccaa ggtaccaaag ttgagatcaa gaggggctcc 3540acatctggga gcgggaaacc gggaagtgga gaaggctcaa ccaaaggtga ggtgcaactt 3600gtagagagtg gtggaggcct ggttcagccg ggagggtccc tcagacttag ttgcgcggct 3660tctggcgttt cactgccgga ctacggggtc agttgggtta ggcaggcgcc tgggaaagga 3720cttgagtgga tcggcgtgat atggggtagt gaaacaacct actacaatag tgctcttaaa 3780tcaaaattca ttatcagccg agacaatgct aagaactccc tctatctcca aatgaatagt 3840cttagagcag aagacacagc cgtttattac tgtgcccgcc attattacta tgggggcagc 3900tacgcaatgg attattgggg ccaagggacc ctggtcacgg tatcaagcta cgtcaccgtc 3960tcttcacagg atcccgccct ggaagagaag aaaggcaatt acgtcgtgac cgaccacggc 4020agctgtgtgc gggcttgtgg cgccgatagc tacgagatgg aagaggacgg cgtgcggaag 4080tgcaagaagt gcgagggccc ctgcagaaaa gtgtgcaacg gcatcggcat cggagagttc 4140aaggatagcc tgagcatcaa cgccaccaac atcaagcact tcaagaactg caccagcatc 4200agcggcgacc tgcacatcct gcccgtggcc tttagaggcg acagcttcac ccacaccccc 4260ccactggatc cccaggaact ggacatcctg aaaaccgtga aagagatcac aggctttctg 4320ctgattcagg cctggcccga gaaccggaca gacctgcacg ccttcgagaa cctggaaatc 4380atcagaggcc ggaccaagca gcacggccag ttttctctgg ccgtggtgtc cctgaacatc 4440accagcctgg gcctgcggag cctgaaagaa atcagcgacg gcgacgtgat catctccggc 4500aacaagaacc tgtgctacgc caacaccatc aattggaaga agctgttcgg cacctccggc 4560cagaaaacaa agatcatctc taaccggggc gagaacagct gcaaagccac cggacaagtg 4620tgccacgccc tgtgtagccc tgagggctgt tggggacccg agcccagaga ttgcgtgtcc 4680tgccggaatg tgtccagagg ccgcgagtgc gtggacaagt gcaacctgct ggaaggcgag 4740ccccgcgagt ttgtggaaaa cagcgagtgc atccagtgcc accccgagtg tctgccccag 4800gccatgaaca ttacctgcac cggcagaggc cccgacaact gtatccagtg cgcccactac 4860atcgacggcc cccactgcgt gaaaacctgt ccagctggcg tgatgggaga gaacaacacc 4920ctcgtgtgga agtacgccga cgccggccat gtgtgccacc tgtgtcaccc caattgcacc 4980tacggctgta ccggccctgg cctggaaggc tgtcctacca acggccccaa gatcccttct 5040aaagatccca aattttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 5100ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 5160agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 5220tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 5280gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 5340agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 5400ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 5460gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 5520ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 5580ctgccccctc gcggaccgca gtgtactaat tatgctctct tgaaattggc tggagatgtt 5640gagagcaatc ccgggcccat gcggatcagc aagccccacc tgcggagcat cagcatccag 5700tgctacctgt gcctgctgct gaacagccac ttcctgaccg aggccggcat ccacgtgttc 5760atcctgggct gcttcagcgc cggactgccc aagaccgagg ccaactgggt gaacgtgatc 5820agcgacctga agaagatcga ggacctgatc cagagcatgc acatcgacgc caccctgtac 5880accgagagcg acgtgcaccc cagctgcaag gtgaccgcca tgaagtgctt tctgctggaa 5940ctgcaggtga tcagcctgga aagcggcgac gccagcatcc acgacaccgt ggagaacctg 6000atcatcctgg ccaacaacag cctgagcagc aacggcaacg tgaccgagag cggctgcaaa 6060gagtgcgagg aactggaaga gaagaacatc aaagagtttc tgcagagctt cgtgcacatc 6120gtgcagatgt tcatcaacac cagctgacgc gtcatcatcg atccggatta gtccaatttg 6180ttaaagacag gatatcagtg gtccaggctc tagttttgac tcaacaatat caccagctga 6240agcctataga gtacgagcca tagataaaat aaaagatttt atttagtctc cagaaaaagg 6300ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca 6360aggcatggaa aaatacataa ctgagaatag agaagttcag atcaaggtca ggaacagatg 6420gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 6480ggccaagaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt 6540cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtccagc cctcagcagt 6600ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct 6660tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag 6720ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg 6780cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct 6840gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcacaca 6900tgcagcatgt atcaaaatta atttggtttt ttttcttaag tatttacatt aaatggccat 6960agtacttaaa gttacattgg cttccttgaa ataaacatgg agtattcaga atgtgtcata 7020aatatttcta attttaagat agtatctcca ttggctttct actttttctt ttattttttt 7080ttgtcctctg tcttccattt gttgttgttg ttgtttgttt gtttgtttgt tggttggttg 7140gttaattttt ttttaaagat cctacactat agttcaagct agactattag ctactctgta 7200acccagggtg accttgaagt catgggtagc ctgctgtttt agccttccca catctaagat 7260tacaggtatg agctatcatt tttggtatat tgattgattg attgattgat gtgtgtgtgt 7320gtgattgtgt ttgtgtgtgt gactgtgaaa atgtgtgtat gggtgtgtgt gaatgtgtgt 7380atgtatgtgt gtgtgtgagt gtgtgtgtgt gtgtgtgcat gtgtgtgtgt gtgactgtgt 7440ctatgtgtat gactgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 7500tgtgaaaaaa tattctatgg tagtgagagc caacgctccg gctcaggtgt caggttggtt 7560tttgagacag agtctttcac ttagcttgga attcactggc cgtcgtttta caacgtcgtg 7620actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 7680gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 7740atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 7800gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 7860acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 7920gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 7980aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 8040taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 8100gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 8160tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 8220ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 8280taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 8340gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 8400aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 8460gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 8520ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 8580ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 8640acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 8700taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 8760tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 8820cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 8880ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 8940gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 9000gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 9060aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 9120aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 9180actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 9240gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 9300atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 9360atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 9420ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 9480gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 9540cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 9600tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 9660cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 9720ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 9780gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 9840tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 9900ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 9960gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 10020cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 10080gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 10140ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 10200acagctatga ccatgattac gccaagcttt gctcttagga gtttcctaat acatcccaaa 10260ctcaaatata taaagcattt gacttgttct atgccctagg gggcgggggg aagctaagcc 10320agcttttttt aacatttaaa atgttaattc cattttaaat gcacagatgt ttttatttca 10380taagggtttc aatgtgcatg aatgctgcaa tattcctgtt accaaagcta gtataaataa 10440aaatagataa acgtggaaat tacttagagt ttctgtcatt aacgtttcct tcctcagttg 10500acaacataaa tgcgctgctg agcaagccag tttgcatctg tcaggatcaa tttcccatta 10560tgccagtcat attaattact agtcaattag ttgattttta tttttgacat atacatgtg 106194010604DNAArtificial SequencepSFG4-CD123-Ev3-CAR 40aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagtttggac tgtcatggct ttttctggtt 3180gctatattga aaggtgtcca atgcagtaga gacgtacaaa taactcaatc ccctagttac 3240ttggctgcgt caccaggaga gaccataaca attaactgta gagcgagcaa atctattagc 3300aaggaccttg catggtacca ggaaaagccc ggaaaaacga ataaacttct tatatactct 3360ggctcaactt tgcaaagtgg aattccgtca agatttagtg gtagcgggtc tggaaccgac 3420ttcacattga ctataagttc cttggagccg gaggattttg cgatgtatta ttgccagcaa 3480cataataaat atccctatac tttcggggga ggaactaaat tggagataaa gggttcaact 3540tctgggtctg gtaaacctgg cagcggcgaa ggtagcacaa aagggcaagt ccaactccag 3600cagcccggcg cggaactcgt gcgacctggc gcttcagtta agctgagctg caaggcctca 3660ggatacacct tcacaagcta ctggatgaat tgggtgaagc agaggcctga tcaaggactc 3720gaatggatag gtagaattga tccctacgac tccgagacgc attataacca gaaatttaag 3780gataaggcga ttcttaccgt ggacaaaagt tcttcaacgg catatatgca actgagcagt 3840ttgacatcag aagacagtgc tgtttattac tgcgcgagag gtaactggga tgactactgg 3900ggccagggca ctaccttggt taccgtgtca agctacgtca ccgtctcttc acaggatccc 3960gccctggaag agaagaaagg caattacgtc gtgaccgacc acggcagctg tgtgcgggct 4020tgtggcgccg atagctacga gatggaagag gacggcgtgc ggaagtgcaa gaagtgcgag 4080ggcccctgca gaaaagtgtg caacggcatc ggcatcggag agttcaagga tagcctgagc 4140atcaacgcca ccaacatcaa gcacttcaag aactgcacca gcatcagcgg cgacctgcac 4200atcctgcccg tggcctttag aggcgacagc ttcacccaca cccccccact ggatccccag 4260gaactggaca tcctgaaaac cgtgaaagag atcacaggct ttctgctgat tcaggcctgg 4320cccgagaacc ggacagacct gcacgccttc gagaacctgg aaatcatcag aggccggacc 4380aagcagcacg gccagttttc tctggccgtg gtgtccctga acatcaccag cctgggcctg 4440cggagcctga aagaaatcag cgacggcgac gtgatcatct ccggcaacaa gaacctgtgc 4500tacgccaaca ccatcaattg gaagaagctg ttcggcacct ccggccagaa aacaaagatc 4560atctctaacc ggggcgagaa cagctgcaaa gccaccggac aagtgtgcca cgccctgtgt 4620agccctgagg gctgttgggg acccgagccc agagattgcg tgtcctgccg gaatgtgtcc 4680agaggccgcg agtgcgtgga caagtgcaac ctgctggaag gcgagccccg cgagtttgtg 4740gaaaacagcg agtgcatcca gtgccacccc gagtgtctgc cccaggccat gaacattacc 4800tgcaccggca gaggccccga caactgtatc cagtgcgccc actacatcga cggcccccac 4860tgcgtgaaaa cctgtccagc tggcgtgatg ggagagaaca acaccctcgt gtggaagtac 4920gccgacgccg gccatgtgtg ccacctgtgt caccccaatt gcacctacgg ctgtaccggc 4980cctggcctgg aaggctgtcc taccaacggc cccaagatcc cttctaaaga tcccaaattt 5040tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 5100tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 5160atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 5220gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 5280cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 5340gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 5400cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 5460attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 5520agtacagcca

ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcgga 5580ccgcagtgta ctaattatgc tctcttgaaa ttggctggag atgttgagag caatcccggg 5640cccatgcgga tcagcaagcc ccacctgcgg agcatcagca tccagtgcta cctgtgcctg 5700ctgctgaaca gccacttcct gaccgaggcc ggcatccacg tgttcatcct gggctgcttc 5760agcgccggac tgcccaagac cgaggccaac tgggtgaacg tgatcagcga cctgaagaag 5820atcgaggacc tgatccagag catgcacatc gacgccaccc tgtacaccga gagcgacgtg 5880caccccagct gcaaggtgac cgccatgaag tgctttctgc tggaactgca ggtgatcagc 5940ctggaaagcg gcgacgccag catccacgac accgtggaga acctgatcat cctggccaac 6000aacagcctga gcagcaacgg caacgtgacc gagagcggct gcaaagagtg cgaggaactg 6060gaagagaaga acatcaaaga gtttctgcag agcttcgtgc acatcgtgca gatgttcatc 6120aacaccagct gacgcgtcat catcgatccg gattagtcca atttgttaaa gacaggatat 6180cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg 6240agccatagat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc 6300ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata 6360cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg 6420ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 6480gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 6540ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc 6600agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 6660atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 6720cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt 6780atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt 6840ctcctctgag tgattgacta cccgtcagcg ggggtctttc acacatgcag catgtatcaa 6900aattaatttg gttttttttc ttaagtattt acattaaatg gccatagtac ttaaagttac 6960attggcttcc ttgaaataaa catggagtat tcagaatgtg tcataaatat ttctaatttt 7020aagatagtat ctccattggc tttctacttt ttcttttatt tttttttgtc ctctgtcttc 7080catttgttgt tgttgttgtt tgtttgtttg tttgttggtt ggttggttaa ttttttttta 7140aagatcctac actatagttc aagctagact attagctact ctgtaaccca gggtgacctt 7200gaagtcatgg gtagcctgct gttttagcct tcccacatct aagattacag gtatgagcta 7260tcatttttgg tatattgatt gattgattga ttgatgtgtg tgtgtgtgat tgtgtttgtg 7320tgtgtgactg tgaaaatgtg tgtatgggtg tgtgtgaatg tgtgtatgta tgtgtgtgtg 7380tgagtgtgtg tgtgtgtgtg tgcatgtgtg tgtgtgtgac tgtgtctatg tgtatgactg 7440tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttgtga aaaaatattc 7500tatggtagtg agagccaacg ctccggctca ggtgtcaggt tggtttttga gacagagtct 7560ttcacttagc ttggaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 7620gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 7680aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 7740tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 7800tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 7860ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 7920tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 7980agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 8040cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 8100tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 8160gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 8220cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 8280atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 8340agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 8400gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 8460ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 8520cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 8580ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 8640atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 8700gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 8760tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 8820gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 8880gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 8940tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 9000ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 9060tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 9120ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 9180ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 9240tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 9300ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 9360tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 9420tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 9480actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 9540cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 9600gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 9660tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 9720ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 9780ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 9840cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 9900cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 9960gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 10020attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 10080ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 10140gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 10200attacgccaa gctttgctct taggagtttc ctaatacatc ccaaactcaa atatataaag 10260catttgactt gttctatgcc ctagggggcg gggggaagct aagccagctt tttttaacat 10320ttaaaatgtt aattccattt taaatgcaca gatgttttta tttcataagg gtttcaatgt 10380gcatgaatgc tgcaatattc ctgttaccaa agctagtata aataaaaata gataaacgtg 10440gaaattactt agagtttctg tcattaacgt ttccttcctc agttgacaac ataaatgcgc 10500tgctgagcaa gccagtttgc atctgtcagg atcaatttcc cattatgcca gtcatattaa 10560ttactagtca attagttgat ttttattttt gacatataca tgtg 106044110604DNAArtificial SequencepSFG4-hCD123-Ev3-CAR 41aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gaattcgggc tttcatggtt gttcctcgtt 3180gcgatactta aaggcgttca gtgcagtaga gacattcaga tgacccaaag ccctagtagc 3240ctctccgcca gtgtgggtga ccgcgtgact attacgtgcc gagcaagcaa gagtattagc 3300aaagatttgg catggtacca gcagaaacca ggcaaggtcc ctaagttgct tatatatagc 3360gggtctacac tccaatctgg agtgccggat cgcttctcag gttccggatc aggaaccgat 3420ttcactctta caatttcctc tctgcaaccg gaggacgtgg caacatacta ttgccagcag 3480cataacaaat atccctatac tttcggtcaa ggtacaaaag tagaaataaa acgaggttcc 3540acctccggga gtgggaagcc cggctccggc gaaggatcca ccaaaggcga ggtacagctc 3600gttgaaagcg ggggcggtct cgtccaacca ggagggtctc ttcgactttc ttgtgctgca 3660agtgggtaca cgtttacttc ctattggatg aattgggttc ggcaagcccc cggtaaaggg 3720cttgaatgga tagggaggat agacccctac gattctgaga cgcattataa tcagaaattc 3780aaagataagt tcataataag cagagacaat gcaaaaaata gtttgtacct ccagatgaat 3840agtcttcggg cagaagacac agcggtatat tactgtgctc ggggtaactg ggatgattac 3900tggggtcagg gaactctggt tactgtgtct agctacgtca ccgtctcttc acaggatccc 3960gccctggaag agaagaaagg caattacgtc gtgaccgacc acggcagctg tgtgcgggct 4020tgtggcgccg atagctacga gatggaagag gacggcgtgc ggaagtgcaa gaagtgcgag 4080ggcccctgca gaaaagtgtg caacggcatc ggcatcggag agttcaagga tagcctgagc 4140atcaacgcca ccaacatcaa gcacttcaag aactgcacca gcatcagcgg cgacctgcac 4200atcctgcccg tggcctttag aggcgacagc ttcacccaca cccccccact ggatccccag 4260gaactggaca tcctgaaaac cgtgaaagag atcacaggct ttctgctgat tcaggcctgg 4320cccgagaacc ggacagacct gcacgccttc gagaacctgg aaatcatcag aggccggacc 4380aagcagcacg gccagttttc tctggccgtg gtgtccctga acatcaccag cctgggcctg 4440cggagcctga aagaaatcag cgacggcgac gtgatcatct ccggcaacaa gaacctgtgc 4500tacgccaaca ccatcaattg gaagaagctg ttcggcacct ccggccagaa aacaaagatc 4560atctctaacc ggggcgagaa cagctgcaaa gccaccggac aagtgtgcca cgccctgtgt 4620agccctgagg gctgttgggg acccgagccc agagattgcg tgtcctgccg gaatgtgtcc 4680agaggccgcg agtgcgtgga caagtgcaac ctgctggaag gcgagccccg cgagtttgtg 4740gaaaacagcg agtgcatcca gtgccacccc gagtgtctgc cccaggccat gaacattacc 4800tgcaccggca gaggccccga caactgtatc cagtgcgccc actacatcga cggcccccac 4860tgcgtgaaaa cctgtccagc tggcgtgatg ggagagaaca acaccctcgt gtggaagtac 4920gccgacgccg gccatgtgtg ccacctgtgt caccccaatt gcacctacgg ctgtaccggc 4980cctggcctgg aaggctgtcc taccaacggc cccaagatcc cttctaaaga tcccaaattt 5040tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 5100tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 5160atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 5220gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 5280cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 5340gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 5400cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 5460attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 5520agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcgga 5580ccgcagtgta ctaattatgc tctcttgaaa ttggctggag atgttgagag caatcccggg 5640cccatgcgga tcagcaagcc ccacctgcgg agcatcagca tccagtgcta cctgtgcctg 5700ctgctgaaca gccacttcct gaccgaggcc ggcatccacg tgttcatcct gggctgcttc 5760agcgccggac tgcccaagac cgaggccaac tgggtgaacg tgatcagcga cctgaagaag 5820atcgaggacc tgatccagag catgcacatc gacgccaccc tgtacaccga gagcgacgtg 5880caccccagct gcaaggtgac cgccatgaag tgctttctgc tggaactgca ggtgatcagc 5940ctggaaagcg gcgacgccag catccacgac accgtggaga acctgatcat cctggccaac 6000aacagcctga gcagcaacgg caacgtgacc gagagcggct gcaaagagtg cgaggaactg 6060gaagagaaga acatcaaaga gtttctgcag agcttcgtgc acatcgtgca gatgttcatc 6120aacaccagct gacgcgtcat catcgatccg gattagtcca atttgttaaa gacaggatat 6180cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg 6240agccatagat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc 6300ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata 6360cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg 6420ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 6480gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 6540ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc 6600agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 6660atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 6720cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt 6780atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt 6840ctcctctgag tgattgacta cccgtcagcg ggggtctttc acacatgcag catgtatcaa 6900aattaatttg gttttttttc ttaagtattt acattaaatg gccatagtac ttaaagttac 6960attggcttcc ttgaaataaa catggagtat tcagaatgtg tcataaatat ttctaatttt 7020aagatagtat ctccattggc tttctacttt ttcttttatt tttttttgtc ctctgtcttc 7080catttgttgt tgttgttgtt tgtttgtttg tttgttggtt ggttggttaa ttttttttta 7140aagatcctac actatagttc aagctagact attagctact ctgtaaccca gggtgacctt 7200gaagtcatgg gtagcctgct gttttagcct tcccacatct aagattacag gtatgagcta 7260tcatttttgg tatattgatt gattgattga ttgatgtgtg tgtgtgtgat tgtgtttgtg 7320tgtgtgactg tgaaaatgtg tgtatgggtg tgtgtgaatg tgtgtatgta tgtgtgtgtg 7380tgagtgtgtg tgtgtgtgtg tgcatgtgtg tgtgtgtgac tgtgtctatg tgtatgactg 7440tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttgtga aaaaatattc 7500tatggtagtg agagccaacg ctccggctca ggtgtcaggt tggtttttga gacagagtct 7560ttcacttagc ttggaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 7620gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 7680aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 7740tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 7800tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 7860ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 7920tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 7980agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 8040cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 8100tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 8160gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 8220cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 8280atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 8340agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 8400gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 8460ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 8520cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 8580ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 8640atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 8700gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 8760tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 8820gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 8880gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 8940tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 9000ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 9060tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 9120ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 9180ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 9240tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 9300ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 9360tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 9420tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 9480actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 9540cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 9600gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 9660tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 9720ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 9780ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 9840cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 9900cctttgagtg

agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 9960gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 10020attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 10080ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 10140gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 10200attacgccaa gctttgctct taggagtttc ctaatacatc ccaaactcaa atatataaag 10260catttgactt gttctatgcc ctagggggcg gggggaagct aagccagctt tttttaacat 10320ttaaaatgtt aattccattt taaatgcaca gatgttttta tttcataagg gtttcaatgt 10380gcatgaatgc tgcaatattc ctgttaccaa agctagtata aataaaaata gataaacgtg 10440gaaattactt agagtttctg tcattaacgt ttccttcctc agttgacaac ataaatgcgc 10500tgctgagcaa gccagtttgc atctgtcagg atcaatttcc cattatgcca gtcatattaa 10560ttactagtca attagttgat ttttattttt gacatataca tgtg 106044210610DNAArtificial SequencepSFG4-Meso-Ev3-CAR 42aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggcc ttagctggct gttccttgtg 3180gccatactca agggcgtcca gtgtagtcgc gatatagagc tgacgcaaag cccggcaatt 3240atgtcagcaa gtccaggaga aaaggtgact atgacttgtt ccgcaagcag ttctgtaagt 3300tacatgcact ggtaccagca aaaaagtgga actagcccta agcggtggat ctatgacacg 3360tctaaattgg cctctggtgt ccccggacga ttttcaggtt caggaagtgg aaacagttac 3420tctcttacta taagtagcgt cgaggcggag gatgacgcca cctactactg ccagcagtgg 3480agcgggtacc ccctcacctt cggggccggg accaagttgg agattaaagg atcaacatcc 3540gggagcggaa agccggggtc aggtgagggc tcaacaaaag ggcaggtaca gctccagcaa 3600agtgggcccg agctggaaaa accgggtgcg agtgtcaaac tttcttgtaa ggccagcggt 3660tactctttca cgggttatac aatgaattgg gtgaaacagt cccacggcaa gagcctcgaa 3720tggataggtc tcatcacacc atacaacgga gcttcctcct ataatcagaa gttccgggga 3780aaggcaaccc tcactgtaga caagtcttcc tcaacagcat atatggatct cctgtcactc 3840acgtccgaag acagcgcggt ttatttttgt gcgagagggg gttatgacgg gcgcgggttc 3900gactactggg gtcaaggcac tacagtaacg gtaagcagct acgtcaccgt ctcttcacag 3960gatcccgccc tggaagagaa gaaaggcaat tacgtcgtga ccgaccacgg cagctgtgtg 4020cgggcttgtg gcgccgatag ctacgagatg gaagaggacg gcgtgcggaa gtgcaagaag 4080tgcgagggcc cctgcagaaa agtgtgcaac ggcatcggca tcggagagtt caaggatagc 4140ctgagcatca acgccaccaa catcaagcac ttcaagaact gcaccagcat cagcggcgac 4200ctgcacatcc tgcccgtggc ctttagaggc gacagcttca cccacacccc cccactggat 4260ccccaggaac tggacatcct gaaaaccgtg aaagagatca caggctttct gctgattcag 4320gcctggcccg agaaccggac agacctgcac gccttcgaga acctggaaat catcagaggc 4380cggaccaagc agcacggcca gttttctctg gccgtggtgt ccctgaacat caccagcctg 4440ggcctgcgga gcctgaaaga aatcagcgac ggcgacgtga tcatctccgg caacaagaac 4500ctgtgctacg ccaacaccat caattggaag aagctgttcg gcacctccgg ccagaaaaca 4560aagatcatct ctaaccgggg cgagaacagc tgcaaagcca ccggacaagt gtgccacgcc 4620ctgtgtagcc ctgagggctg ttggggaccc gagcccagag attgcgtgtc ctgccggaat 4680gtgtccagag gccgcgagtg cgtggacaag tgcaacctgc tggaaggcga gccccgcgag 4740tttgtggaaa acagcgagtg catccagtgc caccccgagt gtctgcccca ggccatgaac 4800attacctgca ccggcagagg ccccgacaac tgtatccagt gcgcccacta catcgacggc 4860ccccactgcg tgaaaacctg tccagctggc gtgatgggag agaacaacac cctcgtgtgg 4920aagtacgccg acgccggcca tgtgtgccac ctgtgtcacc ccaattgcac ctacggctgt 4980accggccctg gcctggaagg ctgtcctacc aacggcccca agatcccttc taaagatccc 5040aaattttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 5100gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 5160atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 5220ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 5280gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 5340tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 5400aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 5460agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 5520ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 5580cgcggaccgc agtgtactaa ttatgctctc ttgaaattgg ctggagatgt tgagagcaat 5640cccgggccca tgcggatcag caagccccac ctgcggagca tcagcatcca gtgctacctg 5700tgcctgctgc tgaacagcca cttcctgacc gaggccggca tccacgtgtt catcctgggc 5760tgcttcagcg ccggactgcc caagaccgag gccaactggg tgaacgtgat cagcgacctg 5820aagaagatcg aggacctgat ccagagcatg cacatcgacg ccaccctgta caccgagagc 5880gacgtgcacc ccagctgcaa ggtgaccgcc atgaagtgct ttctgctgga actgcaggtg 5940atcagcctgg aaagcggcga cgccagcatc cacgacaccg tggagaacct gatcatcctg 6000gccaacaaca gcctgagcag caacggcaac gtgaccgaga gcggctgcaa agagtgcgag 6060gaactggaag agaagaacat caaagagttt ctgcagagct tcgtgcacat cgtgcagatg 6120ttcatcaaca ccagctgacg cgtcatcatc gatccggatt agtccaattt gttaaagaca 6180ggatatcagt ggtccaggct ctagttttga ctcaacaata tcaccagctg aagcctatag 6240agtacgagcc atagataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga 6300aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga 6360aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagctg 6420aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 6480cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg 6540gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga 6600accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac 6660taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa 6720agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac 6780ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg 6840gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcacac atgcagcatg 6900tatcaaaatt aatttggttt tttttcttaa gtatttacat taaatggcca tagtacttaa 6960agttacattg gcttccttga aataaacatg gagtattcag aatgtgtcat aaatatttct 7020aattttaaga tagtatctcc attggctttc tactttttct tttatttttt tttgtcctct 7080gtcttccatt tgttgttgtt gttgtttgtt tgtttgtttg ttggttggtt ggttaatttt 7140tttttaaaga tcctacacta tagttcaagc tagactatta gctactctgt aacccagggt 7200gaccttgaag tcatgggtag cctgctgttt tagccttccc acatctaaga ttacaggtat 7260gagctatcat ttttggtata ttgattgatt gattgattga tgtgtgtgtg tgtgattgtg 7320tttgtgtgtg tgactgtgaa aatgtgtgta tgggtgtgtg tgaatgtgtg tatgtatgtg 7380tgtgtgtgag tgtgtgtgtg tgtgtgtgca tgtgtgtgtg tgtgactgtg tctatgtgta 7440tgactgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg ttgtgaaaaa 7500atattctatg gtagtgagag ccaacgctcc ggctcaggtg tcaggttggt ttttgagaca 7560gagtctttca cttagcttgg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 7620accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 7680atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 7740ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 7800gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 7860cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 7920tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 7980gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 8040cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 8100tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 8160aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 8220ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 8280ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 8340tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 8400tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 8460actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 8520gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 8580acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 8640gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 8700acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 8760gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 8820ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 8880gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 8940cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 9000agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 9060catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 9120tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 9180cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 9240gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 9300taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 9360ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 9420tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 9480ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 9540cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 9600agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 9660gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 9720atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 9780gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 9840gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 9900ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 9960cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 10020cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 10080acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 10140cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 10200accatgatta cgccaagctt tgctcttagg agtttcctaa tacatcccaa actcaaatat 10260ataaagcatt tgacttgttc tatgccctag ggggcggggg gaagctaagc cagctttttt 10320taacatttaa aatgttaatt ccattttaaa tgcacagatg tttttatttc ataagggttt 10380caatgtgcat gaatgctgca atattcctgt taccaaagct agtataaata aaaatagata 10440aacgtggaaa ttacttagag tttctgtcat taacgtttcc ttcctcagtt gacaacataa 10500atgcgctgct gagcaagcca gtttgcatct gtcaggatca atttcccatt atgccagtca 10560tattaattac tagtcaatta gttgattttt atttttgaca tatacatgtg 106104310595DNAArtificial SequencepSFG4-hMeso-Ev3-CAR 43aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggcc tttcttggct gtttttggtc 3180gcaatattga agggcgttca gtgttcaagg gacatacaga tgacgcagtc acctagctct 3240ctttcagcct ctgtgggaga ccgcgttaca attacgtgtt ctgcttctag ttcagtgtct 3300tacatgcatt ggtaccagca aaaacccggc aaggttccaa agctccttat atacgatacg 3360tccaaactgg cttcaggcgt gccagacagg tttagtggat ctgggagcgg cactgacttc 3420acacttacaa tatccagtct gcaacccgaa gatgttgcga catactattg tcagcaatgg 3480agcggctatc cgctcaccgg acaggggaca aaagtggaga taaagcgggg ctccaccagt 3540ggttccggga agcctggtag tggtgagggt tctacgaaag gcgaagtgca gttggtggag 3600tctggtggcg

