Compositions and methods comprising high affinity chimeric antigen receptor (CAR) with cross-reactivity to clinically-relevant EGFR mutated proteins

Abstract

To provide compositions and methods that utilize a high affinity chimeric antigen receptor (CAR) with cross-reactivity to clinically-relevant EGFR mutated proteins.SOLUTION: The present invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain capable of binding multiple isoforms of epidermal growth factor receptor (EGFR), a transmembrane domain, and an intracellular domain.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 833,456, filed April 12, 2019, and U.S. Provisional Patent Application No. 62 / 892,343, filed August 27, 2019, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background of the Invention Glioblastoma (GBM) or glioma grade IV is a devastating cancer with an annual incidence rate of 3.19 per 100,000 people per year (about 10,000 people) and a median survival of 14.6 months after standard treatment of surgery, radiotherapy, and chemotherapy. Little progress in treatment has been achieved in the past 20 years, and the two - year survival rate remains near 25%. In recent years, although low - intensity alternating electric fields have shown some potential, the need for new treatments remains great.

[0003] Research over the past 20 years has demonstrated that cancer is naturally recognized by the immune system and that immune evasion is a central part of carcinogenesis. This is most clearly exemplified by the dramatic clinical responses observed when natural immune checkpoints mediated by programmed death 1 (PD - 1) and cytotoxic T lymphocyte - associated protein 4 (CTLA - 4) are disrupted in cancers such as melanoma and non - small cell lung cancer (NSCLC). In cancers where natural immunity may be more limited, such as acute lymphoblastic leukemia, synthetic chimeric antigen receptor (CAR) - based immunotherapies such as tisagenlecleucel (KYMRIAH®) and axicabtagene ciloleucel (YESCARTA®) have demonstrated the immense cytotoxic potential of T cells to produce sustainable tumor control.

[0004] GBM has a paucity of neoantigens, making it challenging to develop T cell receptor (TCR)-based immunotherapies. Based on Cancer Genome Atlas (TCGA) whole-exome analysis data, the mutation burden in GBM is moderate compared to other tumor types. However, the median mutation frequency in GBM is one to two orders of magnitude lower than that in immunogenic tumors such as melanoma, NSCLC, and bladder cancer, and the available evidence supports that the amount of genetic mutations is one of the most highly correlated factors with the response to immune checkpoint inhibition (ICB). The low amount of genetic mutations and insufficient T cell infiltration, combined with the challenge of delivering large molecules such as antibodies across the blood-brain barrier (BBB), can explain the generally poor responses observed to date with PD-1 inhibitor therapy in GBM.

[0005] Changes within the epidermal growth factor receptor (EGFR) (ErbB1) locus correspond to the highest frequency genetic changes in GBM. Overexpression of EGFR, such as that mediated by local amplification of the EGFR locus as double minute chromosomes in GBM, has long been recognized and is found in 30% of cases. EGFR mutations are also frequent. The oncogenic EGFR variant lacking exons 2-7 (EGFRvIII) is found in approximately 30% of GBM.

[0006] A human clinical trial of autologous EGFRvIII-specific CART cells (CART-EGFRvIII) therapy in recurrent GBM has been completed. In addition to demonstrating safety, biopsies of specific regions of GBM after CART infusion have proven that CART-EGFRvIII cells can penetrate the BBB, infiltrate GBM, and mediate on-target activity. Immunohistochemistry (IHC) analysis and RNA in situ hybridization (ISH) have shown that tumor-infiltrating T cells exhibited an activated phenotype represented by an increase in CD8+ granzyme B+ CD25+ CART cells within the regions of GBM biopsies. These observations were made between 7 and 14 days after CART infusion, consistent with the peripheral engraftment peak seen on days 7 to 10 in the series of patients treated. Region-specific antigen editing and reduction of EGFRvIII were observed in 5 out of 7 treated specimens, which supported the target-specific activity produced by the treatment. Clinical activity of CART cells targeting HER-2 and IL-13Rα2 has also been reported in GBM.

[0007] Tumor heterogeneity and the immunosuppressive tumor microenvironment (TME) are major obstacles to CART therapy in GBM. The site-specific reduction in EGFRvIII after CART therapy is consistent with the observed intratumoral heterogeneity of this change in GBM. Importantly, tissue IHC analysis demonstrated the presence of an adaptive immune response within the GBM TME that closely followed the time course of CART activation. IDO1, PD-L1, IL10, and TGFβ all increased within the tumor tissue proximal to CART cells after treatment. These immunomodulatory pathways are known to play a role in tumor immune escape and are consistent with the emergence of adaptive immune resistance that may further blunt the antitumor activity of CART cells against GBM.

[0008] There remains a need for novel CART therapies that not only target multiple antigens but also avoid immunosuppressive signals within the GBM TME. The present invention addresses this need. SUMMARY OF THE INVENTION

[0009] The present invention is based on the discovery of a high-affinity chimeric antigen receptor (CAR) having cross-reactivity with clinically significant EGFR variant proteins.

[0010] In one aspect, the present invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of epidermal growth factor receptor (EGFR), a transmembrane domain, and an intracellular domain.

[0011] In another aspect, the present invention provides a vector comprising any of the isolated nucleic acids disclosed herein.

[0012] In another aspect, the present invention provides a modified cell comprising a cross-reactive chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain.

[0013] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof. The method comprises administering to the subject any of the modified cells disclosed herein.

[0014] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof. The method comprises administering to the subject a modified cell comprising a CAR. The CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain.

[0015] In various aspects of the above-described or any other aspect of the invention presented herein, the EGFR isoform is selected from the group consisting of wild-type EGFR (wtEGFR), mutant EGFR, EGFRA289V, EGFRA289D, EGFRA289T, EGFRR108K, EGFRR108G, EGFRG598V, EGFRD126Y, EGFRC628F, EGFRR108K / A289V, EGFRR108K / D126Y, EGFRA289V / G598V, EGFRA289V / C628F, and EGFR variant II.

[0016] In certain exemplary aspects, the antigen-binding domain is selected from the group consisting of an antibody, scFv, Fab, or any fragment thereof.

[0017] In certain exemplary aspects, the antigen-binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:31, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, and SEQ ID NO:85. In certain exemplary aspects, the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:32, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:86.

[0018] In certain exemplary aspects, the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:27, and SEQ ID NO:30. In certain exemplary aspects, the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:26, and SEQ ID NO:29.

[0019] In certain exemplary embodiments, the antigen-binding domain comprises a light chain complementarity determining region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, and 7. In certain exemplary embodiments, the antigen-binding domain comprises a heavy chain complementarity determining region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, and 10.

[0020] In certain exemplary embodiments, the CAR further comprises a hinge region. In certain exemplary embodiments, the hinge region is encoded by the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 71. In certain exemplary embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 72.

[0021] In certain exemplary embodiments, the transmembrane domain is encoded by the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 73. In certain exemplary embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 74.

[0022] In certain exemplary embodiments, the intracellular domain is encoded by the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 75. In certain exemplary embodiments, the intracellular domain is encoded by a nucleotide sequence comprising SEQ ID NO: 14 or SEQ ID NO: 77. In certain exemplary embodiments, the intracellular domain is encoded by a nucleotide sequence comprising SEQ ID NOs: 13 and 14 or SEQ ID NOs: 75 and 77. In certain exemplary embodiments, the intracellular domain comprises the amino acid sequence of SEQ ID NO: 76. In certain exemplary embodiments, the intracellular domain comprises the amino acid sequence of SEQ ID NO: 78. In certain exemplary embodiments, the intracellular domain comprises the amino acid sequences of SEQ ID NO: 76 and SEQ ID NO: 78.

[0023] In certain exemplary embodiments, the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, 64, 66, or 68. In certain exemplary embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 65, 67, and 69.

[0024] In certain exemplary embodiments, the transmembrane domain and / or the intracellular domain comprises a killer cell immunoglobulin-like receptor (KIR).

[0025] In certain exemplary embodiments, the nucleic acid further comprises a nucleic acid encoding DAP12.

[0026] In certain exemplary embodiments, the CAR is capable of binding to an EGFR homodimer, an EGFR heterodimer, an EGFR oligomer, and / or an EGFR / ErbB oligomer.

[0027] In certain exemplary embodiments, the cell is a T cell. In certain exemplary embodiments, the cell is an autologous cell. In certain exemplary embodiments, the cell is a human cell.

[0028] In certain exemplary embodiments, the method further comprises administering an additional treatment to the subject. In certain exemplary embodiments, the additional treatment comprises immune checkpoint inhibition (ICB). In certain exemplary embodiments, the ICB is selected from the group consisting of anti-PD-1 treatment, anti-PD-L1 treatment, anti-TIM3 treatment, and anti-CTLA-4 treatment.

[0029] In certain exemplary embodiments, the treatment is delivered locally.

[0030] In certain exemplary embodiments, the modified cell further comprises a minibody. In certain exemplary embodiments, the minibody comprises an scFv specific for PD-1 and a human IgG CH3 domain. In certain exemplary embodiments, the minibody comprises an scFv specific for CTLA-4 and a human IgG CH3 domain. In certain exemplary embodiments, the minibody comprises an scFv specific for TIM-3 and a human IgG CH3 domain. In certain exemplary embodiments, the minibody comprises an scFv specific for PD-L1 and a human IgG CH3 domain.

[0031] In another aspect, the present invention provides a method of treating cancer in a subject in need thereof. The method comprises culturing a plurality of CAR T cells together with GBM organoids (GBOs) derived from the subject, selecting the CAR T cells having the highest efficacy from the plurality of CAR T cells, and administering the CAR T cells having the highest efficacy to the subject, thereby treating cancer in the subject. In certain exemplary embodiments, the plurality of CAR T cells comprises a plurality of modified T cells comprising a plurality of CARs, wherein each CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain. In certain exemplary embodiments, the antigen-binding domain is capable of binding to an antigen selected from the group consisting of CD19, EGFR, multiple isoforms of EGFR (e.g., wild-type EGFR (wtEGFR), mutant EGFR, EGFRA289V, EGFRA289D, EGFRA289T, EGFRR108K, EGFRR108G, EGFRG598V, EGFRD126Y, EGFRC628F, EGFRR108K / A289V, EGFRR108K / D126Y, EGFRA289V / G598V, EGFRA289V / C628F, and EGFR variant II), PSMA, PSCA, and any tumor-associated antigen (TAA).

[0032] In certain exemplary embodiments, the GBO is generated from a biopsy material derived from the subject. The highest efficacy is measured as the highest degree of apoptosis and / or tumor cell death.

[0033] In certain exemplary embodiments, the method further comprises administering an additional treatment to the subject. In certain exemplary embodiments, the additional treatment comprises immune checkpoint inhibition (ICB). In certain exemplary embodiments, the ICB is selected from the group consisting of anti-PD-1 treatment, anti-PD-L1 treatment, anti-TIM3 treatment, and anti-CTLA-4 treatment. [Invention 1001] An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of epidermal growth factor receptor (EGFR), a transmembrane domain, and an intracellular domain. [Invention 1002] The EGFR isoform is selected from the group consisting of wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V , EGFR A289D , EGFR A289T , EGFR R108K , EGFR R108G , EGFR G598V , EGFR D126Y , EGFR C628F , EGFR R108K / A289V , EGFR R108K / D126Y , EGFR A289V / G598V , EGFR A289V / C628F , and the isolated nucleic acid of Invention 1001 selected from the group consisting of EGFR variant II. [Invention 1003] The isolated nucleic acid of Invention 1001, wherein the antigen-binding domain is selected from the group consisting of an antibody, scFv, Fab, or any fragment thereof. [Invention 1004] The isolated nucleic acid of Invention 1001, wherein the antigen-binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:31, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, and SEQ ID NO:85. [Invention 1005] An isolated nucleic acid of the present invention 1001, wherein the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:32, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:86. [The present invention 1006] An isolated nucleic acid of the present invention 1001, wherein the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:27, and SEQ ID NO:30. [The present invention 1007] An isolated nucleic acid of the present invention 1001, wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:26, and SEQ ID NO:29. [The present invention 1008] An isolated nucleic acid of the present invention 1001, wherein the antigen-binding domain comprises a light chain complementarity determining region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, 6, and 7. [The present invention 1009] An isolated nucleic acid of the present invention 1001, wherein the antigen-binding domain comprises a heavy chain complementarity determining region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, 9, and 10. [The present invention 1010] An isolated nucleic acid of the present invention 1001, wherein the CAR further comprises a hinge region. [The present invention 1011] An isolated nucleic acid of the present invention 1010, wherein the hinge region is encoded by the nucleotide sequence of SEQ ID NO:11 or SEQ ID NO:71. [The present invention 1012] An isolated nucleic acid of the present invention 1001, wherein the transmembrane domain is encoded by the nucleotide sequence of SEQ ID NO:12 or SEQ ID NO:73. [The present invention 1013] An isolated nucleic acid of the present invention 1001, wherein the intracellular domain is encoded by the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 75. [The present invention 1014] An isolated nucleic acid of the present invention 1001, wherein the intracellular domain is encoded by a nucleotide sequence comprising SEQ ID NO: 14 or SEQ ID NO: 77. [The present invention 1015] An isolated nucleic acid of the present invention 1001, wherein the intracellular domain is encoded by a nucleotide sequence comprising SEQ ID NO: 13 and SEQ ID NO: 14 or a nucleotide sequence comprising SEQ ID NO: 75 and SEQ ID NO: 77. [The present invention 1016] An isolated nucleic acid of the present invention 1001, wherein the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, 64, 66, or 68. [The present invention 1017] An isolated nucleic acid of the present invention 1001, wherein the transmembrane domain and / or intracellular domain comprises a killer cell immunoglobulin-like receptor (KIR). [The present invention 1018] The isolated nucleic acid of the present invention 1017, further comprising a nucleic acid encoding DAP12. [The present invention 1019] An isolated nucleic acid of the present invention 1001, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 65, 67, and 69. [The present invention 1020] An isolated nucleic acid of the present invention 1001, wherein the CAR is capable of binding to an EGFR homodimer, an EGFR heterodimer, an EGFR oligomer, and / or an EGFR / ErbB oligomer. [The present invention 1021] A vector comprising any of the isolated nucleic acids of the present invention. [The present invention 1022] A modified cell comprising a cross-reactive chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain. [Inventive concept 1023] The modified cell of Inventive concept 1022, wherein the EGFR isoform is selected from the group consisting of wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR R108K EGFR R108G EGFR G598V EGFR D126Y EGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F and EGFR variant II. [Inventive concept 1024] The modified cell of Inventive concept 1022, wherein the antigen-binding domain is selected from the group consisting of an antibody, scFv, Fab, or any fragment thereof. [Inventive concept 1025] The modified cell of Inventive concept 1022, wherein the antigen-binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:31, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, and SEQ ID NO:85. [Inventive concept 1026] The modified cell of Inventive concept 1022, wherein the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:32, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:86. [Inventive concept 1027] The modified cell of Inventive concept 1022, wherein the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:27, and SEQ ID NO:30. [The present invention 1028] The modified cell of the present invention 1022, wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 26, and SEQ ID NO: 29. [The present invention 1029] The modified cell of the present invention 1022, wherein the antigen-binding domain comprises a light chain complementarity-determining region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, and 7. [The present invention 1030] The modified cell of the present invention 1022, wherein the antigen-binding domain comprises a heavy chain complementarity-determining region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 9, and 10. [The present invention 1031] The modified cell of the present invention 1022, wherein the CAR further comprises a hinge region. [The present invention 1032] The modified cell of the present invention 1031, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 72. [The present invention 1033] The modified cell of the present invention 1022, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 74. [The present invention 1034] The modified cell of the present invention 1022, wherein the intracellular domain comprises the amino acid sequence of SEQ ID NO: 76. [The present invention 1035] The modified cell of the present invention 1022, wherein the intracellular domain comprises the amino acid sequence of SEQ ID NO: 78. [The present invention 1036] The modified cell of the present invention 1022, wherein the intracellular domain comprises the amino acid sequences of SEQ ID NO: 76 and SEQ ID NO: 78. [The present invention 1037] The modified cell of the present invention 1022, wherein the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, 64, 66, or 68. [The present invention 1038] A modified cell of the present invention 1022, wherein the transmembrane domain and / or the intracellular domain comprises a killer cell immunoglobulin-like receptor (KIR). [The present invention 1039] A modified cell of the present invention 1038, further comprising a nucleic acid encoding DAP12. [The present invention 1040] A modified cell of the present invention 1022, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 65, 67, and 69. [The present invention 1041] A modified cell of the present invention 1022, wherein the CAR is capable of binding to an EGFR homodimer, an EGFR heterodimer, an EGFR oligomer, and / or an EGFR / ErbB oligomer. [The present invention 1042] A modified cell of the present invention 1022, which is a T cell. [The present invention 1043] A modified cell of the present invention 1022, which is an autologous cell. [The present invention 1044] A modified cell of the present invention 1022, which is a human cell. [The present invention 1045] A method for treating cancer in a subject in need thereof, comprising administering to the subject a modified cell according to any one of the present inventions 1022 to 1044. [The present invention 1046] A method for treating cancer in a subject in need thereof, comprising administering to the subject a modified cell comprising a CAR, wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain. Method. [The present invention 1047] The EGFR isoforms are wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V , EGFR A289D , EGFR A289T , EGFR R108K , EGFR R108G , EGFRG598V , EGFR D126Y , EGFR C628F , EGFR R108K / A289V , EGFR R108K / D126Y , EGFR A289V / G598V , EGFR A289V / C628F , and a method of the present invention 1046 selected from the group consisting of EGFR variant II. [The present invention 1048] The method of the present invention 1046, wherein the antigen-binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 31, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, and SEQ ID NO: 85. [The present invention 1049] The method of the present invention 1046, wherein the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 32, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, and SEQ ID NO: 86. [The present invention 1050] The method of the present invention 1046, wherein the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, and SEQ ID NO: 30. [The present invention 1051] The method of the present invention 1046, wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 26, and SEQ ID NO: 29. [The present invention 1052] The method of the present invention 1046, wherein the antigen-binding domain comprises a light chain complementarity-determining region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, and 7. [The present invention 1053] The method of the present invention 1046, wherein the antigen-binding domain comprises a heavy chain complementarity-determining region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 9, and 10. [The present invention 1054] The method of the present invention 1046, wherein the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, 64, 66, or 68. [The present invention 1055] The method of the present invention 1046, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 65, 67, and 69. [The present invention 1056] The method according to any one of the present inventions 1045 to 1055, further comprising the step of subjecting to an additional treatment. [The present invention 1057] The method of the present invention 1056, wherein the additional treatment comprises immune checkpoint inhibition (ICB). [The present invention 1058] The method of the present invention 1057, wherein the ICB is selected from the group consisting of anti-PD-1 treatment, anti-PD-L1 treatment, anti-TIM3 treatment, and anti-CTLA-4 treatment. [The present invention 1059] The method according to any one of the present inventions 1045 to 1058, wherein the treatment is delivered locally. [The present invention 1060] The method of the present invention 1045 or 1046, wherein the modified cell further comprises a minibody. [The present invention 1061] The method of the present invention 1060, wherein the minibody comprises an scFv specific for PD-1 and a human IgG CH3 domain. [The present invention 1062] The method of the present invention 1060, wherein the minibody comprises an scFv specific for CTLA-4 and a human IgG CH3 domain. [The present invention 1063] The method of the present invention 1060, wherein the minibody comprises an scFv specific for TIM-3 and a human IgG CH3 domain. [The present invention 1064] The method of the present invention 1060, wherein the minibody comprises an scFv specific for PD-L1 and a human IgG CH3 domain. [The present invention 1065] A method of treating cancer in a subject in need thereof, culturing a plurality of CAR T cells together with GBM organoids (GBOs) derived from a subject; selecting, from the plurality of CAR T cells, the CAR T cells having the highest potency; and administering the CAR T cells having the highest potency to the subject, thereby treating cancer in the subject A method comprising: [Invention 1066] The method of Invention 1065, wherein the plurality of CAR T cells comprise a plurality of modified T cells comprising a plurality of CARs, each CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. [Invention 1067] The antigen-binding domain is capable of binding to an antigen selected from the group consisting of CD19, EGFR, multiple isoforms of EGFR (e.g., wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V , EGFR A289D , EGFR A289T , EGFR R108K , EGFR R108G , EGFR G598V , EGFR D126Y , EGFR C628F , EGFR R108K / A289V , EGFR R108K / D126Y , EGFR A289V / G598V , EGFR A289V / C628F , and EGFR variant II), PSMA, PSCA, and any tumor-associated antigen (TAA)), according to the method of Invention 1066. [Invention 1068] The method of Invention 1065, wherein the GBO is generated from a biopsy material from the subject. [Invention 1069] The method of Invention 1065, wherein the highest potency is measured as the highest degree of apoptosis and / or tumor cell death. [Invention 1070] The method of Invention 1065, further comprising administering an additional treatment to the subject. [Invention 1071] The method of Invention 1066, wherein the additional treatment comprises immune checkpoint inhibition (ICB). [The present invention 1072] The method of the present invention 1067, wherein the ICB is selected from the group consisting of anti-PD-1 treatment, anti-PD-L1 treatment, anti-TIM3 treatment, and anti-CTLA-4 treatment.

[0034] The following detailed description of specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, exemplary embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact configurations and means of the embodiments shown in the drawings.

Brief Description of the Drawings

[0035]

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Mode for Carrying Out the Invention

[0036] Detailed Description Definition Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used.

[0037] It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0038] The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

[0039] As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, etc., encompasses variations of ±20% or ±10% from the specified value, more preferably ±5%, still more preferably ±1%, and even more preferably ±0.1% because such variations are appropriate in the practice of the disclosed methods.

[0040] As used herein, “activation” refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term “activated T cell” specifically refers to a T cell that is undergoing cell division.

[0041] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural or recombinant source, or can be an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0042] The term "antibody fragment" refers to a portion of an intact antibody, and refers to the variable antigen-determining region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, as well as multispecific antibodies formed from antibody fragments.

[0043] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an entire antibody molecule in its naturally occurring conformation.

[0044] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an entire antibody molecule in its naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0045] As used herein, the term "synthetic antibody" means an antibody generated using recombinant DNA techniques, such as an antibody expressed by a bacteriophage described herein. This term also refers to an antibody generated by the synthesis of an antibody protein or an antibody-encoding DNA molecule expressing the amino acid sequence defining the antibody, where the DNA sequence or amino acid sequence is obtained using DNA sequence or amino acid sequence synthesis techniques that are available and well-known in the art.

[0046] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can include either or both antibody production and activation of specific immunocompetent cells. One of ordinary skill in the art will understand that virtually any macromolecule, including all proteins or peptides, can act as an antigen. Further, an antigen can be derived from recombinant DNA or genomic DNA. One of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen" as the term is used herein. Further, one of ordinary skill in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Further, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0047] As used herein, the term "self-derived" is intended to mean any material derived from the same individual that is subsequently re-introduced into that individual.

[0048] "Allogeneic" refers to any material derived from different animals of the same species. "Xenogeneic" refers to any material derived from animals of different species.

[0049] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor expressed on an immune effector cell and engineered to specifically bind to an antigen. CARs can be used as a therapy using adoptive cell transfer. T cells are removed from a patient and modified to express a receptor specific for a particular form of an antigen. In some embodiments, the CAR has specificity for a selected target, such as a B cell surface receptor. A CAR may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain that includes a tumor-associated antigen binding region. In some aspects, the CAR includes an extracellular domain that includes an anti-B cell binding domain fused to a CD3 zeta transmembrane and intracellular domain.

[0050] The term "cleave" refers to the breaking of a covalent bond, such as in the backbone of a nucleic acid molecule, or the hydrolysis of a peptide bond. Cleavage can be initiated by a wide variety of methods, including but not limited to enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible. Double-strand cleavage can occur as a result of two separate single-strand cleavage events. Cleavage of DNA can result in the production of either blunt or sticky ends. In certain embodiments, a fusion polypeptide may be used to target cleaved double-stranded DNA.

[0051] As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for its ability to bind antigen using the functional assays described herein.

[0052] "Co-stimulatory ligand", as used herein, refers to a molecule on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by the binding of the TCR / CD3 complex to, for example, an MHC molecule loaded with a peptide. Co-stimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to the Toll ligand receptor, and a ligand that specifically binds to B7-H3. Co-stimulatory ligands can also include, among others, but not limited to, an antibody that specifically binds to a co-stimulatory molecule present on a T cell such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0053] "Co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell including, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and the Toll ligand receptor.

[0054] "Co-stimulatory signal", as used herein, refers to a signal that, in combination with a primary signal such as ligation of TCR / CD3, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0055] "Disease" is the health state of an animal in which the animal cannot maintain homeostasis and its health will continue to deteriorate if the disease is not improved. In contrast, a "disorder" in an animal is a health state in which the animal can maintain homeostasis, but the health state of the animal is less favorable than when there is no disorder. Even if left untreated, a disorder does not necessarily cause a further decline in the health state of the animal.

[0056] As used herein, the term "downregulation" refers to a decrease or loss of gene expression of one or more genes.

[0057] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material or composition described herein that is effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results can include, but are not limited to, antitumor activity determined by any means appropriate in the art.

[0058] "Encoding" refers to the unique property of a particular nucleotide sequence in a polynucleotide such as a gene, cDNA or mRNA, and the biological property resulting therefrom, which serves as a template for the synthesis of other polymers and macromolecules in a biological process and has either a defined nucleotide (i.e., rRNA, tRNA and mRNA) sequence or a defined amino acid sequence. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is identical to the mRNA sequence and is usually the nucleotide sequence shown in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0059] As used herein, "endogenous" refers to any material that is derived from within or produced within a living organism, cell, tissue or system.

[0060] As used herein, the term "exogenous" refers to any material that is introduced from outside or produced outside a living organism, cell, tissue or system.

[0061] As used herein, the term "increasing" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, the T cells increased ex vivo increase in number compared to the number initially present in the culture. In another embodiment, the T cells increased ex vivo increase in number compared to other cell types in the culture. As used herein, the term "ex vivo" refers to cells removed from a living organism (e.g., a human) and propagated outside the living organism (e.g., in a culture dish, test tube, or bioreactor).

[0062] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0063] An "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains cis-acting elements sufficient for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) incorporating the recombinant polynucleotide, and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus).

[0064] As used herein, "homologous" refers to the identity of subunit sequences between two polymer molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If the positions of the subunits in both of the two molecules are occupied by the same monomer subunit; for example, if the positions in each of two DNA molecules are occupied by adenine, then they are homologous at that position. The homology between two sequences is a linear function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., 5 positions in a 10-subunit-long polymer) are homologous, then these two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, then these two sequences are 90% homologous.

[0065] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding portion sequences of an antibody) that contains minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's complementarity-determining regions (CDRs) have been replaced by residues derived from the CDRs of a non-human species (donor antibody) such as a mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some cases, residues of the Fv framework region (FR) of the human immunoglobulin are replaced by the corresponding non-human residues. Furthermore, a humanized antibody can contain residues not found in either the recipient antibody or the introduced CDR or framework sequences. These modifications are made to further refine and optimize the performance of the antibody. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin sequence. A humanized antibody also optionally includes at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0066] "Fully human" refers to immunoglobulins such as antibodies that are of entirely human origin or consist of the same amino acid sequence as the human form of the antibody.

[0067] As used herein, "identity" refers to the identity of the subunit sequences between two polymer molecules, particularly between two amino acid molecules such as between two polypeptide molecules. When two amino acid sequences have the same residue at the same position; for example, if the position in each of two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a linear function of the number of positions that match or are identical; for example, if half of the positions in two sequences (e.g., 5 positions in a 10-amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0068] As used herein, the terms "immunoglobulin" or "Ig" are defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most commonly found circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important for defense against bacteria and viruses. IgD is an immunoglobulin whose function as an antibody is not understood but may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by causing the release of mediators from mast cells and basophils upon exposure to an allergen.

[0069] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen.

[0070] When indicating an "immunologically effective amount", an "autoimmune disease inhibiting effective amount", or a "therapeutic amount", the exact amount of the composition of the invention to be administered can be determined by a physician or researcher taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in condition of the patient (subject).

[0071] As used herein, "explanatory materials" include publications, records, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the invention. The explanatory materials of the kits of the invention may, for example, be affixed to the container containing the nucleic acids, peptides, and / or compositions of the invention, or may be transported together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the explanatory materials may be transported separately from the container with the intention that the explanatory materials and the compound be used synergistically by the recipient.

[0072] As used herein, the term "isoform" means any of two or more functionally similar proteins having similar but not identical amino acid sequences and encoded by different genes or by RNA transcripts from the same gene with different exons removed.

[0073] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment such as, for example, a host cell.

[0074] "KIR" means killer cell immunoglobulin-like receptor. KIRs have been characterized in humans and non-human primates and are polymorphic type I transmembrane molecules present on certain subsets of lymphocytes including NK cells and some T cells. KIRs regulate the killing function of NK cells by interacting with determinants in the alpha 1 and alpha 2 domains of MHC class I molecules. By this interaction, they become able to detect virus-infected cells or tumor cells. Most KIRs are inhibitory, meaning that recognition of MHC by KIRs suppresses the cytotoxic activity of NK cells expressing the KIRs. Only a limited number of KIRs have the ability to activate cells. The KIR gene family consists of at least 15 gene loci (KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3) and two pseudogenes (KIR2DP1 and KIR3DP1) encoded within a 100 - 200 Kb region (19q13.4) of the leukocyte receptor complex (LRC) located on chromosome 19. The LRC constitutes a large 1 Mb dense cluster of rapidly evolving immune genes that contains genes encoding other cell surface molecules with characteristic Ig-like extracellular domains. In addition, the extended LRC contains genes encoding the transmembrane adapter molecules DAP10 and DAP12.

[0075] As used herein, the term "knockdown" refers to a decrease in gene expression of one or more genes.

[0076] As used herein, the term "knockout" refers to the loss of gene expression of one or more genes.

[0077] As used herein, the term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; since lentiviruses can deliver a substantial amount of genetic information into the DNA of the host cell, they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve substantial levels of in vivo gene transfer.