ggcttgttca acccggtggt tctctcaggc ttagctgtgc agcctctggg 3660tacacaatga attgggtaag gcaagcgcct ggaaagggcc ttgagtggat cggtttgata 3720accccataca acggagcttc aagttacaac caaaagttcc gcgggaagtt tattataagc 3780agggataacg caaagaacag cctctatctt caaatgaact cactccgggc tgaagacaca 3840gccgtctatt attgtgcccg ggggggctac gacggacgag ggttcgatta ctggggtcag 3900ggcacactcg tgaccgtgtc aagctacgtc accgtctctt cacaggatcc cgccctggaa 3960gagaagaaag gcaattacgt cgtgaccgac cacggcagct gtgtgcgggc ttgtggcgcc 4020gatagctacg agatggaaga ggacggcgtg cggaagtgca agaagtgcga gggcccctgc 4080agaaaagtgt gcaacggcat cggcatcgga gagttcaagg atagcctgag catcaacgcc 4140accaacatca agcacttcaa gaactgcacc agcatcagcg gcgacctgca catcctgccc 4200gtggccttta gaggcgacag cttcacccac acccccccac tggatcccca ggaactggac 4260atcctgaaaa ccgtgaaaga gatcacaggc tttctgctga ttcaggcctg gcccgagaac 4320cggacagacc tgcacgcctt cgagaacctg gaaatcatca gaggccggac caagcagcac 4380ggccagtttt ctctggccgt ggtgtccctg aacatcacca gcctgggcct gcggagcctg 4440aaagaaatca gcgacggcga cgtgatcatc tccggcaaca agaacctgtg ctacgccaac 4500accatcaatt ggaagaagct gttcggcacc tccggccaga aaacaaagat catctctaac 4560cggggcgaga acagctgcaa agccaccgga caagtgtgcc acgccctgtg tagccctgag 4620ggctgttggg gacccgagcc cagagattgc gtgtcctgcc ggaatgtgtc cagaggccgc 4680gagtgcgtgg acaagtgcaa cctgctggaa ggcgagcccc gcgagtttgt ggaaaacagc 4740gagtgcatcc agtgccaccc cgagtgtctg ccccaggcca tgaacattac ctgcaccggc 4800agaggccccg acaactgtat ccagtgcgcc cactacatcg acggccccca ctgcgtgaaa 4860acctgtccag ctggcgtgat gggagagaac aacaccctcg tgtggaagta cgccgacgcc 4920ggccatgtgt gccacctgtg tcaccccaat tgcacctacg gctgtaccgg ccctggcctg 4980gaaggctgtc ctaccaacgg ccccaagatc ccttctaaag atcccaaatt ttgggtgctg 5040gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 5100ttctgggtga ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 5160cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 5220gcctatcgct ccagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 5280cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 5340aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 5400ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 5460aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 5520accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcgg accgcagtgt 5580actaattatg ctctcttgaa attggctgga gatgttgaga gcaatcccgg gcccatgcgg 5640atcagcaagc cccacctgcg gagcatcagc atccagtgct acctgtgcct gctgctgaac 5700agccacttcc tgaccgaggc cggcatccac gtgttcatcc tgggctgctt cagcgccgga 5760ctgcccaaga ccgaggccaa ctgggtgaac gtgatcagcg acctgaagaa gatcgaggac 5820ctgatccaga gcatgcacat cgacgccacc ctgtacaccg agagcgacgt gcaccccagc 5880tgcaaggtga ccgccatgaa gtgctttctg ctggaactgc aggtgatcag cctggaaagc 5940ggcgacgcca gcatccacga caccgtggag aacctgatca tcctggccaa caacagcctg 6000agcagcaacg gcaacgtgac cgagagcggc tgcaaagagt gcgaggaact ggaagagaag 6060aacatcaaag agtttctgca gagcttcgtg cacatcgtgc agatgttcat caacaccagc 6120tgacgcgtca tcatcgatcc ggattagtcc aatttgttaa agacaggata tcagtggtcc 6180aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac gagccataga 6240taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta 6300ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat acataactga 6360gaatagagaa gttcagatca aggtcaggaa cagatggaac agctgaatat gggccaaaca 6420ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggaacagctg 6480aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 6540cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat cagatgtttc 6600cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg 6660cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc 6720tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg tatccaataa 6780accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga 6840gtgattgact acccgtcagc gggggtcttt cacacatgca gcatgtatca aaattaattt 6900ggtttttttt cttaagtatt tacattaaat ggccatagta cttaaagtta cattggcttc 6960cttgaaataa acatggagta ttcagaatgt gtcataaata tttctaattt taagatagta 7020tctccattgg ctttctactt tttcttttat ttttttttgt cctctgtctt ccatttgttg 7080ttgttgttgt ttgtttgttt gtttgttggt tggttggtta attttttttt aaagatccta 7140cactatagtt caagctagac tattagctac tctgtaaccc agggtgacct tgaagtcatg 7200ggtagcctgc tgttttagcc ttcccacatc taagattaca ggtatgagct atcatttttg 7260gtatattgat tgattgattg attgatgtgt gtgtgtgtga ttgtgtttgt gtgtgtgact 7320gtgaaaatgt gtgtatgggt gtgtgtgaat gtgtgtatgt atgtgtgtgt gtgagtgtgt 7380gtgtgtgtgt gtgcatgtgt gtgtgtgtga ctgtgtctat gtgtatgact gtgtgtgtgt 7440gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgttgtg aaaaaatatt ctatggtagt 7500gagagccaac gctccggctc aggtgtcagg ttggtttttg agacagagtc tttcacttag 7560cttggaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 7620aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 7680gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt 7740attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 7800tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 7860cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 7920gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 7980tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 8040gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 8100atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 8160agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 8220ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 8280gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 8340gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 8400tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 8460acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 8520aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 8580cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 8640gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 8700cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 8760tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 8820tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 8880ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 8940tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 9000gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 9060ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 9120tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 9180agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 9240aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 9300cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 9360agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 9420tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 9480gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 9540gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 9600ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 9660gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 9720ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 9780ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 9840acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 9900gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 9960cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 10020gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 10080gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 10140gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 10200agctttgctc ttaggagttt cctaatacat cccaaactca aatatataaa gcatttgact 10260tgttctatgc cctagggggc ggggggaagc taagccagct ttttttaaca tttaaaatgt 10320taattccatt ttaaatgcac agatgttttt atttcataag ggtttcaatg tgcatgaatg 10380ctgcaatatt cctgttacca aagctagtat aaataaaaat agataaacgt ggaaattact 10440tagagtttct gtcattaacg tttccttcct cagttgacaa cataaatgcg ctgctgagca 10500agccagtttg catctgtcag gatcaatttc ccattatgcc agtcatatta attactagtc 10560aattagttga tttttatttt tgacatatac atgtg 105954410613DNAArtificial SequencepSFG4-ROR1-Ev3-CAR 44aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggtc ttagctggct gttcctcgtg 3180gcaatcctca agggagtaca atgcagcaga gatattaaga tgacgcagtc cccctcttct 3240atgtacgcga gtctgggaga gagggtaacc attacatgta aggcgtctcc cgatataaac 3300tcatatctga gctggtttca acaaaagccg ggcaagagtc cgaagacact catttaccga 3360gcaaaccgcc tggtagacgg tgtgccttcc agattctctg gcggtggcag cgggcaagac 3420tactcactca ccataaattc acttgaatac gaggatatgg gtatatacta ctgcttgcaa 3480tatgatgagt ttccatatac ttttggcggc ggaactaaac ttgaaatgaa agggagcacg 3540tctggttccg gcaagcctgg ttcaggagag ggtagcacca aaggggaagt caaactcgtc 3600gaaagtggtg gtggcttggt gaaacccggc ggatctctga aactcagttg tgccgcttct 3660ggttttacat ttagctccta cgcgatgagt tgggtcaggc aaataccaga gaagagactt 3720gagtgggtcg ctagtattag caggggaggg accacctact atccagactc tgtgaaaggt 3780agattcacta tctctagaga caacgtacgc aatatcctgt atctccaaat gagctcattg 3840aggagcgagg atacggctat gtactattgt ggccgatacg actatgatgg atactatgcg 3900atggattact ggggccaagg cacttctgta acagtttcta gctacgtcac cgtctcttca 3960caggatcccg ccctggaaga gaagaaaggc aattacgtcg tgaccgacca cggcagctgt 4020gtgcgggctt gtggcgccga tagctacgag atggaagagg acggcgtgcg gaagtgcaag 4080aagtgcgagg gcccctgcag aaaagtgtgc aacggcatcg gcatcggaga gttcaaggat 4140agcctgagca tcaacgccac caacatcaag cacttcaaga actgcaccag catcagcggc 4200gacctgcaca tcctgcccgt ggcctttaga ggcgacagct tcacccacac ccccccactg 4260gatccccagg aactggacat cctgaaaacc gtgaaagaga tcacaggctt tctgctgatt 4320caggcctggc ccgagaaccg gacagacctg cacgccttcg agaacctgga aatcatcaga 4380ggccggacca agcagcacgg ccagttttct ctggccgtgg tgtccctgaa catcaccagc 4440ctgggcctgc ggagcctgaa agaaatcagc gacggcgacg tgatcatctc cggcaacaag 4500aacctgtgct acgccaacac catcaattgg aagaagctgt tcggcacctc cggccagaaa 4560acaaagatca tctctaaccg gggcgagaac agctgcaaag ccaccggaca agtgtgccac 4620gccctgtgta gccctgaggg ctgttgggga cccgagccca gagattgcgt gtcctgccgg 4680aatgtgtcca gaggccgcga gtgcgtggac aagtgcaacc tgctggaagg cgagccccgc 4740gagtttgtgg aaaacagcga gtgcatccag tgccaccccg agtgtctgcc ccaggccatg 4800aacattacct gcaccggcag aggccccgac aactgtatcc agtgcgccca ctacatcgac 4860ggcccccact gcgtgaaaac ctgtccagct ggcgtgatgg gagagaacaa caccctcgtg 4920tggaagtacg ccgacgccgg ccatgtgtgc cacctgtgtc accccaattg cacctacggc 4980tgtaccggcc ctggcctgga aggctgtcct accaacggcc ccaagatccc ttctaaagat 5040cccaaatttt gggtgctggt ggtggttggt ggagtcctgg cttgctatag cttgctagta 5100acagtggcct ttattatttt ctgggtgagg agtaagagga gcaggctcct gcacagtgac 5160tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 5220ccaccacgcg acttcgcagc ctatcgctcc agagtgaagt tcagcaggag cgcagacgcc 5280cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 5340gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 5400aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 5460tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 5520cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 5580cctcgcggac cgcagtgtac taattatgct ctcttgaaat tggctggaga tgttgagagc 5640aatcccgggc ccatgcggat cagcaagccc cacctgcgga gcatcagcat ccagtgctac 5700ctgtgcctgc tgctgaacag ccacttcctg accgaggccg gcatccacgt gttcatcctg 5760ggctgcttca gcgccggact gcccaagacc gaggccaact gggtgaacgt gatcagcgac 5820ctgaagaaga tcgaggacct gatccagagc atgcacatcg acgccaccct gtacaccgag 5880agcgacgtgc accccagctg caaggtgacc gccatgaagt gctttctgct ggaactgcag 5940gtgatcagcc tggaaagcgg cgacgccagc atccacgaca ccgtggagaa cctgatcatc 6000ctggccaaca acagcctgag cagcaacggc aacgtgaccg agagcggctg caaagagtgc 6060gaggaactgg aagagaagaa catcaaagag tttctgcaga gcttcgtgca catcgtgcag 6120atgttcatca acaccagctg acgcgtcatc atcgatccgg attagtccaa tttgttaaag 6180acaggatatc agtggtccag gctctagttt tgactcaaca atatcaccag ctgaagccta 6240tagagtacga gccatagata aaataaaaga ttttatttag tctccagaaa aaggggggaa 6300tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 6360ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag 6420ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 6480gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc 6540ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag 6600agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg 6660aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat 6720aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg 6780tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct 6840tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca cacatgcagc 6900atgtatcaaa attaatttgg ttttttttct taagtattta cattaaatgg ccatagtact 6960taaagttaca ttggcttcct tgaaataaac atggagtatt cagaatgtgt cataaatatt 7020tctaatttta agatagtatc tccattggct ttctactttt tcttttattt ttttttgtcc 7080tctgtcttcc atttgttgtt gttgttgttt gtttgtttgt ttgttggttg gttggttaat 7140ttttttttaa agatcctaca ctatagttca agctagacta ttagctactc tgtaacccag 7200ggtgaccttg aagtcatggg tagcctgctg ttttagcctt cccacatcta agattacagg 7260tatgagctat catttttggt atattgattg attgattgat tgatgtgtgt gtgtgtgatt 7320gtgtttgtgt gtgtgactgt gaaaatgtgt gtatgggtgt gtgtgaatgt gtgtatgtat 7380gtgtgtgtgt gagtgtgtgt gtgtgtgtgt gcatgtgtgt gtgtgtgact gtgtctatgt 7440gtatgactgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttgtgaa 7500aaaatattct atggtagtga gagccaacgc tccggctcag gtgtcaggtt ggtttttgag 7560acagagtctt tcacttagct tggaattcac tggccgtcgt tttacaacgt cgtgactggg 7620aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 7680gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 7740aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 7800ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 7860caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 7920ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 7980cgagacgaaa

gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 8040tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 8100ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 8160aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 8220tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 8280atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 8340agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 8400tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 8460tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 8520atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 8580ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 8640tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 8700acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 8760ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 8820aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 8880ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 8940cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 9000gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 9060actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 9120agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 9180cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 9240tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 9300agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 9360ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 9420acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 9480ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 9540gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 9600gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 9660gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 9720tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 9780caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 9840tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 9900gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 9960agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 10020ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 10080gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 10140ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 10200atgaccatga ttacgccaag ctttgctctt aggagtttcc taatacatcc caaactcaaa 10260tatataaagc atttgacttg ttctatgccc tagggggcgg ggggaagcta agccagcttt 10320ttttaacatt taaaatgtta attccatttt aaatgcacag atgtttttat ttcataaggg 10380tttcaatgtg catgaatgct gcaatattcc tgttaccaaa gctagtataa ataaaaatag 10440ataaacgtgg aaattactta gagtttctgt cattaacgtt tccttcctca gttgacaaca 10500taaatgcgct gctgagcaag ccagtttgca tctgtcagga tcaatttccc attatgccag 10560tcatattaat tactagtcaa ttagttgatt tttatttttg acatatacat gtg 106134510616DNAArtificial SequencepSFG4-hROR1-Ev3-CAR 45aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggac ttagttggct tttcctcgtc 3180gccatactca aaggtgtaca atgctcacgc gatatacaaa tgacgcagag tccatcctca 3240ctgtctgcct cagtaggcga cagggtcaca ataacctgta aagcaagccc ggatatcaac 3300tcttacctca gttggttcca acagaaacca ggaaaggtgc cgaagttgtt gatttaccgc 3360gcaaaccgcc tcgttgacgg cgtgccagat aggttttctg gcagcgggag cggcactgac 3420tttacgctca ctatatcaag cctgcagccg gaggacgtag caacttacta ttgcctccaa 3480tacgacgaat tcccttacac gttcgggcaa ggaactaaag ttgaaataaa aagaggaagc 3540acaagtggtt ctggaaaacc cggttccggt gagggcagta ccaaaggaga ggtgcagttg 3600gtagagtctg gtgggggttt ggtgcaaccc ggagggtctc tgaggttgtc ttgcgcggca 3660tccggcttta ctttttcctc atatgcgatg tcatgggtac gacaggcccc aggcaaggga 3720ctggagtgga tcgcatctat ttcacgagga gggacgacat attacccgga ttcagtgaag 3780ggcaaattca taatttcacg cgataacgcc aagaactccc tctacctgca aatgaacagt 3840cttagagcgg aagatacggc tgtgtattac tgtgcgcgat atgactacga tggatattac 3900gcgatggatt attggggaca aggtacactg gtaaccgttt ccagctacgt caccgtctct 3960tcacaggatc ccgccctgga agagaagaaa ggcaattacg tcgtgaccga ccacggcagc 4020tgtgtgcggg cttgtggcgc cgatagctac gagatggaag aggacggcgt gcggaagtgc 4080aagaagtgcg agggcccctg cagaaaagtg tgcaacggca tcggcatcgg agagttcaag 4140gatagcctga gcatcaacgc caccaacatc aagcacttca agaactgcac cagcatcagc 4200ggcgacctgc acatcctgcc cgtggccttt agaggcgaca gcttcaccca caccccccca 4260ctggatcccc aggaactgga catcctgaaa accgtgaaag agatcacagg ctttctgctg 4320attcaggcct ggcccgagaa ccggacagac ctgcacgcct tcgagaacct ggaaatcatc 4380agaggccgga ccaagcagca cggccagttt tctctggccg tggtgtccct gaacatcacc 4440agcctgggcc tgcggagcct gaaagaaatc agcgacggcg acgtgatcat ctccggcaac 4500aagaacctgt gctacgccaa caccatcaat tggaagaagc tgttcggcac ctccggccag 4560aaaacaaaga tcatctctaa ccggggcgag aacagctgca aagccaccgg acaagtgtgc 4620cacgccctgt gtagccctga gggctgttgg ggacccgagc ccagagattg cgtgtcctgc 4680cggaatgtgt ccagaggccg cgagtgcgtg gacaagtgca acctgctgga aggcgagccc 4740cgcgagtttg tggaaaacag cgagtgcatc cagtgccacc ccgagtgtct gccccaggcc 4800atgaacatta cctgcaccgg cagaggcccc gacaactgta tccagtgcgc ccactacatc 4860gacggccccc actgcgtgaa aacctgtcca gctggcgtga tgggagagaa caacaccctc 4920gtgtggaagt acgccgacgc cggccatgtg tgccacctgt gtcaccccaa ttgcacctac 4980ggctgtaccg gccctggcct ggaaggctgt cctaccaacg gccccaagat cccttctaaa 5040gatcccaaat tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta tagcttgcta 5100gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcaggct cctgcacagt 5160gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 5220gccccaccac gcgacttcgc agcctatcgc tccagagtga agttcagcag gagcgcagac 5280gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 5340gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 5400agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 5460gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 5520taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 5580ccccctcgcg gaccgcagtg tactaattat gctctcttga aattggctgg agatgttgag 5640agcaatcccg ggcccatgcg gatcagcaag ccccacctgc ggagcatcag catccagtgc 5700tacctgtgcc tgctgctgaa cagccacttc ctgaccgagg ccggcatcca cgtgttcatc 5760ctgggctgct tcagcgccgg actgcccaag accgaggcca actgggtgaa cgtgatcagc 5820gacctgaaga agatcgagga cctgatccag agcatgcaca tcgacgccac cctgtacacc 5880gagagcgacg tgcaccccag ctgcaaggtg accgccatga agtgctttct gctggaactg 5940caggtgatca gcctggaaag cggcgacgcc agcatccacg acaccgtgga gaacctgatc 6000atcctggcca acaacagcct gagcagcaac ggcaacgtga ccgagagcgg ctgcaaagag 6060tgcgaggaac tggaagagaa gaacatcaaa gagtttctgc agagcttcgt gcacatcgtg 6120cagatgttca tcaacaccag ctgacgcgtc atcatcgatc cggattagtc caatttgtta 6180aagacaggat atcagtggtc caggctctag ttttgactca acaatatcac cagctgaagc 6240ctatagagta cgagccatag ataaaataaa agattttatt tagtctccag aaaaaggggg 6300gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg 6360catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga acagatggaa 6420cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc 6480caagaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct 6540gccccggctc agggccaaga acagatggtc cccagatgcg gtccagccct cagcagtttc 6600tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat 6660ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc 6720aataaaagag cccacaaccc ctcactcggg gcgccagtcc tccgattgac tgagtcgccc 6780gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt 6840ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt tcacacatgc 6900agcatgtatc aaaattaatt tggttttttt tcttaagtat ttacattaaa tggccatagt 6960acttaaagtt acattggctt ccttgaaata aacatggagt attcagaatg tgtcataaat 7020atttctaatt ttaagatagt atctccattg gctttctact ttttctttta tttttttttg 7080tcctctgtct tccatttgtt gttgttgttg tttgtttgtt tgtttgttgg ttggttggtt 7140aatttttttt taaagatcct acactatagt tcaagctaga ctattagcta ctctgtaacc 7200cagggtgacc ttgaagtcat gggtagcctg ctgttttagc cttcccacat ctaagattac 7260aggtatgagc tatcattttt ggtatattga ttgattgatt gattgatgtg tgtgtgtgtg 7320attgtgtttg tgtgtgtgac tgtgaaaatg tgtgtatggg tgtgtgtgaa tgtgtgtatg 7380tatgtgtgtg tgtgagtgtg tgtgtgtgtg tgtgcatgtg tgtgtgtgtg actgtgtcta 7440tgtgtatgac tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgttgt 7500gaaaaaatat tctatggtag tgagagccaa cgctccggct caggtgtcag gttggttttt 7560gagacagagt ctttcactta gcttggaatt cactggccgt cgttttacaa cgtcgtgact 7620gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 7680ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 7740gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 7800tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 7860cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 7920aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 7980gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 8040tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 8100tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 8160ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 8220ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 8280aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 8340gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 8400ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 8460gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 8520cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 8580cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 8640acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 8700caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 8760taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 8820ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 8880aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 8940agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 9000atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 9060tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 9120tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 9180gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 9240taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 9300aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 9360ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 9420catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 9480ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 9540ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 9600agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 9660taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 9720atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 9780cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 9840ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 9900accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 9960gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc 10020gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 10080agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta 10140tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca 10200gctatgacca tgattacgcc aagctttgct cttaggagtt tcctaataca tcccaaactc 10260aaatatataa agcatttgac ttgttctatg ccctaggggg cggggggaag ctaagccagc 10320tttttttaac atttaaaatg ttaattccat tttaaatgca cagatgtttt tatttcataa 10380gggtttcaat gtgcatgaat gctgcaatat tcctgttacc aaagctagta taaataaaaa 10440tagataaacg tggaaattac ttagagtttc tgtcattaac gtttccttcc tcagttgaca 10500acataaatgc gctgctgagc aagccagttt gcatctgtca ggatcaattt cccattatgc 10560cagtcatatt aattactagt caattagttg atttttattt ttgacatata catgtg 106164610610DNAArtificial SequencepSFG4-CD5-Ev3-CAR 46aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac

ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggct tgagttggtt gttccttgtg 3180gcgatactca aaggcgttca atgtagccga gacataaaaa tgacccagtc tccgtcatct 3240atgtatgcaa gcctcggcga gcgagtgacc atcacgtgca aggcgagtca agatataaac 3300agctacttgt catggttcca acaaaaacca gggaaatcac ctaagaccct gatctataga 3360gccaatcgcc tggttgacgg tgtcccctcc cgctttagcg gctccggaag cggtcaagat 3420tactctctca caatttcttc cttggattat gaagacatgg ggatctacta ttgtcaacag 3480tatgacgaat ccccgtggac tttcggtggc ggtaccaaat tggaaataaa gggctctaca 3540agcggctcag gaaaacctgg atcaggcgaa gggtctacga agggccagat acaactcgtt 3600caaagtgggc cagaactcaa aaaaccggga gaaacagtga aaatttcttg taaggcatca 3660ggatacacat tcacaaacta cgggatgaat tgggtcaaac aagcacccgg aaaggggctg 3720cgctggatgg ggtggatcaa cacacatact ggggaaccta cttacgcaga cgatttcaag 3780ggcagattcg ccttttcttt ggagacctcc gcctctactg catacttgca gataaacaac 3840ctgaagaatg aagataccgc cacctacttc tgtacgcgca ggggctacga ttggtatttt 3900gatgtatggg gggcaggcac cactgttact gtgtcaagct acgtcaccgt ctcttcacag 3960gatcccgccc tggaagagaa gaaaggcaat tacgtcgtga ccgaccacgg cagctgtgtg 4020cgggcttgtg gcgccgatag ctacgagatg gaagaggacg gcgtgcggaa gtgcaagaag 4080tgcgagggcc cctgcagaaa agtgtgcaac ggcatcggca tcggagagtt caaggatagc 4140ctgagcatca acgccaccaa catcaagcac ttcaagaact gcaccagcat cagcggcgac 4200ctgcacatcc tgcccgtggc ctttagaggc gacagcttca cccacacccc cccactggat 4260ccccaggaac tggacatcct gaaaaccgtg aaagagatca caggctttct gctgattcag 4320gcctggcccg agaaccggac agacctgcac gccttcgaga acctggaaat catcagaggc 4380cggaccaagc agcacggcca gttttctctg gccgtggtgt ccctgaacat caccagcctg 4440ggcctgcgga gcctgaaaga aatcagcgac ggcgacgtga tcatctccgg caacaagaac 4500ctgtgctacg ccaacaccat caattggaag aagctgttcg gcacctccgg ccagaaaaca 4560aagatcatct ctaaccgggg cgagaacagc tgcaaagcca ccggacaagt gtgccacgcc 4620ctgtgtagcc ctgagggctg ttggggaccc gagcccagag attgcgtgtc ctgccggaat 4680gtgtccagag gccgcgagtg cgtggacaag tgcaacctgc tggaaggcga gccccgcgag 4740tttgtggaaa acagcgagtg catccagtgc caccccgagt gtctgcccca ggccatgaac 4800attacctgca ccggcagagg ccccgacaac tgtatccagt gcgcccacta catcgacggc 4860ccccactgcg tgaaaacctg tccagctggc gtgatgggag agaacaacac cctcgtgtgg 4920aagtacgccg acgccggcca tgtgtgccac ctgtgtcacc ccaattgcac ctacggctgt 4980accggccctg gcctggaagg ctgtcctacc aacggcccca agatcccttc taaagatccc 5040aaattttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 5100gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 5160atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 5220ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 5280gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 5340tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 5400aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 5460agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 5520ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 5580cgcggaccgc agtgtactaa ttatgctctc ttgaaattgg ctggagatgt tgagagcaat 5640cccgggccca tgcggatcag caagccccac ctgcggagca tcagcatcca gtgctacctg 5700tgcctgctgc tgaacagcca cttcctgacc gaggccggca tccacgtgtt catcctgggc 5760tgcttcagcg ccggactgcc caagaccgag gccaactggg tgaacgtgat cagcgacctg 5820aagaagatcg aggacctgat ccagagcatg cacatcgacg ccaccctgta caccgagagc 5880gacgtgcacc ccagctgcaa ggtgaccgcc atgaagtgct ttctgctgga actgcaggtg 5940atcagcctgg aaagcggcga cgccagcatc cacgacaccg tggagaacct gatcatcctg 6000gccaacaaca gcctgagcag caacggcaac gtgaccgaga gcggctgcaa agagtgcgag 6060gaactggaag agaagaacat caaagagttt ctgcagagct tcgtgcacat cgtgcagatg 6120ttcatcaaca ccagctgacg cgtcatcatc gatccggatt agtccaattt gttaaagaca 6180ggatatcagt ggtccaggct ctagttttga ctcaacaata tcaccagctg aagcctatag 6240agtacgagcc atagataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga 6300aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga 6360aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagctg 6420aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 6480cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg 6540gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga 6600accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac 6660taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa 6720agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac 6780ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg 6840gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcacac atgcagcatg 6900tatcaaaatt aatttggttt tttttcttaa gtatttacat taaatggcca tagtacttaa 6960agttacattg gcttccttga aataaacatg gagtattcag aatgtgtcat aaatatttct 7020aattttaaga tagtatctcc attggctttc tactttttct tttatttttt tttgtcctct 7080gtcttccatt tgttgttgtt gttgtttgtt tgtttgtttg ttggttggtt ggttaatttt 7140tttttaaaga tcctacacta tagttcaagc tagactatta gctactctgt aacccagggt 7200gaccttgaag tcatgggtag cctgctgttt tagccttccc acatctaaga ttacaggtat 7260gagctatcat ttttggtata ttgattgatt gattgattga tgtgtgtgtg tgtgattgtg 7320tttgtgtgtg tgactgtgaa aatgtgtgta tgggtgtgtg tgaatgtgtg tatgtatgtg 7380tgtgtgtgag tgtgtgtgtg tgtgtgtgca tgtgtgtgtg tgtgactgtg tctatgtgta 7440tgactgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg ttgtgaaaaa 7500atattctatg gtagtgagag ccaacgctcc ggctcaggtg tcaggttggt ttttgagaca 7560gagtctttca cttagcttgg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 7620accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 7680atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 7740ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 7800gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 7860cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 7920tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 7980gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 8040cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 8100tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 8160aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 8220ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 8280ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 8340tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 8400tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 8460actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 8520gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 8580acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 8640gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 8700acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 8760gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 8820ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 8880gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 8940cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 9000agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 9060catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 9120tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 9180cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 9240gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 9300taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 9360ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 9420tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 9480ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 9540cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 9600agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 9660gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 9720atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 9780gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 9840gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 9900ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 9960cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 10020cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 10080acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 10140cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 10200accatgatta cgccaagctt tgctcttagg agtttcctaa tacatcccaa actcaaatat 10260ataaagcatt tgacttgttc tatgccctag ggggcggggg gaagctaagc cagctttttt 10320taacatttaa aatgttaatt ccattttaaa tgcacagatg tttttatttc ataagggttt 10380caatgtgcat gaatgctgca atattcctgt taccaaagct agtataaata aaaatagata 10440aacgtggaaa ttacttagag tttctgtcat taacgtttcc ttcctcagtt gacaacataa 10500atgcgctgct gagcaagcca gtttgcatct gtcaggatca atttcccatt atgccagtca 10560tattaattac tagtcaatta gttgattttt atttttgaca tatacatgtg 106104710610DNAArtificial SequencepSFG4-hCD5-Ev3-CAR 47aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagttcggtc tcagttggct gtttttggtt 3180gctatcttga agggcgtcca atgcagccgg gacatccaga tgacccagtc tccctctagc 3240atgtcagcga gtcttggtga tcgagtgacg attacctgca gagcctctca agatataaac 3300agctatcttt catggttcca acagaagccg gggaagtccc caaaaactct catatacagg 3360gcgaatcgac tcgtagacgg tgtgccttca aggttttccg ggagtggtag tggcacagat 3420tacacactta caatctcttc attgcagtat gaggatttcg ggatctacta ctgtcaacag 3480tacgacgaat ccccatggac gtttgggggc gggaccaaac ttgagataaa agggagcaca 3540tctggaagtg gtaaacctgg gtcaggggag ggttccacaa aaggacaaat tcaacttgtc 3600caaagcggtc ctggtcttaa gaagcctgga gggtctgtca ggataagttg tgcggcatcc 3660ggctacacct tcaccaacta tgggatgaac tgggtgaaac aagcgcctgg gaaaggtctt 3720cgatggatgg gctggattaa tacccacact ggagagccca cttacgctga tgatttcaaa 3780ggacgattta ccttctcctt ggatacttcc aagagtaccg cgtacttgca aatcaacagt 3840ctccgggctg aagacacggc cacatacttc tgtacgcgga gagggtatga ctggtatttt 3900gatgtgtggg gtcagggaac aaccgtgact gtttcaagct acgtcaccgt ctcttcacag 3960gatcccgccc tggaagagaa gaaaggcaat tacgtcgtga ccgaccacgg cagctgtgtg 4020cgggcttgtg gcgccgatag ctacgagatg gaagaggacg gcgtgcggaa gtgcaagaag 4080tgcgagggcc cctgcagaaa agtgtgcaac ggcatcggca tcggagagtt caaggatagc 4140ctgagcatca acgccaccaa catcaagcac ttcaagaact gcaccagcat cagcggcgac 4200ctgcacatcc tgcccgtggc ctttagaggc gacagcttca cccacacccc cccactggat 4260ccccaggaac tggacatcct gaaaaccgtg aaagagatca caggctttct gctgattcag 4320gcctggcccg agaaccggac agacctgcac gccttcgaga acctggaaat catcagaggc 4380cggaccaagc agcacggcca gttttctctg gccgtggtgt ccctgaacat caccagcctg 4440ggcctgcgga gcctgaaaga aatcagcgac ggcgacgtga tcatctccgg caacaagaac 4500ctgtgctacg ccaacaccat caattggaag aagctgttcg gcacctccgg ccagaaaaca 4560aagatcatct ctaaccgggg cgagaacagc tgcaaagcca ccggacaagt gtgccacgcc 4620ctgtgtagcc ctgagggctg ttggggaccc gagcccagag attgcgtgtc ctgccggaat 4680gtgtccagag gccgcgagtg cgtggacaag tgcaacctgc tggaaggcga gccccgcgag 4740tttgtggaaa acagcgagtg catccagtgc caccccgagt gtctgcccca ggccatgaac 4800attacctgca ccggcagagg ccccgacaac tgtatccagt gcgcccacta catcgacggc 4860ccccactgcg tgaaaacctg tccagctggc gtgatgggag agaacaacac cctcgtgtgg 4920aagtacgccg acgccggcca tgtgtgccac ctgtgtcacc ccaattgcac ctacggctgt 4980accggccctg gcctggaagg ctgtcctacc aacggcccca agatcccttc taaagatccc 5040aaattttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 5100gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 5160atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 5220ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 5280gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 5340tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 5400aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 5460agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 5520ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 5580cgcggaccgc agtgtactaa ttatgctctc ttgaaattgg ctggagatgt tgagagcaat 5640cccgggccca tgcggatcag caagccccac ctgcggagca tcagcatcca gtgctacctg 5700tgcctgctgc tgaacagcca cttcctgacc gaggccggca tccacgtgtt catcctgggc 5760tgcttcagcg ccggactgcc caagaccgag gccaactggg tgaacgtgat cagcgacctg 5820aagaagatcg aggacctgat ccagagcatg cacatcgacg ccaccctgta caccgagagc 5880gacgtgcacc ccagctgcaa ggtgaccgcc atgaagtgct ttctgctgga actgcaggtg 5940atcagcctgg aaagcggcga cgccagcatc cacgacaccg tggagaacct gatcatcctg 6000gccaacaaca gcctgagcag caacggcaac gtgaccgaga gcggctgcaa agagtgcgag 6060gaactggaag