[0078] As used herein, the term "limited toxicity" refers to the peptides, polynucleotides, cells and / or antibodies of the present invention that clearly demonstrate a substantially negative biological effect, anti-tumor effect, or absence of substantially negative physiological symptoms on healthy cells, non-tumor cells, non-affected cells, non-target cells or populations of such cells, either in vitro or in vivo.

[0079] As used herein, the term "modified" means an altered state or structure of the molecule or cell of the present invention. A molecule can be modified in many ways, such as chemically, structurally, and functionally. A cell can be modified by the introduction of nucleic acids.

[0080] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of response in a subject as compared to the level of response in the subject in the absence of treatment or compound and / or as compared to the level of response in a subject that is otherwise identical but not receiving treatment. This term encompasses disturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0081] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0082] Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns as long as the nucleotide sequence encoding the protein may include introns depending on the type.

[0083] The term "functionally linked" refers to a functional association between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, when a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. Generally, functionally linked DNA sequences are adjacent and, if necessary, connect two protein-coding regions within the same reading frame.

[0084] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate that the expression of the tumor antigen in cells from a disease area such as a solid tumor in a particular tissue or organ of a patient is at an abnormal level compared to the expression level in normal cells from that tissue or organ. A patient having a solid tumor or hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0085] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0086] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, the nucleic acids and polynucleotides used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and that they can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., recombinant libraries using conventional cloning techniques and PCR (trademark), etc., or cloning of nucleic acid sequences from cell genomes, as well as synthetic means.

[0087] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of a protein or peptide. Polypeptides include any peptide or protein containing two or more amino acids joined together by peptide bonds. As used herein, this term refers to both short chains, which are generally also referred to as peptides, oligopeptides and oligomers in the art, and long chains, which are generally referred to as proteins in the art, and there are many types among them. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0088] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of a cell or an introduced synthetic machinery that is required to initiate the specific transcription of a polynucleotide sequence.

[0089] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for the expression of a gene product that is operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence can, for example, be one that expresses the gene product in a tissue-specific manner.

[0090] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly under most or all physiological conditions of the cell.

[0091] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly only substantially when an inducer corresponding to the promoter is present intracellularly.

[0092] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes the gene product to be produced intracellularly only substantially when the cell is a cell of the tissue type corresponding to the promoter.

[0093] "Signal transduction pathway" refers to the biochemical relationships among a diverse array of signal transduction molecules that play a role in the propagation of a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes that can receive a signal and propagate the signal through the plasma membrane of the cell.

[0094] As used herein with respect to antibodies, the term "specifically binds" means an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such cross-reactivity by itself does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different genotypes of that antigen. However, such cross-reactivity by itself does not change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used in the context of the interaction of an antibody, protein or peptide with a second chemical species to mean that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction mixture containing the labeled "A" and that antibody will reduce the amount of labeled A bound to the antibody.

[0095] The term "stimulate" means a primary response induced by a stimulatory molecule (e.g., the TCR / CD3 complex) binding to its cognate ligand and thereby mediating a signaling event, including, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate a change in the expression of certain molecules, such as down-regulation of TGF-beta, and / or rearrangement of the cytoskeletal structure.

[0096] As used herein, the term "stimulatory molecule" means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0097] As used herein, a "stimulatory ligand" means a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0098] The term "subject" is intended to include organisms (e.g., mammals) capable of eliciting an immune response. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cows, pigs, dogs, cats, and murine mammals. Preferably, the subject is human.

[0099] As used herein, a "substantially purified" cell is a cell that essentially lacks other cell types. Substantially purified cells also refer to cells that have been separated from other cell types that are normally associated with them in their native state. In some instances, a population of substantially purified cells refers to a homogeneous population of cells. In other instances, the term simply refers to cells that have been separated from cells that were originally associated with them in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0100] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0101] As used herein, the term "T cell receptor" or "TCR" refers to a membrane protein complex involved in the activation of T cells in response to antigen presentation. The TCR is responsible for recognizing an antigen bound to a major histocompatibility complex molecule. Although the TCR is composed of a heterodimer of alpha (α) and beta (β) chains, in some cells, the TCR consists of gamma and delta (γ / δ) chains. The TCR may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, namely variable and constant domains. In some embodiments, the TCR may be modified in any cell that includes a TCR, such as, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0102] As used herein, the term "therapeutic" means treatment and / or prevention. A therapeutic effect is obtained by suppression, remission or eradication of a disease state.

[0103] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. This includes the primary subject cell and its progeny.

[0104] To "treat" a disease, as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder that a subject is suffering from.

[0105] As used herein, "local" or "locally" when used to refer to delivery of treatment means intrathecal, intratumoral, or other forms of treatment delivery that are not parenteral.

[0106] As used herein, the phrases "under transcriptional control" or "functionally linked" mean that a promoter is in the correct position and orientation with respect to a polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0107] A "vector" is a composition of materials that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides complexed with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the entry of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0108] Range: Throughout this disclosure, various aspects of the invention can be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a description of a range should be considered to specifically disclose all the sub-ranges and individual numerical values within that range. For example, a range description such as 1 - 6 should be considered to specifically disclose sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0109] Explanation The present invention provides a high-affinity chimeric antigen receptor (CAR) having cross-reactivity with clinically significant EGFR variant proteins and methods of using the same. In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of the epidermal growth factor receptor (EGFR), a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the CAR is a humanized CAR. In certain embodiments, the CAR is an affinity matured humanized CAR. In certain embodiments, the CAR is a KIR CAR. In certain aspects, the present invention includes a method of treating cancer in a subject in need thereof by administering the CAR of the present invention.

[0110] Chimeric antigen receptor (CAR) The present invention provides a chimeric antigen receptor (CAR) capable of binding to / having affinity for multiple EGFR isoforms. Also provided are nucleic acids encoding the CAR, vectors encoding the nucleic acids, and modified cells (e.g., modified T cells) comprising the CAR, vector, or nucleic acid.

[0111] The subject CAR of the present invention comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain. The subject CAR of the present invention may optionally include a hinge domain. Thus, the subject CAR of the present invention comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a hinge domain, a transmembrane domain, and an intracellular domain. In certain embodiments, each of the domains of the subject CAR is separated by a linker.

[0112] The antigen-binding domain may be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, each described elsewhere herein for expression in a cell. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is operably linked to a second nucleic acid encoding the transmembrane domain and further operably linked to a third nucleic acid sequence encoding a co-stimulatory signaling domain.

[0113] The antigen-binding domains described herein can be combined with any of the transmembrane domains, any of the intracellular domains, or any of the other domains described herein that can be included in the CARs of the present invention.

[0114] In one aspect, the present invention includes an isolated CAR comprising an antigen-binding domain capable of binding to multiple isoforms of epidermal growth factor receptor (EGFR), a transmembrane domain, and an intracellular domain. The EGFR isoforms to which the CAR can bind include wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V , EGFR A289D , EGFR A289T , EGFR R108K , EGFR R108G , EGFR G598V , EGFR D126Y , EGFR C628F , EGFR R108K / A289V , EGFR R108K / D126Y , EGFR A289V / G598V , EGFR A289V / C628F , and including, but not limited to, EGFR variant II.

[0115] In one aspect, the present invention includes an isolated CAR comprising an antigen-binding domain capable of binding to multiple isoforms of EGFR, a CD8 hinge domain, a CD8 transmembrane domain, and a 4-1BBZ intracellular domain. In another aspect, the present invention includes an isolated nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a CD8 hinge domain, a CD8 transmembrane domain, and a 4-1BBZ intracellular domain. Another aspect of the present invention is an isolated polypeptide comprising a CAR, wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a CD8 hinge domain, a CD8 transmembrane domain, and a 4-1BBZ intracellular domain.

[0116] In one aspect, the present invention includes a killer cell immunoglobulin-like receptor (KIR)-based chimeric antigen receptor (CAR). The KIR-CAR is based on the killer cell immunoglobulin-like receptor (KIR) normally expressed by natural killer (NK) cells. In certain embodiments, the present invention includes a KIR-CAR comprising an antigen-binding domain capable of binding to multiple isoforms of EGFR, a KIR transmembrane domain, and a KIR intracellular (cytoplasmic) domain. In certain embodiments, the present invention includes an isolated nucleic acid encoding a KIR-CAR, wherein the KIR-CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a KIR transmembrane domain, and a KIR intracellular (cytoplasmic) domain. In certain embodiments, the present invention includes an isolated polypeptide comprising a KIR-CAR, wherein the KIR-CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a KIR transmembrane domain, and a KIR intracellular (cytoplasmic) domain.

[0117] In one aspect, the present invention includes a humanized EGFR CAR. In one aspect, the present invention includes an affinity matured humanized EGFR CAR.

[0118] (Table 1) Amino acid sequences and nucleotide sequences TIFF2025102823000001.tif41157TIFF2025102823000002.tif243157TIFF2025102823000003.tif245157TIFF2025102823000004.tif243157TIFF2025102823000005.tif243157TIFF2025102823000006.tif243157TIFF2025102823000007.tif253157TIFF2025102823000008.tif234157TIFF2025102823000009.tif243157TIFF2025102823000010.tif243157TIFF2025102823000011.tif198157

[0119] Antigen-binding domain The antigen-binding domain of the CAR is the extracellular region of the CAR for binding to specific target antigens including proteins, carbohydrates, and glycolipids. In some embodiments, the CAR comprises an affinity for a target antigen (e.g., a tumor-associated antigen) on a target cell (e.g., a cancer cell). The target antigen can include any type of protein or its epitope associated with the target cell. For example, the CAR can comprise an affinity for a target antigen on a target cell that indicates a specific state of the target cell.

[0120] In certain embodiments, the CAR of the present invention comprises an antigen-binding domain capable of binding to multiple isoforms of epidermal growth factor receptor (EGFR). In certain embodiments, the antigen-binding domain is cross-reactive with multiple isoforms of EGFR. In certain embodiments, the antigen-binding domain specifically binds to multiple isoforms of EGFR. In certain embodiments, the antigen-binding domain of the present invention comprises an antibody or a fragment thereof capable of binding to multiple EGFR molecules. Preferably, the antigen-binding domain is a scFv antibody capable of binding to multiple EGFR isoforms. EGFR isoforms include, but are not limited to, wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V and EGFR R108K Including, but not limited to. In certain embodiments, the subject CAR is wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR R108K EGFR R108G EGFR G598V EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F And is capable of binding to one or more EGFR isoforms selected from the group consisting of EGFR variant II.

[0121] In certain embodiments, the antigen-binding domain of the CAR binds to dimerized EGFR. For example, the CAR can bind to an EGFR homodimer containing two identical EGFR isoforms. The CAR can also bind to an EGFR heterodimer containing two different EGFR isoforms, or an oligomer containing multiple copies of the same or different EGFR isoforms. In certain embodiments, the CAR can bind to an EGFR / ErbB heterodimer or oligomer.

[0122] As described herein, the CARs of the disclosure that have affinity for a specific target antigen (e.g., EGFR) on a target cell can include a target-specific binding domain. In some embodiments, the target-specific binding domain is a murine target-specific binding domain, e.g., the target-specific binding domain is of murine origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin. In an exemplary embodiment, the CARs of the disclosure that have affinity for EGFR on a target cell can include an EGFR binding domain. In some embodiments, the Tn-MUC1 binding domain is a murine EGFR binding domain, e.g., the EGFR binding domain is of murine origin. In some embodiments, the EGFR binding domain is a humanized EGFR binding domain. In some embodiments, the EGFR binding domain is a human EGFR binding domain, e.g., the EGFR binding domain is of human origin.

[0123] The antigen-binding domain can include any domain that binds to an antigen and can include, but is not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. Thus, in one aspect, the antigen-binding domain portion includes a mammalian antibody or a fragment thereof. In another aspect, the antigen-binding domain of the CAR is selected from the group consisting of an anti-EGFR antibody or a fragment thereof. In some aspects, the antigen-binding domain is selected from the group consisting of an antibody, an antigen-binding fragment (Fab), and a single-chain variable fragment (scFv). In some aspects, the EGFR-binding domain of the present invention is selected from the group consisting of an EGFR-specific antibody, an EGFR-specific Fab, and an EGFR-specific scFv. In one aspect, the EGFR-binding domain is an EGFR-specific antibody. In one aspect, the EGFR-binding domain is an EGFR-specific Fab. In one aspect, the EGFR-binding domain is an EGFR-specific scFv.

[0124] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin (e.g., mouse or human) are covalently linked to form a VH::VL heterodimer. The heavy chain (VH) and light chain (VL) are either directly linked or linked by a linker or spacer that encodes a peptide that connects the N-terminus of VH to the C-terminus of VL or the C-terminus of VH to the N-terminus of VL. The terms "linker" and "spacer" are used interchangeably herein. In some aspects, the antigen-binding domain (e.g., the Tn-MUC1-binding domain) includes an scFv having a VH-linker-VL arrangement from the N-terminus to the C-terminus. In some aspects, the antigen-binding domain (e.g., the Tn-MUC1-binding domain) includes an scFv having a VL-linker-VH arrangement from the N-terminus to the C-terminus. One of ordinary skill in the art will be able to select an arrangement suitable for use in the present invention.

[0125] Linkers typically are rich in serine or threonine for solubility in addition to glycine for flexibility. Linkers can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and International Publication No. 2014 / 087010, the contents of which are hereby incorporated by reference in their entirety. Various linker sequences are known in the art, including, but not limited to, glycine-serine (GS) linkers such as (GS) n , (GSGGS) n (SEQ ID NO:36), (GGGS) n (SEQ ID NO:37), and (GGGGS) n (SEQ ID NO:38) [wherein n represents an integer of at least 1]. Exemplary linker sequences can include, but are not limited to, amino acid sequences such as GGSG (SEQ ID NO:39), GGSGG (SEQ ID NO:40), GSGSG (SEQ ID NO:41), GSGGG (SEQ ID NO:42), GGGSG (SEQ ID NO:43), GSSSG (SEQ ID NO:44), GGGGS (SEQ ID NO:45), GGGGSGGGGSGGGGS (SEQ ID NO:46), etc. One of ordinary skill in the art will be able to select a linker sequence suitable for use in the present invention. In one aspect, the antigen-binding domain of the present invention (e.g., the EGFR-binding domain) includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are separated by a linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:46) encoded by the nucleic acid sequence TIFF2025102823000012.tif5128. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:48. In certain embodiments, the linker is encoded by the nucleic acid sequence of SEQ ID NO:49.

[0126] As used herein, "Fab" refers to a fragment of an antibody structure that binds to an antigen, is monovalent, and has no Fc portion. For example, an antibody digested with the enzyme papain yields two Fab fragments and an Fc fragment (e.g., the heavy (H) chain constant region; the Fc region that does not bind to the antigen).

[0127] As used herein, "F(ab')2" refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, where this fragment has two antigen-binding (ab') (bivalent) regions, and each (ab') region contains a part of the H chain and two separate amino acid chains in which the light (L) chains are linked by an S-S bond for binding to the antigen, and the remaining H chain portions are linked together. The "F(ab')2" fragment can be split into two individual Fab' fragments.

[0128] In some examples, the antigen-binding domain may be derived from the same species as the species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may include a human antibody or a fragment thereof described elsewhere herein. In some embodiments, non-human antibodies are humanized, in which specific sequences or regions of the antibody are modified to increase similarity to antibodies naturally produced in humans. In one embodiment, the antigen-binding domain portion is humanized.

[0129] Humanized antibodies are prepared by CDR grafting (see, e.g., EP 239,400, International Publication No. WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering or resurfacing (see, e.g., EP 592,106 and EP 519,596, Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498, Studnicka et al., 1994, Protein Engineering, 7(6):805-814, and Roguska et al., 1994, PNAS, 91:969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein by reference in its entirety), and, for example, U.S. Patent Application Publication No. 2005 / 0042664, U.S. Patent Application Publication No. 2005 / 0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 93 / 17105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al.It can be produced using a variety of techniques known in the art, including, but not limited to, the techniques disclosed in , J. Mol. Biol., 235(3):959-73 (1994). In many cases, framework residues in the framework region will be replaced by the corresponding residues from the CDR donor antibody in order to vary, e.g., improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling the interaction of CDR and framework residues to identify framework residues important for antigen binding, as well as by sequence comparison to identify unusual framework residues at specific positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089, which is hereby incorporated by reference in its entirety; and Riechmann et al., 1988, Nature, 332:323).

[0130] A humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "imported" residues that are typically taken from the "import" variable domain. Thus, a humanized antibody contains one or more CDRs from a non-human immunoglobulin molecule and a framework region from a human. Humanization of antibodies is well known in the art and can be essentially performed by substituting rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, i.e., by CDR grafting (EP 239,400, PCT publication number WO91 / 09967, and U.S. Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized chimeric antibodies, those that are substantially less than intact human variable domains are replaced by the corresponding sequences from non-human species. In practice, a humanized antibody is typically a human antibody in which some CDR residues and sometimes some framework (FR) residues are replaced by residues from similar sites in a rodent antibody.

[0131] In certain embodiments, the CAR of the present invention includes an EGFR binding domain capable of binding to multiple EGFR isoforms, such as an EGFR-specific scFv. In certain embodiments, the antigen-binding domain includes an antibody or a fragment thereof derived from the monoclonal antibody mAb 806 (Binder et al. (2018) Cancer cell, 34(1), pp.163-177). In certain embodiments, the EGFR binding domain includes the amino acid sequence shown in SEQ ID NO:2. In certain embodiments, the EGFR binding domain includes the amino acid sequence shown in SEQ ID NO:32. In certain embodiments, the EGFR binding domain is encoded by the nucleotide sequence shown in SEQ ID NO:1. In certain embodiments, the EGFR binding domain is encoded by the nucleotide sequence shown in SEQ ID NO:31.

[0132] In certain embodiments, the CAR of the present invention includes a humanized EGFR binding domain capable of binding to multiple EGFRs. In certain embodiments, the antigen-binding domain includes an antibody or a fragment thereof derived from the humanized monoclonal antibody mAb 806. In certain embodiments, the EGFR binding domain generally includes the amino acid sequence shown in SEQ ID NO:28. In certain embodiments, the EGFR binding domain includes the amino acid sequence shown in SEQ ID NO:80. In certain embodiments, the EGFR binding domain includes the amino acid sequence shown in SEQ ID NO:82. In certain embodiments, the EGFR binding domain is generally encoded by the nucleotide sequence shown in SEQ ID NO:25. In certain embodiments, the EGFR binding domain is encoded by the nucleotide sequence shown in SEQ ID NO:79. In certain embodiments, the EGFR binding domain is encoded by the nucleotide sequence shown in SEQ ID NO:81.

[0133] In certain embodiments, the CAR of the invention comprises an EGFR binding domain that is affinity matured and humanized and capable of binding to multiple EGFRs. In certain embodiments, the antigen binding domain comprises an antibody or fragment thereof derived from the monoclonal antibody mAb 806 that is affinity matured and humanized. In certain embodiments, the EGFR binding domain comprises the amino acid sequence set forth in SEQ ID NO:84. In certain embodiments, the EGFR binding domain comprises the amino acid sequence set forth in SEQ ID NO:86. In certain embodiments, the EGFR binding domain is encoded by the nucleotide sequence set forth in SEQ ID NO:83. In certain embodiments, the EGFR binding domain is encoded by the nucleotide sequence set forth in SEQ ID NO:85.

[0134] In certain embodiments, the antigen binding domain comprises a variable light chain region (VL) comprising the amino acid sequence set forth in SEQ ID NO:3 encoded by SEQ ID NO:33. The variable light chain region of the antigen binding domain comprises three light chain complementarity determining regions (CDRs). As used herein, "complementarity determining region" or "CDR" refers to the region of the variable chain of an antigen binding molecule that binds to a particular antigen. Thus, the EGFR binding domain may comprise a variable light chain region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6; and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7.

[0135] In certain embodiments, the antigen binding domain comprises a variable light chain region comprising the amino acid sequence set forth in SEQ ID NO:27 encoded by the nucleic acid sequence set forth in SEQ ID NO:24. In certain embodiments, the antigen binding domain comprises a variable light chain region comprising the amino acid sequence set forth in SEQ ID NO:30.

[0136] In certain embodiments, the antigen-binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 4, encoded by SEQ ID NO: 34. The EGFR-binding domain may comprise a heavy chain variable region comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 8; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10.

[0137] In certain embodiments, the antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26, encoded by the nucleic acid sequence shown in SEQ ID NO: 23. In certain embodiments, the antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29.

[0138] In certain embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 62 and / or a VL comprising the amino acid sequence shown in SEQ ID NO: 63.

[0139] In certain embodiments, the antigen-binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of ID NOs: 4, 26, and 29 and / or a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 27, and 30. In certain embodiments, the antigen-binding domain comprises a heavy chain variable region (VH) encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 43, 23, and 62 and / or a light chain variable region (VL) encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 33, 24, and 63.

[0140] Permissible variations of the EGFR binding domain while maintaining specific binding to EGFR would be known to those of ordinary skill in the art. For example, in some embodiments, the EGFR binding domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any of the amino acid sequences set forth in SEQ ID NO: 2-10, 26-30, 32, 80, 82, 84, or 86. In some embodiments, the EGFR binding domain is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the nucleic acid sequences set forth in SEQ ID NO: 1, 23-25, 31, 33, 34, 62-63, 79, 81, 83, or 85.

[0141] The antigen-binding domain can be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, described elsewhere herein. In one embodiment, the nucleic acid encoding the antigen-binding domain is operably linked to the nucleic acid encoding the transmembrane domain and the nucleic acid encoding the intracellular domain.

[0142] An antigen-binding domain described herein, such as an antibody or fragment thereof that binds to EGFR, can be combined with any of the transmembrane domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that can be included in a CAR.

[0143] Transmembrane domain With respect to the transmembrane domain, the CARs of the invention (e.g., cross-reactive EGFR CARs) can be designed to include a transmembrane domain that connects the antigen-binding domain to the intracellular domain. The transmembrane domain of a subject CAR is a region capable of traversing the plasma membrane of a cell (e.g., an immune cell or a progenitor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is sandwiched between the antigen-binding domain and the intracellular domain of the CAR.

[0144] In one embodiment, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some examples, the transmembrane domain can be modified by selection or amino acid substitution to avoid binding to such domains and transmembrane domains of the same or different surface membrane proteins, minimizing interaction with other members of the receptor complex.

[0145] The transmembrane domain may be derived from either a natural source or a synthetic source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, such as a type I transmembrane protein. When the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into the cell membrane, such as an artificial hydrophobic sequence. Examples of transmembrane regions for particular uses in the present invention include, without limitation, transmembrane domains derived from the alpha, beta, or zeta chains of the T cell receptor, CD2, CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), KIR, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9 (i.e., including at least the transmembrane regions thereof). In some embodiments, the transmembrane domain may be synthetic, in which case it will predominantly contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0146] The transmembrane domains described herein can be combined with any of the antigen-binding domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein that may be included in the CAR of a subject.

[0147] In some embodiments, the transmembrane domain further includes a hinge region. The CARs of interest of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain facilitates protein folding suitable for the CAR. The hinge region is an optional component of the CAR. The hinge region may include a domain selected from the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, an artificial hinge composed of a polypeptide that can be as small as about three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0148] In some embodiments, the CARs of interest of the present disclosure include a hinge region that connects the antigen-binding domain to the transmembrane domain, which in turn connects to the intracellular domain. The hinge region preferably enables the antigen-binding domain to recognize and bind to a target antigen on the target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, such that the antigen-binding domain can have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell. The flexibility of the hinge region allows the hinge region to assume many different conformations.

[0149] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0150] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa.

[0151] A suitable hinge region can be easily selected and can be any of 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including some suitable lengths, for example, 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0152] For example, the hinge region is a glycine polymer (G) n , a glycine - serine polymer (e.g., (GS) n , (GSGGS) n (SEQ ID NO:36) and (GGGS) n(comprising (SEQ ID NO:37), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine polymers and glycine-serine polymers can be used; both Gly and Ser are relatively conformationally flexible and can thus serve as neutral tethers between components. Glycine polymers can be used; glycine accesses significantly more φ-ψ space than alanine and is much less restricted than residues with longer side chains (see, for example, Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:39), GGSGG (SEQ ID NO:40), GSGSG (SEQ ID NO:41), GSGGG (SEQ ID NO:42), GGGSG (SEQ ID NO:43), GSSSG (SEQ ID NO:44), etc.

[0153] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Amino acid sequences of immunoglobulin hinge regions are known in the art; see, for example, Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, immunoglobulin hinge regions can have the following amino acid sequences: TIFF2025102823000013.tif5162 (see, for example, Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); TIFF2025102823000014.tif41152, etc.

[0154] The hinge region can comprise the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 hinge region. In one aspect, the hinge region can comprise one or more amino acid substitutions and / or insertions and / or deletions as compared to the wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be substituted by Tyr, whereby the hinge region comprises the sequence including TIFF2025102823000015.tif4128; see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one aspect, the hinge region can comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0155] In certain aspects, the transmembrane domain comprises the CD8α transmembrane domain. In certain aspects, the transmembrane domain comprises the CD8α hinge domain and the CD8α transmembrane domain. In certain aspects, the subject CAR comprises the CD8α transmembrane domain encoded by the nucleic acid sequence set forth in SEQ ID NO:12. In certain aspects, the subject CAR comprises the hinge domain encoded by the nucleic acid sequence of SEQ ID NO:11 and the transmembrane domain encoded by the nucleic acid sequence set forth in SEQ ID NO:12.

[0156] In certain aspects, the transmembrane domain comprises a KIR domain. In certain aspects, the transmembrane domain comprises a KIRS2 domain.

[0157] Permissible variations of the transmembrane and / or hinge domains while maintaining their intended function will be known to those skilled in the art. For example, in some embodiments, the transmembrane domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any of the amino acid sequences shown in SEQ ID NO:11. For example, in some embodiments, the hinge transmembrane domain is encoded by a nucleic acid sequence comprising a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any of the nucleic acid sequences shown in SEQ ID NO:12.

[0158] The transmembrane domain may be combined with any hinge domain and / or may comprise one or more transmembrane domains described herein. The transmembrane domains described herein can be combined with any of the antigen-binding domains described herein, any of the co-stimulatory signaling domains or intracellular or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR.

[0159] In one aspect, the transmembrane domain can be synthetic, in which case it will comprise mainly hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0160] A spacer domain can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that functions to link the transmembrane domain to either the extracellular domain or the intracellular domain within the polypeptide chain. The spacer domain can contain up to 300 amino acids, for example, 10 - 100 amino acids, or 25 - 50 amino acids. In some aspects, the spacer domain can be a short-chain oligopeptide or polypeptide linker, for example, 2 to 10 amino acids in length. For example, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of the subject CAR.

[0161] Intracellular domain The subject CARs of the invention also include an intracellular domain. The intracellular domain of the CAR is responsible for activating at least one of the effector functions of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular domain transmits an effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., to damage and / or destroy the target cell.

[0162] The intracellular domain or, alternatively, the cytoplasmic domain of the CAR is responsible for the activation of the cells in which the CAR is expressed. Examples of intracellular domains for use in the present invention include the cytoplasmic portions of surface receptors, costimulatory molecules, and any molecule that acts in concert to initiate signal transduction in T cells, in addition to any derivatives or variants of these elements and any synthetic sequences having the same functional properties, but are not limited thereto.

[0163] In certain embodiments, the intracellular domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. In certain embodiments, the intracellular domain comprises 4-1BB and CD3 zeta. In certain embodiments, the intracellular domain comprises 4-1BB. In certain embodiments, the intracellular domain comprises CD3 zeta. In certain embodiments, the intracellular domain comprises KIRS2.

[0164] In one embodiment, the intracellular domain of the CAR comprises a costimulatory signaling domain comprising any portion of one or more costimulatory molecules, e.g., CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivatives or variants thereof, any synthetic sequences having the same functional properties, and at least one signaling domain from any combination thereof.

[0165] Examples of intracellular signaling domains include, without limitation, either the ζ chain of the T cell receptor complex or its homolog, such as the η chain, FcsRIγ and β chains, MB1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5 and CD28. In one aspect, the intracellular signaling domain can be the human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, immunoreceptor tyrosine activation motif (ITAM)-bearing cytoplasmic receptors, and combinations thereof.

[0166] Other examples of intracellular domains include ligands that specifically bind to TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CD8, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTOne or more molecules or receptors including, but not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, any KIR, e.g., KIR2, KIRS2, KIR2DS2, other costimulatory molecules described herein, any derivatives, variants, or fragments thereof, any synthetic sequences of costimulatory molecules having the same functional ability, and any combinations thereof, and fragments or domains derived from one or more of such molecules or receptors are included.

[0167] Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several different other immune signaling receptors, including, without limitation, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory receptors, and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, the intracellular signaling domains can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), e.g., NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), and can include the signaling domains of NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0168] Intracellular signaling domains suitable for use in the CARs of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., an increase in the production of one or more cytokines by the cell; a change in the transcription of a target gene; a change in the activity of a protein; a change in cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response, etc.) in response to activation of the CAR (i.e., activation by an antigen and a dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular signaling domain includes a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain diffuses into the cytoplasm instead of covalently binding to the membrane-bound CAR.

[0169] Intracellular signaling domains suitable for use in the CARs of the present invention include immunoreceptor tyrosine activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, where the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the subject CAR includes three ITAM motifs. In some embodiments, the intracellular signaling domain includes, without limitation, the signaling domains of human immunoglobulin receptors that contain an immunoreceptor tyrosine activation motif (ITAM), such as Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5, etc. (see, e.g., Gillis et al., Front. (2014) Immunol. 5:254).