agaagaacat caaagagttt ctgcagagct tcgtgcacat cgtgcagatg 6120ttcatcaaca ccagctgacg cgtcatcatc gatccggatt agtccaattt gttaaagaca 6180ggatatcagt ggtccaggct ctagttttga ctcaacaata tcaccagctg aagcctatag 6240agtacgagcc atagataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga 6300aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga 6360aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagctg 6420aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 6480cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg 6540gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga 6600accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac 6660taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa 6720agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac 6780ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg 6840gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcacac atgcagcatg 6900tatcaaaatt aatttggttt tttttcttaa gtatttacat taaatggcca tagtacttaa 6960agttacattg gcttccttga aataaacatg gagtattcag aatgtgtcat aaatatttct 7020aattttaaga tagtatctcc attggctttc tactttttct tttatttttt tttgtcctct 7080gtcttccatt tgttgttgtt gttgtttgtt tgtttgtttg ttggttggtt ggttaatttt 7140tttttaaaga tcctacacta tagttcaagc tagactatta gctactctgt aacccagggt 7200gaccttgaag tcatgggtag cctgctgttt tagccttccc acatctaaga ttacaggtat 7260gagctatcat ttttggtata ttgattgatt gattgattga tgtgtgtgtg tgtgattgtg 7320tttgtgtgtg tgactgtgaa aatgtgtgta tgggtgtgtg tgaatgtgtg tatgtatgtg 7380tgtgtgtgag tgtgtgtgtg tgtgtgtgca tgtgtgtgtg tgtgactgtg tctatgtgta 7440tgactgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg ttgtgaaaaa 7500atattctatg gtagtgagag ccaacgctcc ggctcaggtg tcaggttggt ttttgagaca 7560gagtctttca cttagcttgg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 7620accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 7680atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 7740ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 7800gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 7860cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 7920tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 7980gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 8040cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 8100tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 8160aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 8220ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 8280ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 8340tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 8400tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 8460actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 8520gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 8580acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 8640gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 8700acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 8760gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 8820ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 8880gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 8940cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 9000agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 9060catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 9120tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 9180cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 9240gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 9300taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 9360ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 9420tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 9480ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 9540cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 9600agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 9660gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 9720atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 9780gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 9840gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 9900ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 9960cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 10020cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 10080acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 10140cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 10200accatgatta cgccaagctt tgctcttagg agtttcctaa tacatcccaa actcaaatat 10260ataaagcatt tgacttgttc tatgccctag ggggcggggg gaagctaagc cagctttttt 10320taacatttaa aatgttaatt ccattttaaa tgcacagatg tttttatttc ataagggttt 10380caatgtgcat gaatgctgca atattcctgt taccaaagct agtataaata aaaatagata 10440aacgtggaaa ttacttagag tttctgtcat taacgtttcc ttcctcagtt gacaacataa 10500atgcgctgct gagcaagcca gtttgcatct gtcaggatca atttcccatt atgccagtca 10560tattaattac tagtcaatta gttgattttt atttttgaca tatacatgtg 106104810274DNAArtificial SequencepSFG4-CD19-DAP12-CAR 48aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catgctcgag ggagtgcagg tggaaaccat ctccccagga 1920gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt 1980gaagatggaa agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta 2040ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag 2100agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc 2160atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga atctggcggt 2220ggatccggag tcgacggatt tggtgatgtc ggtgctcttg agagtttgag gggaaatgca 2280gatttggctt acatcctgag catggagccc tgtggccact gcctcattat caacaatgtg 2340aacttctgcc gtgagtccgg gctccgcacc cgcactggct ccaacatcga ctgtgagaag 2400ttgcggcgtc gcttctcctc gctgcatttc atggtggagg tgaagggcga cctgactgcc 2460aagaaaatgg tgctggcttt gctggagctg gcgcagcagg accacggtgc tctggactgc 2520tgcgtggtgg tcattctctc tcacggctgt caggccagcc acctgcagtt cccaggggct 2580gtctacggca cagatggatg ccctgtgtcg gtcgagaaga ttgtgaacat cttcaatggg 2640accagctgcc ccagcctggg agggaagccc aagctctttt tcatccaggc ctgtggtggg 2700gagcagaaag accatgggtt tgaggtggcc tccacttccc ctgaagacga gtcccctggc 2760agtaaccccg agccagatgc caccccgttc caggaaggtt tgaggacctt cgaccagctg 2820gacgccatat ctagtttgcc cacacccagt gacatctttg tgtcctactc tactttccca 2880ggttttgttt cctggaggga ccccaagagt ggctcctggt acgttgagac cctggacgac 2940atctttgagc agtgggctca ctctgaagac ctgcagtccc tcctgcttag ggtcgctaat 3000gctgtttcgg tgaaagggat ttataaacag atgcctggtt gctttaattt cctccggaaa 3060aaacttttct ttaaaacatc agctagcaga gccgagggca ggggaagtct tctaacatgc 3120ggggacgtgg aggaaaatcc cgggcccatg gagtttgggc tgagctggct ttttcttgtg 3180gctattttaa aaggtgtcca gtgctctaga gacatccaga tgacacagac tacatcctcc 3240ctgtctgcct ctctgggaga cagagtcacc atcagttgca gggcaagtca ggacattagt 3300aaatatttaa attggtatca gcagaaacca gatggaactg ttaaactcct gatctaccat 3360acatcaagat tacactcagg agtcccatca aggttcagtg gcagtgggtc tggaacagat 3420tattctctca ccattagcaa cctggagcaa gaagatattg ccacttactt ttgccaacag 3480ggtaatacgc ttccgtacac gttcggaggg gggaccaagc tggagctgaa acgtggtggt 3540ggtggttctg gtggtggtgg ttctggcggc ggcggctccg gtggtggtgg atccgaggtg 3600cagctgcagc agtctggacc tggcctggtg gcgccctcac agagcctgtc cgtcacatgc 3660actgtctcag gggtctcatt acccgactat ggtgtaagct ggattcgcca gcctccacga 3720aagggtctgg agtggctggg agtaatatgg ggtagtgaaa ccacatacta taattcagct 3780ctcaaatcca gactgaccat catcaaggac aactccaaga gccaagtttt cttaaaaatg 3840aacagtctgc aaactgatga cacagccatt tactactgtg ccaaacatta ttactacggt 3900ggtagctatg ctatggacta ctggggccaa gggaccacgg tcaccgtctc ctcgtacgtc 3960accgtctctt cacaggatcc cgccgagccc aaatctcctg acaaaactca cacatgccca 4020ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 4080aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 4140cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 4200aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 4260gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 4320ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 4380gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 4440ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcaaccg 4500gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 4560agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 4620atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 4680aaagatccca aaggcgtgct ggccggaatc gtgatgggcg acctggtgct gacagtgctg 4740atcgccctgg ctgtgtactt cctgggcaga ctggtgccca gaggaagagg cgctgccgaa 4800gccgccaccc ggaagcagag aatcaccgag acagagagcc cctatcagga actgcagggc 4860cagcggagcg acgtgtacag cgacctgaat acccagcggc cctactacaa gagagtgaag 4920ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 4980ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 5040gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 5100aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 5160aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 5220cttcacatgc aggccctgcc ccctcgcgga ccgcagtgta ctaattatgc tctcttgaaa 5280ttggctggag atgttgagag caatcccggg cccatgcgga tcagcaagcc ccacctgcgg 5340agcatcagca tccagtgcta cctgtgcctg ctgctgaaca gccacttcct gaccgaggcc 5400ggcatccacg tgttcatcct gggctgcttc agcgccggac tgcccaagac cgaggccaac 5460tgggtgaacg tgatcagcga cctgaagaag atcgaggacc tgatccagag catgcacatc 5520gacgccaccc tgtacaccga gagcgacgtg caccccagct gcaaggtgac cgccatgaag 5580tgctttctgc tggaactgca ggtgatcagc ctggaaagcg gcgacgccag catccacgac 5640accgtggaga acctgatcat cctggccaac aacagcctga gcagcaacgg caacgtgacc 5700gagagcggct gcaaagagtg cgaggaactg gaagagaaga acatcaaaga gtttctgcag 5760agcttcgtgc acatcgtgca gatgttcatc aacaccagct gacgcgtcat catcgatccg 5820gattagtcca atttgttaaa gacaggatat cagtggtcca ggctctagtt ttgactcaac 5880aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag attttattta 5940gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag 6000taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa 6060ggtcaggaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt 6120cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca aacaggatat 6180ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt 6240ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga 6300aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg 6360cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc gccagtcctc 6420cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg 6480acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg 6540ggggtctttc acacatgcag catgtatcaa aattaatttg gttttttttc ttaagtattt 6600acattaaatg gccatagtac ttaaagttac attggcttcc ttgaaataaa catggagtat 6660tcagaatgtg tcataaatat ttctaatttt aagatagtat ctccattggc tttctacttt 6720ttcttttatt tttttttgtc ctctgtcttc catttgttgt tgttgttgtt tgtttgtttg 6780tttgttggtt ggttggttaa ttttttttta aagatcctac actatagttc aagctagact 6840attagctact ctgtaaccca gggtgacctt gaagtcatgg gtagcctgct gttttagcct 6900tcccacatct aagattacag gtatgagcta tcatttttgg tatattgatt gattgattga 6960ttgatgtgtg tgtgtgtgat tgtgtttgtg tgtgtgactg tgaaaatgtg tgtatgggtg 7020tgtgtgaatg tgtgtatgta tgtgtgtgtg tgagtgtgtg tgtgtgtgtg tgcatgtgtg 7080tgtgtgtgac tgtgtctatg tgtatgactg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 7140tgtgtgtgtg tgtgttgtga aaaaatattc tatggtagtg agagccaacg ctccggctca 7200ggtgtcaggt tggtttttga gacagagtct ttcacttagc ttggaattca ctggccgtcg 7260ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 7320atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 7380agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 7440gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 7500taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 7560cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 7620caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 7680ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 7740gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 7800aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 7860tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 7920aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 7980aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 8040tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 8100aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 8160tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 8220ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 8280taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 8340agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 8400caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 8460tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 8520gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 8580cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 8640caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 8700ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 8760aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 8820gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 8880atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 8940tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 9000gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 9060actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 9120gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 9180agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 9240ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 9300aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 9360cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 9420gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 9480cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 9540cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 9600gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 9660aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 9720actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 9780cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 9840aatttcacac aggaaacagc tatgaccatg attacgccaa gctttgctct taggagtttc 9900ctaatacatc ccaaactcaa atatataaag catttgactt gttctatgcc ctagggggcg 9960gggggaagct aagccagctt tttttaacat ttaaaatgtt aattccattt taaatgcaca 10020gatgttttta tttcataagg gtttcaatgt gcatgaatgc tgcaatattc ctgttaccaa 10080agctagtata aataaaaata gataaacgtg gaaattactt agagtttctg tcattaacgt 10140ttccttcctc agttgacaac ataaatgcgc tgctgagcaa gccagtttgc atctgtcagg 10200atcaatttcc cattatgcca gtcatattaa ttactagtca attagttgat ttttattttt 10260gacatataca tgtg 10274499374DNAArtificial SequencepSFG4-CD19-DAP12-Ev3-CAR 2.0 49aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc

60atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac 120agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttatgctc cccgagctca 420ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 480ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc 540cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg 600ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 660tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc 720ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 780gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt 840tgtggcccga cctgagtcct aaaatcccga tcgtttagga ctctttggtg cacccccctt 900agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc 960tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat 1020cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg 1080ctagcctgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat 1140cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac 1260ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtggggta 1320catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca 1380ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat 1500atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga 1560catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt 1620ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt 1680ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa 1740cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa 1800agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg 1860tggaccatcc tctagactgc catggagttt gggctgagct ggctttttct tgtggctatt 1920ttaaaaggtg tccagtgctc tagagacatc cagatgacac agactacatc ctccctgtct 1980gcctctctgg gagacagagt caccatcagt tgcagggcaa gtcaggacat tagtaaatat 2040ttaaattggt atcagcagaa accagatgga actgttaaac tcctgatcta ccatacatca 2100agattacact caggagtccc atcaaggttc agtggcagtg ggtctggaac agattattct 2160ctcaccatta gcaacctgga gcaagaagat attgccactt acttttgcca acagggtaat 2220acgcttccgt acacgttcgg aggggggacc aagctggagc tgaaacgtgg tggtggtggt 2280tctggtggtg gtggttctgg cggcggcggc tccggtggtg gtggatccga ggtgcagctg 2340cagcagtctg gacctggcct ggtggcgccc tcacagagcc tgtccgtcac atgcactgtc 2400tcaggggtct cattacccga ctatggtgta agctggattc gccagcctcc acgaaagggt 2460ctggagtggc tgggagtaat atggggtagt gaaaccacat actataattc agctctcaaa 2520tccagactga ccatcatcaa ggacaactcc aagagccaag ttttcttaaa aatgaacagt 2580ctgcaaactg atgacacagc catttactac tgtgccaaac attattacta cggtggtagc 2640tatgctatgg actactgggg ccaagggacc acggtcaccg tctcctcgta cgtcaccgtc 2700tcttcacagg atcccgccct ggaagagaag aaaggcaatt acgtcgtgac cgaccacggc 2760agctgtgtgc gggcttgtgg cgccgatagc tacgagatgg aagaggacgg cgtgcggaag 2820tgcaagaagt gcgagggccc ctgcagaaaa gtgtgcaacg gcatcggcat cggagagttc 2880aaggatagcc tgagcatcaa cgccaccaac atcaagcact tcaagaactg caccagcatc 2940agcggcgacc tgcacatcct gcccgtggcc tttagaggcg acagcttcac ccacaccccc 3000ccactggatc cccaggaact ggacatcctg aaaaccgtga aagagatcac aggctttctg 3060ctgattcagg cctggcccga gaaccggaca gacctgcacg ccttcgagaa cctggaaatc 3120atcagaggcc ggaccaagca gcacggccag ttttctctgg ccgtggtgtc cctgaacatc 3180accagcctgg gcctgcggag cctgaaagaa atcagcgacg gcgacgtgat catctccggc 3240aacaagaacc tgtgctacgc caacaccatc aattggaaga agctgttcgg cacctccggc 3300cagaaaacaa agatcatctc taaccggggc gagaacagct gcaaagccac cggacaagtg 3360tgccacgccc tgtgtagccc tgagggctgt tggggacccg agcccagaga ttgcgtgtcc 3420tgccggaatg tgtccagagg ccgcgagtgc gtggacaagt gcaacctgct ggaaggcgag 3480ccccgcgagt ttgtggaaaa cagcgagtgc atccagtgcc accccgagtg tctgccccag 3540gccatgaaca ttacctgcac cggcagaggc cccgacaact gtatccagtg cgcccactac 3600atcgacggcc cccactgcgt gaaaacctgt ccagctggcg tgatgggaga gaacaacacc 3660ctcgtgtgga agtacgccga cgccggccat gtgtgccacc tgtgtcaccc caattgcacc 3720tacggctgta ccggccctgg cctggaaggc tgtcctacca acggccccaa gatccctagc 3780aaggatccta agggagtgct ggccggaatc gtgatgggcg acctggtgct gacagtgctg 3840atcgccctgg ctgtgtactt cctgggcaga ctggtgccca gaggaagagg cgctgccgaa 3900gccgccaccc ggaagcagag aatcaccgag acagagagcc cctatcagga actgcagggc 3960cagcggagcg acgtgtacag cgacctgaat acccagcggc cctactacaa gagagtgaag 4020ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 4080ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 4140gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 4200aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 4260aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 4320cttcacatgc aggccctgcc ccctcgcgga ccgcagtgta ctaattatgc tctcttgaaa 4380ttggctggag atgttgagag caatcccggg cccatgcgga tcagcaagcc ccacctgcgg 4440agcatcagca tccagtgcta cctgtgcctg ctgctgaaca gccacttcct gaccgaggcc 4500ggcatccacg tgttcatcct gggctgcttc agcgccggac tgcccaagac cgaggccaac 4560tgggtgaacg tgatcagcga cctgaagaag atcgaggacc tgatccagag catgcacatc 4620gacgccaccc tgtacaccga gagcgacgtg caccccagct gcaaggtgac cgccatgaag 4680tgctttctgc tggaactgca ggtgatcagc ctggaaagcg gcgacgccag catccacgac 4740accgtggaga acctgatcat cctggccaac aacagcctga gcagcaacgg caacgtgacc 4800gagagcggct gcaaagagtg cgaggaactg gaagagaaga acatcaaaga gtttctgcag 4860agcttcgtgc acatcgtgca gatgttcatc aacaccagct gacgcgtcat catcgatccg 4920gattagtcca atttgttaaa gacaggatat cagtggtcca ggctctagtt ttgactcaac 4980aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag attttattta 5040gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag 5100taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa 5160ggtcaggaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt 5220cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca aacaggatat 5280ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt 5340ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga 5400aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg 5460cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc gccagtcctc 5520cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg 5580acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg 5640ggggtctttc acacatgcag catgtatcaa aattaatttg gttttttttc ttaagtattt 5700acattaaatg gccatagtac ttaaagttac attggcttcc ttgaaataaa catggagtat 5760tcagaatgtg tcataaatat ttctaatttt aagatagtat ctccattggc tttctacttt 5820ttcttttatt tttttttgtc ctctgtcttc catttgttgt tgttgttgtt tgtttgtttg 5880tttgttggtt ggttggttaa ttttttttta aagatcctac actatagttc aagctagact 5940attagctact ctgtaaccca gggtgacctt gaagtcatgg gtagcctgct gttttagcct 6000tcccacatct aagattacag gtatgagcta tcatttttgg tatattgatt gattgattga 6060ttgatgtgtg tgtgtgtgat tgtgtttgtg tgtgtgactg tgaaaatgtg tgtatgggtg 6120tgtgtgaatg tgtgtatgta tgtgtgtgtg tgagtgtgtg tgtgtgtgtg tgcatgtgtg 6180tgtgtgtgac tgtgtctatg tgtatgactg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 6240tgtgtgtgtg tgtgttgtga aaaaatattc tatggtagtg agagccaacg ctccggctca 6300ggtgtcaggt tggtttttga gacagagtct ttcacttagc ttggaattca ctggccgtcg 6360ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 6420atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 6480agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 6540gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 6600taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 6660cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 6720caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 6780ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 6840gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 6900aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 6960tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 7020aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 7080aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 7140tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 7200aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 7260tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 7320ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 7380taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 7440agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 7500caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 7560tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 7620gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 7680cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 7740caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 7800ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 7860aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 7920gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 7980atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 8040tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 8100gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 8160actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 8220gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 8280agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 8340ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 8400aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 8460cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 8520gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 8580cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 8640cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 8700gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 8760aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 8820actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 8880cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 8940aatttcacac aggaaacagc tatgaccatg attacgccaa gctttgctct taggagtttc 9000ctaatacatc ccaaactcaa atatataaag catttgactt gttctatgcc ctagggggcg 9060gggggaagct aagccagctt tttttaacat ttaaaatgtt aattccattt taaatgcaca 9120gatgttttta tttcataagg gtttcaatgt gcatgaatgc tgcaatattc ctgttaccaa 9180agctagtata aataaaaata gataaacgtg gaaattactt agagtttctg tcattaacgt 9240ttccttcctc agttgacaac ataaatgcgc tgctgagcaa gccagtttgc atctgtcagg 9300atcaatttcc cattatgcca gtcatattaa ttactagtca attagttgat ttttattttt 9360gacatataca tgtg 9374501006PRTArtificial SequencepSFG4-CD19-DAP12-Ev3-CAR 2.0 50Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 115 120 125Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu145 150 155 160Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val 165 170 175Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys 180 185 190Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr 195 200 205Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys 210 215 220Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala225 230 235 240Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met 245 250 255Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr 260 265 270Val Ser Ser Gln Asp Pro Ala Leu Glu Glu Lys Lys Gly Asn Tyr Val 275 280 285Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr 290 295 300Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro305 310 315 320Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser 325 330 335Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser 340 345 350Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser 355 360 365Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys 370 375 380Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu385 390 395 400Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly 405 410 415Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn 420 425 430Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp 435 440 445Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn 450 455 460Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser465 470 475 480Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala 485 490 495Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val 500 505 510Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn 515 520 525Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile 530 535 540Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr545 550 555 560Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 565 570 575Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn 580 585 590Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys 595 600 605His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys 610 615 620Pro Thr Asn Gly Pro Lys Ile Pro Ser Lys Asp Pro Lys Gly Val Leu625 630 635 640Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu 645 650 655Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala 660 665 670Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr 675 680 685Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr 690 695 700Gln Arg Pro Tyr Tyr Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala705 710 715 720Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 725 730 735Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 740 745 750Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 755 760 765Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 770 775 780Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr785 790 795 800Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 805 810 815Gln Ala Leu Pro Pro Arg Gly Pro Gln Cys Thr Asn Tyr Ala Leu Leu 820 825 830Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Arg Ile Ser 835 840 845Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr Leu Cys Leu Leu 850 855 860Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His Val Phe Ile Leu865 870 875 880Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala Asn Trp Val Asn 885 890 895Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His 900 905 910Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys 915 920 925Val

Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu 930 935 940Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile945 950 955 960Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly 965 970 975Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu 980 985 990Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 995 1000 100551219DNAArtificial SequenceDAP12 51ggcgtgctgg ccggaatcgt gatgggcgac ctggtgctga cagtgctgat cgccctggct 60gtgtacttcc tgggcagact ggtgcccaga ggaagaggcg ctgccgaagc cgccacccgg 120aagcagagaa tcaccgagac agagagcccc tatcaggaac tgcagggcca gcggagcgac 180gtgtacagcg acctgaatac ccagcggccc tactacaag 2195273PRTArtificial SequenceDAP12 52Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu1 5 10 15Ile Ala Leu Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg 20 25 30Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu 35 40 45Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp 50 55 60Leu Asn Thr Gln Arg Pro Tyr Tyr Lys65 7053342DNAArtificial SequenceCD3zeta 53agagtgaagt 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 cctcgcggac cg 34254114PRTArtificial SequenceCD3zeta 54Arg 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 110Gly Pro55489DNAArtificial SequenceIL15 55atgcggatca gcaagcccca cctgcggagc atcagcatcc agtgctacct gtgcctgctg 60ctgaacagcc acttcctgac cgaggccggc atccacgtgt tcatcctggg ctgcttcagc 120gccggactgc ccaagaccga ggccaactgg gtgaacgtga tcagcgacct gaagaagatc 180gaggacctga tccagagcat gcacatcgac gccaccctgt acaccgagag cgacgtgcac 240cccagctgca aggtgaccgc catgaagtgc tttctgctgg aactgcaggt gatcagcctg 300gaaagcggcg acgccagcat ccacgacacc gtggagaacc tgatcatcct ggccaacaac 360agcctgagca gcaacggcaa cgtgaccgag agcggctgca aagagtgcga ggaactggaa 420gagaagaaca tcaaagagtt tctgcagagc ttcgtgcaca tcgtgcagat gttcatcaac 480accagctga 48956162PRTArtificial SequenceIL15 56Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr1 5 10 15Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His65 70 75 80Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn145 150 155 160Thr Ser57819DNAArtificial SequencehCLL-1 scFv 57atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgctcg 60cgagacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg cgacagagtg 120accatcacct gcagagccag cagcaacgtg atcagcagct acgtgcactg gtaccagcag 180aagcccggca aggcccccaa gctgctgatc tacagcacca gcaacctggc cagcggcgtg 240cccagcagat tcagcggcag cagaagcggc accgacttca ccctgaccat cagcagcctg 300cagcccgagg acttcgccac ctactactgc cagcagtaca gcggctaccc cctgaccttc 360ggccagggca ccaaggtgga gatcaagaga ggtggtggtg gttctggtgg tggtggttct 420ggcggcggcg gctccggtgg tggtggatcc gaggtgcagc tggtggagag cggcggcggc 480ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggcta cagcatcacc 540agcgcctact actggaactg ggtgagacag gcccccggca agggcctgga gtgggtggcc 600tacatcagct acgacggcag aaacaactac aaccccagcc tgaagaacag attcaccatc 660agcgccgaca ccagcaagaa caccgcctac ctgcagatga acagcctgag agccgaggac 720accgccgtgt actactgcag cagagagggc gactacgacg tgggcaacta ctacgccatg 780gactactggg gccagggcac cctggtgacc gtgagctcg 81958273PRTArtificial SequencehCLL-1 scFv 58Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser 35 40 45Asn Val Ile Ser Ser Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly145 150 155 160Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 165 170 175Tyr Ser Ile Thr Ser Ala Tyr Tyr Trp Asn Trp Val Arg Gln Ala Pro 180 185 190Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Tyr Asp Gly Arg Asn 195 200 205Asn Tyr Asn Pro Ser Leu Lys Asn Arg Phe Thr Ile Ser Ala Asp Thr 210 215 220Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp225 230 235 240Thr Ala Val Tyr Tyr Cys Ser Arg Glu Gly Asp Tyr Asp Val Gly Asn 245 250 255Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 260 265 270Ser59109PRTArtificial SequencehCLL1 VH 59Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Asn Val Ile Ser Ser 20 25 30Tyr Val His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 10560123PRTArtificial SequencehCLL1 VL 60Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Ala 20 25 30Tyr Tyr Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45Val Ala Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu 50 55 60Lys Asn Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Glu Gly Asp Tyr Asp Val Gly Asn Tyr Tyr Ala Met Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12061804DNAArtificial SequenceCD99 scFv 61atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgctct 60agaagcagcg agctgaccca ggaccccgcc gtgagcgtgg ccctgggcca gaccgtgaga 120atcacctgcc agggcgacag cctgagaagc tactacgcca gctggtacca gcagaagccc 180ggccaggccc ccgtgctggt gatctacggc aagaacaaca gacccagcgg catccccgac 240agattcagcg gcagcagcag cggcaacacc gccagcctga ccatcaccgg cgcccaggcc 300gaggacgagg ccgactacta ctgcaacagc agcttcccca gaaccagcag cgtggtgttc 360ggcggcggca ccaagctgac cgtgctgggc ggtggtggtg gttctggtgg tggtggttct 420ggcggcggcg gctccggtgg tggtggatcc atggccgagg tgcagctggt ggagagcggc 480ggcggcctgg tgagacccgg cggcagcctg agactgagct gcgccgccag cggcttcacc 540ttcagcagct acgccatgag ctgggtgaga caggcccccg gcaagggcct ggagtgggtg 600agcgccatca gcggcagcgg cggcagcacc tactacgccg acagcgtgaa gggcagattc 660accatcagca gagacaacag caagaacacc ctgtacctgc agatgaacag cctgagagcc 720gaggacaccg ccgtgtacta ctgcgccaag agccacaaga gattcgacta ctggggccag 780ggcaccctgg tgaccgtgag caga 80462268PRTArtificial SequenceCD99 scFv 62Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Ser Arg Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser 20 25 30Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu 35 40 45Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp65 70 75 80Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr 85 90 95Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ser Phe 100 105 110Pro Arg Thr Ser Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val 115 120 125Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Met Ala Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 165 170 175Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala 180 185 190Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly 195 200 205Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 210 215 220Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala225 230 235 240Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Ser His Lys Arg Phe Asp 245 250 255Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Arg 260 26563108PRTArtificial SequenceCD99 VH 63Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ser Phe Pro Arg Thr Ser Ser 85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 10564118PRTArtificial SequenceCD99 VL 64Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Lys Ser His Lys Arg Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Arg 115

Claims

1. An isolated antigen-specific humanized single chain variable fragment (scFv) comprising a light chain variable region (V.sub.L) and a heavy chain variable region (V.sub.H), wherein the scFv is CD5-specific and wherein the V.sub.L comprises an amino acid sequence of SEQ ID NO:31 and the V.sub.H comprises an amino acid sequence of SEQ ID NO:32, or sequences with 90% sequence identity to framework regions of SEQ ID NO:31 and SEQ ID NO:32, respectively.

2. The humanized scFv of claim 1, wherein the CD5-specific scFv comprises an amino acid sequence of SEQ ID NO:30.

3. The humanized scFv of claim 1, wherein the scFv comprises an amino acid sequence with at least 95% sequence identity to framework regions in an amino acid sequence of SEQ ID NO: 30.

4. The humanized scFv of claim 1, wherein the scFv is comprised in a chimeric antigen receptor (CAR).

5. The humanized scFv of claim 4, wherein the CAR comprises at least one signaling domain selected from the group consisting of CD3, CD28, OX40/CD134, 4-1BB/CD137, Fc.epsilon.RI.gamma., ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP10, DAP12, CD70, CD40, and a combination thereof.

6. The humanized scFv of claim 5, wherein the at least one signaling domain comprises DAP10 or DAP12.

7. The humanized scFv of claim 4, wherein the CAR is co-expressed with IL-15.

8. An isolated polynucleotide encoding the isolated humanized scFv of claim 1.

9. The polynucleotide of claim 8, wherein the polynucleotide comprises nucleotide sequence encoding a CAR that comprises the scFv.

10. An expression vector comprising the isolated polynucleotide of claim 9.

11. The vector of claim 10, wherein the vector expresses the CAR and expresses IL-15.

12. The vector of claim 10, wherein the vector is further defined as a viral vector.

13. A host cell engineered to express the polynucleotide of claim 9.

14. The cell of claim 13, wherein the host cell is further defined as an immune cell.

15. The cell of claim 14, wherein the immune cell is a T cell, peripheral blood lymphocyte, NK cell, invariant NK cell, NKT cell, or stem cell.

16. The cell of claim 14, wherein the immune cell is isolated from cord blood.

17. A pharmaceutical composition comprising a population of cells according to claim 13.

18. A method of treating an immune-related disorder in a subject comprising administering an effective amount of cells of claim 13 to the subject.

19. The method of claim 18, wherein the immune-related disorder is a cancer, autoimmune disorder, graft versus host disease, allograft rejection, or inflammatory condition.