[0170] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the whole protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0171] In one aspect, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase-binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activation receptor-associated protein; killer activation receptor-associated protein, etc.). In one aspect, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRl gamma; high-affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high-affinity gamma chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G; T cell receptor T3 gamma chain; CD3-GAMMA; T3G; gamma polypeptide (TiT3 complex), etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z; T cell receptor T3 zeta chain; CD247; CD3-ZETA; CD3H; CD3Q; T3Z; TCRZ, etc.). In one aspect, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-related alpha); MB-1 membrane glycoprotein; Ig-alpha; membrane-bound immunoglobulin-related protein; surface IgM-related protein, etc.).In one aspect, the intracellular signaling domain suitable for use in the CARs of the present disclosure includes a DAP10 / CD28 type signaling chain. In one aspect, the intracellular signaling domain suitable for use in the CARs of the present disclosure includes a ZAP70 polypeptide. In some aspects, the intracellular signaling domain includes the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b or CD66d. In one aspect, the intracellular signaling domain in the CAR includes the cytoplasmic signaling domain of human CD3 zeta.

[0172] Generally, the entire intracellular signaling domain can be used, but in many cases, it is not necessarily required to use the entire chain. To the extent that truncated portions of the intracellular signaling domain are used, such truncated portions can be used in place of the intact chain as long as they transmit effector function signals. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0173] The intracellular domains described herein can be combined with any of the antigen-binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0174] In certain embodiments, the intracellular domain of the subject CAR comprises a 4-1BB co-stimulatory domain. In certain embodiments, the intracellular domain is encoded by the nucleic acid sequence set forth in SEQ ID NO:13. In certain embodiments, the intracellular domain of the subject CAR comprises a CD3 zeta intracellular signaling domain. In certain embodiments, the intracellular domain is encoded by the nucleic acid sequence set forth in SEQ ID NO:14. In certain embodiments, the intracellular domain of the subject CAR comprises a 4-1BB domain and a CD3 zeta domain. In certain embodiments, the intracellular domain is encoded by a nucleic acid sequence comprising SEQ ID NO:13 and SEQ ID NO:14.

[0175] In certain embodiments, the intracellular domain comprises a DAP12 domain. In certain embodiments, the intracellular domain comprises a KIR domain. The intracellular domain may comprise one or more intracellular domains described herein. In certain embodiments, the CAR comprises a KIRS2 transmembrane and intracellular domain. In certain embodiments, the intracellular domain is encoded by a nucleotide sequence comprising SEQ ID NO:18.

[0176] Permissible variations of the intracellular domain while maintaining specific activity will be known to those of skill in the art. For example, in some embodiments, the intracellular domain is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any of the nucleic acid sequences set forth in SEQ ID NO:13, 14, and / or 18.

[0177] In another aspect, a spacer domain may be incorporated between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to either the antigen-binding domain or the intracellular domain within the polypeptide chain. In one aspect, the spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids. In another aspect, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, may form a linkage between the transmembrane domain and the intracellular domain of the CAR. Examples of linkers include glycine-serine doublets.

[0178] KIR-CAR In certain aspects, the invention relates to a "KIR-CAR", which is a CAR design that includes components of receptors found on natural killer (NK) cells. The KIR-CAR provided herein includes an antigen-binding domain capable of binding to multiple isoforms of the epidermal growth factor receptor (EGFR), and a KIR transmembrane domain and / or a KIR intracellular (cytoplasmic) domain.

[0179] Accordingly, the invention provides a composition comprising a KIR-CAR, an isolated nucleic acid comprising a KIR-CAR, an isolated polypeptide comprising a KIR-CAR, and a recombinant T cell comprising a KIR-CAR; wherein the KIR-CAR comprises an antigen-binding domain capable of binding to multiple isoforms of the EGFR.

[0180] NK cells are mononuclear cells that originate from lymphocyte progenitor cells in the bone marrow. Morphological features typically include the expression of cluster determinants (CD) CD16, CD56, and / or CD57 on the cell surface and the absence of the alpha / beta or gamma / delta TCR complex. Biological properties typically include the ability to bind to and kill target cells that do not express "self" major histocompatibility complex (MHC) / human leukocyte antigen (HLA) proteins, and the ability to kill tumor cells or other diseased cells that express ligands for activating NK receptors. NK cells are characterized by their ability to bind to and kill several types of tumor cell lines without the need for prior immunization or activation. NK cells can also release soluble proteins and cytokines that exert a regulatory effect on the immune system; undergo multiple rounds of cell division and produce daughter cells with biological properties similar to those of the parent cell. Upon activation by interferon and / or cytokines, NK cells mediate the lysis of tumor cells and cells infected with intracellular pathogens through a mechanism that requires direct physical contact between the NK cell and the target cell. Lysis of the target cell involves the release of cytotoxic granules from the NK cell onto the bound target surface, as well as effector proteins such as perforin and granzyme B that penetrate the target's plasma membrane and induce apoptosis or programmed cell death. Normally, healthy cells are protected from lysis by NK cells. NK cell activity is regulated by a complex mechanism involving both stimulatory and inhibitory signals.

[0181] Briefly, the lytic activity of NK cells is regulated by various cell surface receptors that transmit either positive or negative intracellular signals upon interaction with ligands on target cells. The balance between the positive and negative signals propagated through these receptors determines whether the target cells are lysed (killed) by NK cells. NK cell stimulatory signals can be mediated by natural cytotoxicity receptors (NCR) such as NKp30, NKp44, and NKp46; as well as NKG2C receptor, NKG2D receptor, certain activating killer cell immunoglobulin-like receptors (KIR), and other activating NK receptors (Lanier, Annual Review of Immunology 2005; 23:225-74). NK cell inhibitory signals can be mediated by receptors such as Ly49, CD94 / NKG2A, and certain inhibitory KIR that recognize major histocompatibility complex (MHC) class I molecules (Karre et al., Nature 1986; 319:675-8; Ohlen et al, Science 1989; 246:666-8). These inhibitory receptors bind to polymorphic determinants of MHC class I molecules (including HLA class I) present on other cells and inhibit NK cell-mediated lysis.

[0182] KIR, also known as killer cell immunoglobulin-like receptor, is a polymorphic type I transmembrane molecule present on certain subsets of lymphocytes including NK cells and some T cells, and is characterized in humans and non-human primates. KIR interacts with determinants in the alpha 1 and 2 domains of MHC class I molecules, and as described elsewhere herein, different KIRs can be either stimulatory or inhibitory to NK cells.

[0183] The nomenclature for KIRs is based on the number of extracellular domains (KIR2D and KIR3D have two and three extracellular Ig domains, respectively) and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). The presence or absence of a given KIR varies from NK cell to NK cell within the NK population present in a single individual. Among humans, there is also a relatively high level of polymorphism of the KIR genes, such that certain KIR genes are present in some, but not all, individuals. The expression of KIR alleles on NK cells is regulated probabilistically, meaning that in a given individual, a given lymphocyte may express one, two, or more different KIRs depending on the individual's genotype. The NK cells of a single individual typically express different combinations of KIRs, providing a repertoire of NK cells with different specificities for MHC class I molecules.

[0184] Certain KIR gene products cause stimulation of lymphocyte activity when bound to appropriate ligands. Activating KIR all have short cytoplasmic tails with charged transmembrane residues that associate with adapter molecules having immunoreceptor activation tyrosine motifs (ITAMs) that transmit stimulatory signals to NK cells. In contrast, inhibitory KIR have long cytoplasmic tails that contain immunoreceptor inhibitory tyrosine motifs (ITIMs) that transmit inhibitory signals to NK cells upon binding to their MHC class I ligands. Known inhibitory KIR include members of the KIR2DL and KIR3DL subfamilies. Inhibitory KIR having two Ig domains (KIR2DL) recognize HLA-C allotypes, and KIR2DL2 (previous name p58.2) and the closely related allelic product KIR2DL3 both recognize “group 1” HLA-C allotypes (including HLA-Cw1, -3, -7, and -8), while KIR2DL1 (p58.1) recognizes “group 2” HLA-C allotypes (HLA-Cw2, -4, -5, and -6). Recognition by KIR2DL1 is dictated by the presence of a Lys residue at position 80 of the HLA-C allele. Recognition by KIR2DL2 and KIR2DL3 is dictated by the presence of an Asn residue at position 80 of HLA-C. Importantly, most HLA-C alleles have either an Asn or a Lys residue at position 80. Thus, KIR2DL1, -2, and -3 together recognize essentially all HLA-C allotypes found in humans. One KIR having three Ig domains, KIR3DL1 (p70), recognizes an epitope shared by HLA-Bw4 alleles. Finally, the homodimer of the molecule having three Ig domains, KIR3DL2 (p140), recognizes HLA-A3 and -A11.

[0185] However, the present invention should not be limited to inhibitory KIRs containing a cytoplasmic tail containing an ITIM. Rather, any inhibitory protein having a cytoplasmic domain associated with an inhibitory signal can be used in the construction of the CARs of the present invention. Non-limiting examples of inhibitory proteins include, but are not limited to, CTLA-4, PD-1, etc. These proteins are known to inhibit T cell activation.

[0186] Accordingly, the present invention provides a KIR-CAR comprising an extracellular domain comprising a target-specific binding element, also referred to as an antigen-binding domain, optionally fused to a KIR or a fragment thereof. In one aspect, the KIR is an activating KIR comprising a short cytoplasmic tail that associates with an adapter molecule having an immunoreceptor activation tyrosine motif (ITAM) that transmits a stimulatory signal to NK cells.

[0187] In some cases, it may be desirable to remove / include no hinge region when constructing the KIR-CAR. Without wishing to be bound by any particular theory, removal of the hinge region of the KIR-CAR may result in increased cytolytic activity.

[0188] In certain aspects, the present invention provides an isolated nucleic acid comprising an EF1 alpha sequence, a DAP12 sequence, a T2A sequence, an 806-scFv sequence, a KIR transmembrane domain sequence, and a KIR cytoplasmic (intracellular) domain sequence.

[0189] Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, for example, by screening a library from cells expressing the gene, by deriving the gene from a vector known to contain it, or by directly isolating it from cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than by cloning.

[0190] In certain embodiments, the invention provides a KIR-CAR comprising an antigen-binding domain comprising 806-scFv, a KIR transmembrane domain, and / or a KIR intracellular domain. The KIR-CAR may optionally comprise a DAP12 domain, or the KIR-CAR may be co-expressed with DAP12.

[0191] In certain embodiments, the KIR is selected from the group consisting of KIRS2, KIR2DS2, and KIR2. In certain embodiments, the linker is a short glycine-serine linker.

[0192] Genetically modified cells comprising a KIR-CAR capable of binding to multiple isoforms of EGFR are also included in the present invention. In certain embodiments, the KIR-CAR comprises 806-scFv, a KIR transmembrane domain, and / or a KIR intracellular domain.

[0193] CAR sequence The subject CARs of the invention are CARs that have affinity for multiple isoforms of EFGR (e.g., wtEGFR, mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR R108K EGFR R108G EGFR G598V ). In one embodiment, the EGFR CAR of the invention comprises the amino acid sequence shown in SEQ ID NO:20, which may be encoded by the nucleic acid sequence shown in SEQ ID NO:19.

[0194] In another aspect, the CAR comprises the amino acid sequence set forth in SEQ ID NO:22, which can be encoded by the nucleic acid sequence set forth in SEQ ID NO:21. In another aspect, the CAR comprises the amino acid sequence set forth in SEQ ID NO:65, which can be encoded by the nucleic acid sequence set forth in SEQ ID NO:64. In another aspect, the CAR comprises the amino acid sequence set forth in SEQ ID NO:67, which can be encoded by the nucleic acid sequence set forth in SEQ ID NO:66. In another aspect, the CAR comprises the amino acid sequence set forth in SEQ ID NO:69, which can be encoded by the nucleic acid sequence set forth in SEQ ID NO:68.

[0195] Permissible variations of the CAR while maintaining specific activity will be known to those skilled in the art. For example, in some embodiments, the CAR comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:20, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69. For example, in some embodiments, the CAR is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68.

[0196] Accordingly, the CARs of interest in the present invention include an antigen-binding domain capable of binding to multiple isoforms of EGFR and a transmembrane domain. In one aspect, the CAR includes an antigen-binding domain capable of binding to multiple isoforms of EGFR and a transmembrane domain, wherein the transmembrane domain includes a CD8 hinge region. In one aspect, the CAR includes an antigen-binding domain capable of binding to multiple isoforms of EGFR and a transmembrane domain, wherein the transmembrane domain includes a CD8 transmembrane domain. In one aspect, the CAR includes an antigen-binding domain capable of binding to multiple isoforms of EGFR and a transmembrane domain, wherein the transmembrane domain includes a CD8 hinge region and a CD8 transmembrane domain. In one aspect, the CAR includes an antigen-binding domain capable of binding to multiple isoforms of EGFR and a transmembrane domain, wherein the transmembrane domain includes a KIR transmembrane domain.

[0197] Accordingly, the CARs of interest in the present invention include an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain. In one aspect, the CAR includes an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain, wherein the intracellular domain includes a 4-1BB domain. In one aspect, the CAR includes an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain, wherein the intracellular domain includes a CD3 zeta domain. In one aspect, the CAR includes an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain, wherein the intracellular domain includes a 4-1BB domain and a CD3 zeta domain. In one aspect, the CAR includes an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain, wherein the intracellular domain includes a KIR domain.

[0198] Accordingly, the present invention provides a modified immune cell or a progenitor cell thereof, such as a modified T cell, comprising a chimeric antigen receptor (CAR) having an affinity for a plurality of isoforms of EGFR described herein.

[0199] Treatment method The modified cells described herein (e.g., T cells comprising a CAR capable of binding to a plurality of isoforms of EGFR) may be included in a composition for immunotherapy. The composition may comprise a pharmaceutical composition and may further comprise a pharmaceutically acceptable carrier. A therapeutically effective amount of a pharmaceutical composition comprising the modified T cells may be administered.

[0200] In one aspect, the present invention includes a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising any of the modified T cells of the present invention.

[0201] In another aspect, the present invention is a method of treating cancer in a subject in need thereof, comprising: a) culturing a plurality of CAR T cells with GBM organoids (GBOs); b) selecting the CAR T cells having the highest efficacy from the plurality of CAR T cells; and c) administering the CAR T cells having the highest efficacy to the subject, thereby treating cancer in the subject. In certain embodiments, the plurality of CAR T cells comprise a plurality of modified T cells comprising a plurality of CARs, wherein each CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the antigen-binding domain is capable of binding to an antigen selected from the group consisting of CD19, EGFR, multiple isoforms of EGFR (e.g., wild-type EGFR (wtEGFR), mutant EGFR, EGFRA289V, EGFRA289D, EGFRA289T, EGFRR108K, EGFRR108G, EGFRG598V, EGFRD126Y, EGFRC628F, EGFRR108K / A289V, EGFRR108K / D126Y, EGFRA289V / G598V, EGFRA289V / C628F, and EGFR variant II), PSMA, PSCA, and any tumor-associated antigen (TAA). In certain embodiments, the GBO is generated from a biopsy material derived from the subject. In such embodiments, the GBO is subject-specific, thereby individualizing the treatment of cancer (e.g., CAR selection) for that subject. The CART treatment can be combined with a secondary treatment (e.g., immune checkpoint inhibition (ICB)). The secondary treatment can be administered before, during, or after the CART treatment.

[0202] In certain embodiments, the cancer to be treated is glioblastoma (GBM). In certain embodiments, treating GBM by the methods described herein helps to overcome intratumoral antigen heterogeneity and / or adaptive immune resistance of GBM.

[0203] The modified cells of the present invention (e.g., CAR T cells) can bind to cells (e.g., cancer / tumor cells) that express one or more isoforms of EGFR, thereby treating diseases or disorders associated with the expression of EGFR (e.g., cancer). The EGFR isoforms on the cells to which the CAR T cells can bind include wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR R108K EGFR R108G EGFR G598V EGFR D126Y EGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F and including, but not limited to, EGFR variant II.

[0204] Methods for the administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy is described, e.g., in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by autologous transplantation in which cells are isolated from a subject who is to receive the cell therapy or a sample derived from such a subject and / or prepared by other means. Thus, in some aspects, the cells are derived from a subject in need of treatment, e.g., a patient, and the cells are administered to the same subject after isolation and processing.

[0205] In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by allogeneic transplantation in which cells are isolated from a subject other than the subject who is to receive or will ultimately receive the cell therapy, e.g., a first subject, and / or prepared by other means. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0206] In some embodiments, the subject has been treated with a therapeutic agent that targets a disease or condition, such as a tumor, prior to administration of the cell or composition containing the cell. In some aspects, the subject is resistant or non-responsive to other therapeutic agents. In some embodiments, the subject has a persistent or recurrent disease after treatment with another therapeutic intervention, such as chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. In some embodiments, the administration effectively treats the subject despite the subject becoming resistant to another treatment.

[0207] In some embodiments, the subject is responsive to other therapeutic agents and treatment with the therapeutic agent reduces the disease burden. In some aspects, the subject is initially responsive to the therapeutic agent but demonstrates recurrence of the disease or condition over time. In some embodiments, the subject has not recurred. In some such embodiments, the subject has been determined to be at risk of recurrence, such as having a high risk of recurrence, and thus the cells are administered prophylactically, for example to reduce the likelihood of recurrence or prevent recurrence. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0208] The modified immune cells of the present invention can be administered to an animal, preferably a mammal, more preferably a human, for treating cancer. In addition, if it is desirable to treat or alleviate a disease, the cells of the present invention can be used for treating any condition related to cancer, particularly for a cell-mediated immune response against tumor cells. The types of cancer to be treated with the modified cells or pharmaceutical compositions of the present invention include carcinomas, sarcomas, and blastomas, as well as certain leukemias or lymphoid tumors, benign and malignant tumors, and malignant lesions, such as sarcomas, carcinomas, and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, cervical cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancer can be a non-solid tumor (such as a hematological tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included. In one aspect, the cancer is a solid tumor or a hematological tumor. In one aspect, the cancer is a carcinoma. In one aspect, the cancer is a sarcoma. In one aspect, the cancer is leukemia. In one aspect, the cancer is a solid tumor. In one aspect, the cancer is breast cancer. In one aspect, the cancer is GBM.

[0209] The cells of the present invention can be administered at a dosage and by a route and at a time to be determined in appropriate preclinical and clinical experiments and trials. The cell composition may be administered multiple times at dosages within these ranges. The administration of the cells of the present invention can be combined with other methods useful for treating the desired disease or condition as determined by one of ordinary skill in the art. The cells of the present invention to be administered can be autologous with respect to the subject being treated.

[0210] Administration of the cells and compositions of the present invention may be carried out by any convenient method known to those skilled in the art. The cells and compositions of the present invention may be administered to a subject by aerosol inhalation, injection, oral ingestion, infusion, implantation or transplantation. The compositions described herein may be administered to a patient by transarterial, subcutaneous, intradermal, intratumoral, intraarticular, intramedullary, intramuscular, intrathecal, by intravenous (i.v.) injection, or intraperitoneally. In other examples, the cells of the present invention are directly injected into an inflammatory site in a subject, a local disease site in a subject, a lymph node, an organ, a tumor, etc.

[0211] In some embodiments, the cells are administered at a desired dosage that includes the desired dose or number of cells or cell types and / or the desired ratio of cell types in several aspects. Thus, the dosage of the cells is based in some embodiments on the total number of cells (or number per kg of body weight) and the desired ratio of individual populations or subtypes such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of the cells is based on the desired total number of cells (or number per kg of body weight) in an individual population or individual cell type. In some embodiments, the dosage is based on a combination of features such as the desired total number of cells, the desired ratio, and the desired total number of cells in an individual population.

[0212] The appropriate dosage for the prevention or treatment of a disease depends on the type of disease to be treated, the type of cell or recombinant receptor, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, the treatment history, the clinical history and response of the subject to the cells, and the judgment of the attending physician. The compositions and cells are appropriately administered to a subject in some embodiments once or over a series of treatments.

[0213] In certain embodiments, a secondary treatment is provided to the subject. Secondary treatments include, but are not limited to, chemotherapy, radiation, surgery, and medication.

[0214] In certain embodiments, the cells are administered as part of a combinatorial treatment, either simultaneously with or sequentially in any order with another therapeutic intervention, such as an antibody or an agent such as an engineered cell or receptor or cytotoxic or therapeutic agent. The cells are co-administered, either simultaneously with or sequentially in any order, with one or more additional therapeutic agents or in connection with another therapeutic intervention, in some embodiments. In some situations, the cells are co-administered with another therapy at a time close enough for the cell population to enhance or reverse the effect of one or more additional therapeutic agents. In some embodiments, the cells are administered prior to one or more additional therapeutic agents. In some embodiments, the cells are administered after one or more additional therapeutic agents. In some embodiments, one or more additional agents include cytokines, such as IL-2, to enhance persistence, for example. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0215] In certain embodiments, the modified cells comprising a CAR can be administered to a subject in combination with an immune checkpoint inhibitor (e.g., an inhibitor of PD-1, CTLA-4, PD-L1, or TIM-3). For example, the modified cells may be administered in combination with an antibody or antibody fragment that targets, e.g., PD-1 (programmed death 1 protein). Examples of anti-PD-1 antibodies include pembrolizumab (KEYTRUDA®, formerly known as lambrolizumab, also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or antigen-binding fragments thereof, but are not limited thereto. In certain embodiments, the modified cells may be administered in combination with an anti-PD-L1 antibody or antigen-binding fragment thereof. Examples of anti-PD-L1 antibodies include BMS-936559, MPDL3280A (TECENTRIQ®, atezolizumab), and MEDI4736 (durvalumab, Imfinzi), but are not limited thereto. In certain embodiments, the modified cells may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof. Examples of anti-CTLA-4 antibodies include ipilimumab (trade name Yervoy), but are not limited thereto. In certain embodiments, the modified cells may be administered in combination with an anti-T cell inhibitory receptor Tim-3 (T cell immunoglobulin and mucin domain-containing-3) antibody. Other types of immune checkpoint inhibitors may also be used, including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. The immune checkpoint inhibitor may be administered before, after, or simultaneously with the modified cells comprising a CAR.

[0216] In certain embodiments, the CART cells of the present invention can serve as a local delivery vehicle for crossing the blood-brain barrier (BBB). A "minibody", or blocking protein, is a combination of an scFv sequence targeting a checkpoint molecule (e.g., PD-1, CTLA-4, TIM-3) with the human IgG CH3 region, which can be encoded in a CAR lentivirus. The CART cells can be redirected to neoantigens in GBM and can locally secrete the blocking protein, thereby overcoming potential limitations in the uptake of checkpoint inhibitory molecules in the central nervous system. Since T cells have the ability to efficiently cross the BBB, endowing T cells with the ability to produce protein therapeutics is a particularly attractive strategy for overcoming otherwise limited drug permeability into the central nervous system. This targeted delivery strategy also reduces the risk of systemic off-target effects of ICB.

[0217] Introduction of nucleic acid Methods for introducing nucleic acids into cells include physical, biological, and chemical methods. Physical methods for introducing polynucleotides such as RNA into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. RNA can be introduced into target cells using commercially available methods including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX)(Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany)). RNA can also be introduced into cells using lipofection, using polymer encapsulation, using peptide-mediated transfection, or using a biolistic particle delivery system such as a "gene gun" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0218] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammals, for example, human cells. Other viral vectors can be derived from, for example, lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0219] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., artificial membrane vesicles).

[0220] Suitable lipids can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristoyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipid chloroform or chloroform / methanol solutions can be stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. “Liposome” is a general term encompassing a variety of single and multi-membrane lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. Prior to the formation of the closed structure, the lipid components undergo self-reorganization, confining water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures different from the normal vesicular structure in solution state are also included. For example, the lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0221] Regardless of the method used to introduce exogenous nucleic acids into host cells or to expose cells to the inhibitors of the invention by other means, various assays may be performed to confirm the presence of nucleic acids in host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art such as Southern blotting, Northern blotting, RT-PCR and PCR; "biochemical" assays such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot), or by the assays described herein for identifying agents falling within the scope of the invention.

[0222] Moreover, the nucleic acids may be introduced by any means such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells. One nucleic acid may be introduced by one method, and another nucleic acid may be introduced into T cells by another method.

[0223] RNA In one aspect, the nucleic acid introduced into the host cell is RNA. In another aspect, the RNA is mRNA including in vitro transcribed RNA or synthetic RNA. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted by PCR, using appropriate primers and RNA polymerase, into a template for in vitro mRNA synthesis. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences or any other suitable DNA source. The desired template for in vitro transcription is a chimeric membrane protein. As an example, the template encodes an antibody, a fragment of an antibody or a portion of an antibody. As another example, the template includes an extracellular domain including a single-chain variable domain of an antibody such as anti-CD3, and an intracellular domain of a co-stimulatory molecule. In one aspect, the template for the RNA chimeric membrane protein encodes a chimeric membrane protein including an extracellular domain including an antigen-binding domain derived from an antibody against a co-stimulatory molecule, and an intracellular domain derived from a portion of the intracellular domains of CD28 and 4-1BB.

[0224] PCR may be used to generate a template for in vitro transcription of mRNA, which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to the DNA regions to be used as templates for PCR. As used herein, "substantially complementary" refers to a nucleotide sequence in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. Substantially complementary sequences are capable of annealing or hybridizing to the target DNA under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, primers can be designed to amplify gene portions (open reading frames) that are normally transcribed in cells, including the 5' UTR and 3' UTR. Primers may also be designed to amplify portions of genes that encode specific domains of interest. In one aspect, the primers are designed to amplify the coding region of human cDNA, including all or part of the 5' UTR and 3' UTR. Primers useful for PCR are generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a nucleotide region that is substantially complementary to a nucleotide on the DNA template that is upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the 5' position of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a nucleotide region that is substantially complementary to the double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the 3' position of the DNA sequence to be amplified relative to the coding strand.

[0225] Chemical structures that have the ability to promote RNA stability and / or translation efficiency may also be used. The RNA preferably has a 5' UTR and a 3' UTR. In one embodiment, the 5' UTR is 0 to 3000 nucleotides in length. The lengths of the 5' UTR sequence and the 3' UTR sequence to be added to the coding region can be varied by different methods, including but not limited to designing PCR primers that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can modify the lengths of the 5' UTR and the 3' UTR necessary to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0226] The 5' UTR and the 3' UTR can be the native endogenous 5' UTR and 3' UTR for the gene of interest. Alternatively, a UTR sequence that is not endogenous to the gene of interest can be added by incorporating the UTR sequence into the forward and reverse primers or by any other modification of the template. The use of a UTR sequence that is not endogenous to the gene of interest can be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3' UTR sequence can decrease mRNA stability. Thus, the 3’ UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0227] In one aspect, the 5' UTR can contain the Kozak sequence of an endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. The Kozak sequence can increase the translation efficiency of some RNA transcripts, but it does not seem to be necessary for all RNAs to enable efficient translation. The need for the Kozak sequence for many mRNAs is known in the art. In other aspects, the 5' UTR can be derived from an RNA genome of a stable RNA virus in the cell. In other aspects, various nucleotide analogs can be used in the 3' UTR or 5' UTR to interfere with exonucleolytic degradation of the mRNA.

[0228] To enable RNA synthesis from a DNA template without the need for gene cloning, the transcription promoter should be linked to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated upstream of the open reading frame to be transcribed in the PCR product. In one aspect, the promoter is a T7 polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences for T7, T3, and SP6 promoters are known in the art.

[0229] In one aspect, the mRNA has both a cap on the 5' end and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and the stability of the mRNA in the cell. On a circular DNA template, such as plasmid DNA, RNA polymerase produces long concatemer products that are not suitable for expression in eukaryotic cells. Transcription of linearized plasmid DNA at the end of the 3' UTR yields a normally sized mRNA that is not effective for eukaryotic transfection even when polyadenylated post-transcriptionally.

[0230] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the final base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0231] The conventional method for the incorporation of poly A / T stretches into a DNA template is molecular cloning. However, poly A / T sequences incorporated into plasmid DNA can cause plasmid instability, which is why deletions and other abnormalities often contaminate plasmid DNA templates obtained from bacterial cells at high levels. This not only makes the cloning procedure time-consuming and laborious but often reduces its reliability. Therefore, a method that enables the construction of DNA templates with 3' stretches of poly A / T without cloning is highly desirable.

[0232] The poly A / T segment of the transcribed DNA template can be produced during PCR by using a reverse primer containing a poly T tail such as a 100T tail (the size can be 50 - 5000T), or after PCR by any other method including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tails also provide stability to the RNAs and reduce their degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 - 5000 adenosines.

[0233] The poly(A) tail of the RNA can be further elongated by using a poly(A) polymerase such as E. coli poly A polymerase (E-PAP) after in vitro transcription. In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300 - 400 nucleotides results in approximately a two-fold increase in the translation efficiency of the RNA. Additionally, the addition of different chemical groups to the 3' end can increase the stability of the mRNA. Such additions can contain modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0234] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. The 5' cap is provided using techniques known in the art and the techniques described herein (Cougot, et al., Trends in Biochem. Sci., 29:436 - 444 (2001); Stepinski, et al., RNA, 7:1468 - 95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958 - 966 (2005)).

[0235] The RNA produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence within the sequence. The IRES sequence can be any viral, chromosomal, or artificially engineered sequence that initiates cap-independent ribosome binding to the mRNA and promotes the start of translation. Any solute suitable for cell electroporation can be included, which can contain factors such as sugars, peptides, lipids, proteins, antioxidants, and surfactants that promote cell permeability and viability.

[0236] In some embodiments, the RNA, such as in vitro transcribed RNA, is electroporated into cells.

[0237] The disclosed methods are applicable to the modulation of T cell activity in basic research and therapies in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the evaluation of the ability of genetically modified T cells to kill target cancer cells.

[0238] These methods also provide the ability to control expression levels over a wide range, for example, by varying the promoter or the amount of input RNA, and to individually regulate the expression levels. Furthermore, PCR-based mRNA production techniques greatly facilitate the design of mRNAs with different structures and combinations of their domains.

[0239] One advantage of the RNA transfection method of the present invention is that RNA transfection is essentially transient and does not use vectors. The transgene can be delivered to lymphocytes and expressed therein as a minimal expression cassette after short-term in vitro cell activation without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely to occur. Due to the transfection efficiency of RNA and the ability of RNA to uniformly modify the entire lymphocyte population, cell cloning is unnecessary.

[0240] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies that have both been tested successfully in various animal models. In vitro transcribed RNA is transfected into cells by lipofection or electroporation. To achieve long-term expression of the transferred IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0241] Several IVT vectors that are standardly used as templates for in vitro transcription and are genetically modified to produce stabilized RNA transcripts are known from the literature. Currently, the protocols used in the art are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest flanked by untranslated regions (UTRs) either 3' and / or 5', and a 3' polyadenylation cassette containing 50 - 70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and either extend or mask the poly(A) sequence at the 3' end. Whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs is not clear.

[0242] RNA has several advantages compared to more conventional plasmid or viral approaches. Gene expression from RNA origin does not require transcription and the protein product is produced rapidly after transfection. Furthermore, since RNA only has to access the cytoplasm rather than the nucleus, extremely high transfection rates occur with typical transfection methods. In addition, plasmid-based approaches require that the promoter driving the expression of the gene of interest be active in the cells under test.

[0243] In another aspect, the RNA construct is delivered into cells by electroporation. For example, reference is made to the formulation and methodology of electroporation of nucleic acid constructs into mammalian cells, as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1. Various parameters, including the strength of the electric field required for electroporation of any known cell type, are generally known from the relevant research literature as well as numerous patents and applications in the art. See, for example, U.S. Patent Nos. 6,678,556, 7,171,264, and 7,173,116. Devices for the therapeutic application of electroporation are commercially available, for example, the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Patent Nos. 6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482; electroporation may also be used, for example, as described in US20070128708A1, for transfection of cells in vitro. Electroporation may also be utilized to deliver nucleic acids intracellularly in vitro. Thus, electroporation-mediated administration of nucleic acids containing an expression construct, using any of a number of available devices and electroporation systems known to those of skill in the art, presents an exciting new means for delivering the RNA of interest to target cells.

[0244] Source of T cells In certain embodiments, the source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is human. T cells can be obtained from several sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain embodiments, any number of T cell lines available in the art may be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as Ficoll separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis are washed to remove the plasma fraction and the cells may be placed in an appropriate buffer or medium such as phosphate buffered saline (PBS) or a wash solution, which may be calcium-free and magnesium-free or lacking in many if not all divalent cations for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers such as calcium-free, magnesium-free PBS. Alternatively, unwanted components of the apheresis sample may be removed and the cells may be resuspended directly in the medium.

[0245] In another embodiment, T cells are isolated from peripheral blood, for example, by centrifugation on a PERCOLL™ gradient by lysing red blood cells and depleting monocytes. Alternatively, T cells can be isolated from the umbilical cord. In any event, a particular subset of T cells can be further isolated by positive or negative selection techniques.

[0246] The umbilical cord blood mononuclear cells isolated as such can deplete cells expressing certain antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be achieved using isolated antibodies, biological samples containing antibodies such as ascites, antibodies bound to physical supports, and antibodies bound to cells.

[0247] Enrichment of the T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the cells undergoing negative selection. Preferred methods are negative magnetic immunoadherence or cell sorting and / or selection via flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells undergoing negative selection. For example, to enrich for CD4 + cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0248] To isolate a desired population of cells by positive or negative selection, the cell concentration and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and the beads. For example, in one embodiment, a cell concentration of 2 billion cells / ml is used. In one embodiment, a cell concentration of 1 billion cells / ml is used. In further embodiments, more than 100 million cells / ml are used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 1 billion cells / ml are used. In further embodiments, cell concentrations of 125 million or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0249] T cells can also be frozen after a washing step that does not require a monocyte removal step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and some monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. Although numerous freezing solutions and parameters are known in the art and would be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing and immediate non-controlled freezing in -20°C or liquid nitrogen may also be used.

[0250] In one aspect, the T cell population is contained in cells such as peripheral blood mononuclear cells, cord blood cells, purified populations of T cells, and T cell lines. In another aspect, the peripheral blood mononuclear cells contain the T cell population. In yet another aspect, the purified T cells contain the T cell population.

[0251] Expansion of T cells In certain exemplary aspects, the modified cells disclosed herein can be multiplied by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, and any and all whole integers and partial integers therebetween. In one aspect, the T cells increase in the range of about 20-fold to about 50-fold.

[0252] Following culturing, the modified cells can be incubated in the cell culture medium placed in the culture device for a certain period of time, or until the cells reach confluence or a high cell density for optimal passage before subculturing the cells into another culture device. The culture device can be any culture device commonly used for in vitro culturing of cells. Preferably, the confluence level is 70% or greater before subculturing the cells into another culture device. More preferably, the confluence level is 90% or greater. The period can be any time suitable for in vitro cell culture. The T cell medium may be replaced during culturing of the T cells at any time. Preferably, the T cell medium is replaced every about 2 - 3 days. The T cells are then recovered from the culture device and can be used immediately therefrom, or cryopreserved and stored for later use. In one aspect, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed before introducing nucleic acid into the T cells.

[0253] In another aspect, the method includes the steps of isolating T cells and expanding the T cells. In another aspect, the invention further includes the step of cryopreserving the T cells prior to expansion. In yet another aspect, the cryopreserved T cells are thawed for electroporation with RNA encoding the chimeric membrane protein.

[0254] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942, which is incorporated herein by reference. Expansion as described in U.S. Patent No. 5,199,942 can be an alternative or an addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells includes addition of cell growth factors or other factors such as those described in U.S. Patent No. 5,199,942, for example flt3-L, IL-1, IL-3, and c-kit ligand. In one aspect, expanding the T cells includes culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0255] The step of culturing as described herein (after contact or electroporation with an agent as described herein) is very short, for example, less than 24 hours, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The step of culturing as further described herein (contact with an agent as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or more days.

[0256] Various terms are used to describe cultured cells. A cell culture generally refers to cells obtained from a living organism and grown under controlled conditions. A primary cell culture is a culture of cells, tissues, or organs obtained directly from an organism and is the culture before the first subculture. When cells are placed in a growth medium under conditions that facilitate cell growth and / or division to yield a larger cell population, the cells are expanded in culture. When cells are expanded in culture, the growth rate of the cells is typically measured by the time required for the cells to double in number, a time known as the doubling time, another way of putting it.

[0257] Each round of subculture is referred to as a passage. When cells are subcultured, they are said to have been passaged. A particular cell population or cell line is sometimes referred to or characterized by the number of passages it has undergone. For example, a cultured cell population that has been passaged 10 times may be referred to as a P10 culture. The primary culture, i.e., the first culture after cells are isolated from a tissue, is called P0. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that there can be multiple population doublings during the passage period; thus, the population doubling number of a culture is greater than the passage number. The increase in cells (i.e., the population doubling number) during the period between passages depends on a number of factors including, but not limited to, seeding density, substrate, medium, and passage interval.

[0258] In one aspect, the cells may be cultured for several hours (about 3 hours) to about 14 days or any integer value of time in between. Conditions suitable for T cell culture include a suitable medium (e.g., Minimal Essential Medium or RPMI Medium 1640 or X-vivo 15, (Lonza)) containing factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-alpha, or any other additive for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium may be supplemented with amino acids, sodium pyruvate, and vitamins and can be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines sufficient for T cell growth and expansion, including RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells to be injected into a subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0259] The medium used for culturing T cells may contain agents that can costimulate T cells. For example, an agent that can stimulate CD3 is an antibody against CD3, and an agent that can stimulate CD28 is an antibody against CD28. Because, as shown by the data disclosed herein, the cells isolated by the methods disclosed herein can be expanded by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more. In one aspect, T cells are expanded by culturing an electroporated population by about 20-fold to about 50-fold, or more.

[0260] In one aspect, a method of expanding T cells can further include isolating the expanded T cells for further application. In another aspect, the method of expanding can further include subsequent electroporation of the expanded T cells followed by culturing. Subsequent electroporation can include introducing a nucleic acid encoding an agent, e.g., transducing the expanded T cells, transfecting the expanded T cells, or electroporating a nucleic acid into the expanded T cells to make a population of expanded T cells, where the agent further stimulates the T cells. The agent can stimulate the T cells, for example, by stimulating further expansion, effector function, or another T cell function.

[0261] Pharmaceutical composition The pharmaceutical composition of the present invention may comprise the modified T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0262] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the patient's condition and the type and severity of the patient's disease, but appropriate dosages can be determined by clinical trials.

[0263] The cells of the present invention to be administered can be autologous, allogeneic or xenogeneic to the subject receiving the therapy.

[0264] The cells of the present invention can be administered at a dosage, route and time determined in appropriate preclinical and clinical experiments and tests. The cell composition can be administered multiple times at dosages within these ranges. Administration of the cells of the present invention may be combined with other methods useful for treating the desired disease or condition as determined by those skilled in the art.

[0265] Generally, a pharmaceutical composition comprising the modified T cells described herein is 10 4 ~10 9 cells / kg body weight, and in some cases, 10 5 ~10 6It can be defined that it can be administered at a dose of cells / kg body weight (including all integer values within these ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by one of ordinary skill in the medical field by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0266] The administration of the modified T cells of the present invention can be carried out by any convenient method known to those skilled in the art. The cells of the present invention can be administered to a subject by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient by transarterial, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous (i.v.) injection, or intraperitoneal injection. In other cases, the cells of the present invention are directly injected into the site of inflammation in the subject, the local disease site in the subject, lymph nodes, organs, tumors, etc.

[0267] It should be understood that the methods and compositions that would be useful in the present invention are not limited to the specific formulations shown in the examples. The following examples are presented for the purpose of providing those skilled in the art with a thorough disclosure and explanation of how to make and use the cells, expansion and culture methods, and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0268] The practice of the present invention, unless otherwise indicated, employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of the art. Such techniques are fully explained in references such as "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and may thus be considered in the formulation and practice of the present invention. Techniques that are particularly useful for certain embodiments are discussed in subsequent sections.

Examples

[0269] Experimental Examples The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only and the present invention is not limited to these examples, but includes all variations that are apparent as a result of the teachings provided herein.

[0270] Example 1: Expression of EGFR missense mutants in U87 cells Changes within the epidermal growth factor receptor (EGFR) (ErbB1) locus correspond to the highest frequency of genetic changes in GBM. Overexpression of EGFR, such as that mediated by focal amplification of the EGFR locus as double minute chromosomes in GBM, has long been recognized and is found in 60% of cases. Mutations in EGFR are also frequent. The oncogenic EGFR variant lacking exons 2-7 (EGFRvIII) is found in approximately 30% of GBM. Using TCGA data in addition to next-generation sequencing data of GBM cases, missense mutations with oncogenic activity were identified at positions 108, 289, and 598 in the extracellular domain (ECD) of EGFR (Figure 7). Retrospective analysis showed that patients with these mutations exhibited poor overall survival. More than 60% of GBMs with amplified EGFR showed mutations in the ECD, and these mutations could be targeted by cross-reactive CART cells.

[0271] To test the function of EGFR-specific CAR T cells, a lentiviral expression system encoding EGFR variants relevant to GBM was generated (Figure 8). EGFR missense mutations were introduced into the EGFR gene by Geneart gene synthesis and site-directed mutagenesis (Thermo fisher). Lentiviral vectors co-expressing CFP and EGFR mutations were transduced into the U87 wtEGFR cell line and the U87 MG cell line, and CFP-positive cells were sorted by fluorescence-activated cell sorting. Co-expression of EGFR variants and CFP in the U87 MG cell line transduced with wtEGFR (U87 wtEGFR) is shown in Figure 9. Co-expression of EGFR variants and CFP in U87 MG GBM cells is shown in Figure 10.

[0272] Example 2: Targeted cell lysis of GBM cells by cross-reactive EGFR-specific CAR T cells Chimeric antigen receptor (CAR) T cells that induce broad specificity against multiple EGFR isoforms were generated in this specification. A lentiviral expression vector encoding an scFv derived from the monoclonal antibody mAb806, a CD8 hinge domain, a CD8 transmembrane domain, and a 4-1BB intracellular signaling domain was constructed (Figure 1A). Primary human CD4+ T cells and CD8+ T cells were transduced with the lentiviral vector encoding the CAR. After a 6-day incubation, approximately 60% of the T cells expressed the EGFR-specific 806-4-1BB CAR on the cell surface (Figure 1B).

[0273] The monoclonal antibody mAb806 detects structural features shared by multiple EGFR missense mutations (Binder et al. (2018) Cancer Cell, 34, 1; 163-177). Therefore, CAR T cells incorporating 806scFv should enable broad specificity and targeting of heterogeneous EGFR tumor cell populations. The antigen-specific cytolytic activity of 806-41BB CAR T cells was tested against human GBM cell lines (Figure 2). These GBM cell lines were based on the U87 MG parental GBM cell line with a basal level of EGFR, into which wild-type EGFR (U87 wtEGFR) or its variant EGFRvIII (U87 wtEGFR / EGFRvIII) or EGFR A289V (U87 wtEGFR / EGFR A289V ) had been transduced. Antigen-specific cytolytic activity was measured using various CAR T cell to tumor cell ratios in a 4-hour chromium release assay. 2173 CAR specific for EGFRvIII and cetuximab (C225) CAR specific for wild-type EGFR were used as positive controls. CD19 CAR T cells were used as a negative control. 806-41BB CAR T cells showed antigen-specific cytolytic activity against multiple EGFR isoforms (Figure 2).

[0274] In vitro cell lysis by 806 4-1BB CAR T cells was demonstrated in U87 parental cell lines transduced with EGFR missense mutations R108K and A289V and EGFR variant VIII in a 4-hour chromium release assay. EGFR wild-type specific C10 4-1BB, and VIII-specific 2173, 4-1BB CARs were used as positive controls. CD19 4-1BB CAR was used as a negative control. 806 CAR T cells were able to specifically lyse wild-type and mutant EGFR-expressing U87 cells, whereas the control Nalm6 cells, a precursor B cell line that does not express EGFR, were not targeted (Figure 3). Collectively, these data demonstrate that 806-41BB CAR T cells can specifically target and kill diverse EGFR-expressing cells.

[0275] The sequence of the humanized form of mAb806, ABT-806, is shown in Figures 11 (DNA) and 12 (amino acid).

[0276] Example 3: Targeting of EGFR-specific cells by 806 KIR CAR T cells KIR CARs that induce broad specificity for multiple EGFR isoforms were also generated in this specification. A lentiviral vector containing the 806-scFv, KIR transmembrane and intracellular domains, and the DAP12 sequence was constructed (Figure 4A). Primary human T cells were stimulated with anti-CD3 / anti-CD28 T cell activation beads for 24 hours. The T cells were then transduced with the 806-KIR lentiviral vector and expanded in vitro for 10 days. When analyzed by flow cytometry using biotinylated goat-anti-mouse F(ab)2 followed by streptavidin-APC, approximately 44% of the T cells expressed the 806 KIR CAR (Figure 4B). The antigen-specific cell lysis activity of 806-KIR CAR T against EGFR-expressing GBM cell lines and their variant vIII-expressing GBM cell lines and A289V-expressing GBM cell lines was measured using luciferase as a reporter gene for viable cells (Figure 5). The 806 KIR CAR T cells were able to lyse the U87 MG, U87 MG EGFRvIII, U87 wtEGFR, U87 wtEGFR / EGFRvIII, and U87 wtEGFR / EGFR A289V cell lines. The EGFRvIII-specific 2173 CAR and cetuximab (C225) CAR that recognize EGFRvIII and wild-type EGFR were used as positive controls. The data demonstrated the antigen-specific cell lysis activity of the 806 KIR CAR T cells.

[0277] Example 4: Targeted cell lysis of GBM cross-reactive EGFR-specific humanized CAR T cells A lentiviral expression vector encoding a humanized ABT806 scFv in a 4-1BBz CAR and another vector encoding a humanized ABT806 scFv in a KIR-CAR were created. The humanized sequences correspond to SEQ ID NOs. 23-28. Primary human CD4+ and CD8+ T cells were transduced with the vectors, and significant positivity was demonstrated after 6 days of incubation. Both the humanized monoclonal ABT806 4-1BBz CAR and the humanized monoclonal ABT806 KIR CAR showed cytolytic activity against the U87 MG cell line modified with an EGFR variant or the U87 wtEGFR cell line modified with an EGFR variant.

[0278] Example 5: Combination treatment with humanized 806-41BB CAR Subcutaneous animal combination experiments were performed using the humanized 806-41BBz CAR, and anti-PD-1 inhibitors were tested against U87 wtEGFR / EGFRvIII (Figures 16A-16B). To achieve the best clinical efficacy, multiple injections were used to compare the dosing regimens of both anti-PD-1 inhibition and CAR T cells. An orthotopic tumor model was used for additional tumor growth inhibition studies of 806-41BB CAR T cells. The delivery routes were compared using intravenous and intrathecal administration of 806 41BBz and 806 KIR CAR.

[0279] Using the scFvs of pembrolizumab, nivolumab, and atezolizumab, two versions of PD-1 / PD-L1 blockers were generated, resulting in six constructs of each mini-body and the signaling domain of IFNγ in cis with the scFv. A plasmid encoding "PD-1 / PD-L1 blocker-secreting 806BB" was transfected into 293T cells. The supernatant was collected 72 hours after transfection and used for a PD-1 / PD-L1 binding assay by direct ELISA.

[0280] Collect the supernatant from 806 BBz CAR T cells secreting PD-1 / PD-L1 blocker and use it for the PD-1 / PD-L1 binding assay by direct ELISA. CAR expression of 806 BBz CAR is detected on the T cells transduced with 806 BBz CAR T cells secreting PD-1 / PD-L1 blocker.

[0281] Co-culture 806 BBz CAR T cells secreting PD-1 / PD-L1 blocker with U87 wtEGFR / EGFRvIII positive target cells. Evaluate cytokine secretion by flow-based intracellular cytokine staining 16 hours after co-culture.

[0282] After subcutaneous implantation of tumors, inject UTD T cells, 806 BBz CAR T cells, 806 BBz CAR T cells secreting blocker or blocker-secreting T cells via the tail vein. Tumor size and BLI signal transduction indicate an increase in the tumor growth inhibitory activity of 806 BBz CAR T cells secreting blocker.

[0283] Example 6: In vivo and in vitro administration of CAR T cell combination therapy for human GBM Treat subcutaneous tumors with combination therapy of 806 BBz CAR and anti-PD1 antibody (Figs. 16A - 16B). Treat subcutaneous U87 wtEGFR / EGFRvIII cell line with a combination of either PBS or anti-PD-1 antibody and non-transduced T cells or 806 BBz CAR T cells. A greater decrease was demonstrated in the relative tumor change as determined by bioluminescence with combination therapy (Fig. 16A). The tumor change rate relative to PBS + non-transduced (UTD) cells 16 days after CAR T injection is shown in Fig. 16B.

[0284] The in vivo antitumor activity of 806 KIR CAR against U87 wtEGFR (Figure 17A) and U87 wtEGFR / EGFRvIII (Figure 17B) flank tumors was demonstrated. The tumor models had overexpression of wild-type EGFR either alone or in the presence of concomitant EGFR mutations. This pairing is a more significant physiological representation than the single expression of EGFR mutations in the absence of overexpression of wild-type EGFR. The in vivo antitumor activity of 806 BBz against U87 wtEGFR / EGFRvIII flank tumors was also demonstrated (Figure 17C).

[0285] The in vitro potency of 806 CAR T cells was also demonstrated (Figures 18A - 18B and 19A - 19C). The antigen-specific cell lysis activities of 806 and 2173 CAR T cells in U87MG and U87 wtEGFR expressing EGFR and its variant EGFRvIII, EGFR R108K / G and EGFR A289D / T / V were shown in cell lines at an effector-to-target ratio of display in a 24-hour luciferase assay (Figure 18A). C225 BBz and C225 KIR CAR, which recognize wtEGFR, EGFRvIII, and its mutant variants, were used as positive controls, and CD19 BBz CAR was used as a negative control. The antigen-specific cell lysis activities of 806 and 2173 CAR T cells in K562 cells expressing EGFR and its variants were demonstrated in a 4-hour chromium release assay at the effector-to-target ratio of display (Figure 18B). K562 cells do not express basal EGFR and provide a clean background against which antigen specificity should be tested.

[0286] K562 cells expressing wtEGFR, EGFRvIII, or EGFR mutants were co-cultured with 806 CAR T cells for 48 hours, and the secretion of IFN-γ, TNF-α, and IL2 was measured by ELISA (19A - 19B). The CD107a degranulation of CAR T cells was measured when co-cultured with K562 cells expressing wtEGFR, EGFRvIII, or EGFR mutants for 4 hours. The results were CD3 +Represented as the percentage of CD107a expression on the cells (Figure 19C).

[0287] The antitumor efficacy of 806 CAR T cells was demonstrated in primary stellate cells and keratinocytes (Figures 20A - 20C). The surface expression of EGFR on human primary stellate cells and keratinocytes was evaluated by flow cytometry (Figure 20A). Primary stellate cells and keratinocytes were co - cultured with 806 CAR T cells at the indicated ratio in a 4 - hour chromium assay (Figure 20B). 806 CAR T cells and primary stellate cells and keratinocytes were co - cultured at an effector - to - target ratio of 1:5, and IFN - γ was measured from the supernatant after incubation at 37°C for 24 hours (Figure 20C).

[0288] Example 7: Development of an advanced biomarker platform for predicting the clinical efficacy of CAR T treatment in real time GBM organoids (GBOs) were co - cultured with CAR T cells (Figures 21A - 21C). GBOs are described in detail in Jacob et al. (2019) Cell 180:1;188 - 204.e22, the content of which is hereby incorporated by reference in its entirety. Briefly, GBOs were generated by the following method.

[0289] Glioblastoma tissue samples and peripheral blood samples were collected from glioblastoma patients. Fresh glioblastoma tissue obtained by surgical resection was placed in sterile phosphate-buffered saline and immediately removed, and the attending neuropathologist confirmed a preliminary diagnosis of high-grade glioma. When a large mass of tissue was available, the tissue was anatomically subdivided into separate small regions for analysis of intratumoral heterogeneity. After the preliminary diagnosis of glioblastoma was confirmed, the tissue was distributed and placed in Hibernate A medium (BrainBits) maintained at 4°C. Since the reliability of GBO production decreased with an extended time between surgical removal and tissue processing, it was inevitable to process the tissue immediately for reliable organoid generation. For dissection under a stereomicroscope (Zeiss) in a laminar flow biosafety cabinet, the tissue was transferred to a sterile glass dish containing H+GPSA medium supplemented with Hibernate A, 1× GlutaMax (Thermo Fisher Scientific), 1× PenStrep (Thermo Fisher Scientific), and 1× amphotericin B (Thermo Fisher Scientific). The amount of glioblastoma tissue received ranged from 0.5 to 2 mL in volume. The excised tumor was minced into small pieces approximately 0.5 to 1 mm in diameter using fine dissection scissors (Fine Science Tools) and washed with H+GPSA medium to remove cell debris. Small pieces containing a significant amount of necrosis or peripheral brain tissue were removed. The tumor pieces were incubated in 1× RBC lysis buffer (Thermo Fisher Scientific) at room temperature for 10 minutes with gentle rotation to lyse most of the contaminating red blood cells. The RBC lysis buffer was aspirated, and the tumor pieces were washed with H+GPSA medium. Some tumor pieces were snap-frozen for bulk RNA sequencing and whole exome analysis. For histological studies, some tumor pieces were directly placed in methanol-free formaldehyde (Polysciences) diluted to 4% in DPBS (Thermo Fisher Scientific) and gently rotated at room temperature for 1 hour. After fixation, the tumor pieces were placed in plastic molds (Electron Microscopy Sciences) and snap-frozen in tissue freezing medium (General Data) on dry ice.The frozen tissue was stored at -80 °C until processing.

[0290] The remaining tumor pieces not set aside for RNA sequencing, whole exome analysis, or histology were dispensed into an ultra-low attachment 6-well culture plate (Corning) with GBO medium containing 4 mL per well of 50% DMEM:F12 (Thermo Fisher Scientific), 50% Neurobasal (Thermo Fisher Scientific), 1× GlutaMax (Thermo Fisher Scientific), 1× NEAAs (Thermo Fisher Scientific), 1× PenStrep (Thermo Fisher Scientific), 1× N2 supplement (Thermo Fisher Scientific), 1× B27 w / o vitamin A supplement (Thermo Fisher Scientific), 1× 2-mercaptoethanol (Thermo Fisher Scientific), and 2.5 mg / ml human insulin (Sigma), and placed on an orbital shaker rotating at 120 rpm in a sterile incubator at 37 °C, 5% CO2, and 90% humidity. Approximately 75% of the medium was replaced every 48 hours by tilting the plate at a 45° angle and aspirating the medium above the sunken GBO. In cultures within 1 week, cell debris and blood debris often detached from the tumor pieces and the medium became slightly turbid. The detachment stopped promptly, and the tumor pieces generally formed spherical organoids within 1 - 2 weeks depending on the tissue quality and patient-specific tumor growth characteristics. The criterion for successful establishment of GBO from a given patient's tumor was that the microdissected tumor pieces survived for 2 weeks, developed a spherical morphology, and grew continuously in culture. GBOs cultured for a long period (>1 month) were routinely cut into small pieces approximately 200 - 500 mm in diameter using fine dissection scissors to prevent substantial necrosis in the center due to limitations in nutrient and oxygen diffusion. GBOs were harvested for RNA sequencing, whole exome analysis, and histology.

[0291] After 24 and 72 hours of co - culture with 806 BBZ, 2173 BBz, and CD19 BBz CAR T cells, immunofluorescent staining of GBO was performed (Figure 21A). Cell staining for CD3 and quantification of cleaved caspase 3 are shown in Figure 21B and Figure 21C, respectively. Although there was no significant difference in CD3 expression, caspase activity was significantly different, demonstrating that 806 BBz CAR T cells led to an increase in tumor cell death compared to 2173 BBz CAR T cells. 8167 GBO expressed endogenous amplified wtEGFR, EGFRvIII, and EGFR A289V and depicted a greater physiological representation of GBM than standard glioblastoma stem cell lines.

[0292] Other aspects The detailed listing of elements in any definition of a variable herein includes the definition of that variable as any single element or combination (or sub - combination) of the recited elements. The detailed description of aspects herein includes aspects as any single aspect or aspects in combination with any other aspect or portion thereof.

[0293] The disclosures of patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Although the invention has been disclosed in relation to specific aspects, it is clear that other aspects and variations of the invention may be devised by other skilled artisans without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

[0294] Sequence Information SEQUENCE LISTING <110> The Trustees of the University of Pennsylvania <120> Compositions and Methods Comprising a High Affinity Chimeric Antigen Receptor (CAR) with Cross-Reactivity to Clinically-Relevant EGFR Mutated Proteins <150> US 62 / 833,456 <151> 2019-04-12 <150> US 62 / 892,343 <151> 2019-08-27 <160> 86 <170> PatentIn version 3.5 <210> 1 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> 806 scFv <400> 1 gatgtccagc tgcaagagtc tggccctagc ctggtcaagc ctagccagag cctgagcctg 60 acatgtaccg tgaccggcta cagcatcacc agcgacttcg cctggaactg gatcagacag 120 ttccccggca acaagctgga atggatgggc tacatcagct acagcggcaa cacccggtac 180 aaccccagcc tgaagtcccg gatctccatc accagagaca ccagcaagaa ccagttcttc 240 ctgcagctga acagcgtgac catcgaggac accgccacct actactgtgt gacagccggc 300 agaggcttcc cttattgggg acagggaacc ctggtcacag tgtctgctgg tggcggagga 360 tctggcggag gcggatcttc tggcggtggc tctgatatcc tgatgacaca gagccccagc 420 agcatgtctg tgtccctggg cgataccgtg tccatcacct gtcacagcag ccaggacatc 480 aacagcaaca tcggctggct gcagcagagg cctggcaagt cttttaaggg cctgatctac 540 cacggcacca acctggatga tgaggtgccc agcagatttt ccggctctgg aagcggagcc 600 gactactccc tgacaatcag cagcctggaa agcgaggact tcgccgatta ctactgcgtg 660 cagtacgccc agtttccttg gacctttgga ggcggcacaa agctggaaat caagcgg 717 <210> 2 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> 806 scFv <400> 2 Asp Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly 115 120 125 Gly Gly Ser Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val 130 135 140 Ser Leu Gly Asp Thr Val Ser Ile Thr Cys His Ser Ser Gln Asp Ile 145 150 155 160 Asn Ser Asn Ile Gly Trp Leu Gln Gln Arg Pro Gly Lys Ser Phe Lys 165 170 175 Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp Glu Val Pro Ser Arg 180 185 190 Phe Ser Gly Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser 195 200 205 Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln 210 215 220 Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 225 230 235 <210> 3 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> 806 VL <400> 3 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly 1 5 10 15 Asp Thr Val Ser Ile Thr Cys His Ser Ser Gln Asp Ile Asn Ser Asn 20 25 30 Ile Gly Trp Leu Gln Gln Arg Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly Thr Asn Leu Asp Asp Glu Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 4 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> 806 VH <400> 4 Asp Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 5 His Ser Ser Gln Asp Ile Asn Ser Asn Ile Gly 1 5 10 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 6 His Gly Thr Asn Leu Asp Asp 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 7 Val Gln Tyr Ala Gln Phe Pro Trp Thr 1 5 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 8 Gly Tyr Ser Ile Thr Ser Asp Phe Ala Trp Asn 1 5 10 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 9 Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg Tyr Asn Pro Ser Leu Lys 1 5 10 15 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 10 Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp 1 5 10 <210> 11 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> CD8 hinge <400> 11 accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60 tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120 gacttcgcct gcgat 135 <210> 12 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> CD8 transmembrane <400> 12 atctacattt gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc 60 actctttact gt 72 <210> 13 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 41BB <400> 13 aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 60 actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 120 gaactg 126 <210> 14 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CD3zeta <400> 14 cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60 tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120 cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180 gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240 agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300 tatgacgctc ttcacatgca ggccctgccg cctcgg 336 <210> 15 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> CD8 SRP <400> 15 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 16 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> DAP12 <400> 16 atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60 ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120 ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180 gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240 aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300 gtctacagcg acctcaacac acagaggccg tattacaaa 339 <210> 17 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> T2A <400> 17 gtcgagggcg gcggagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat 60 cccggcccta gg 72 <210> 18 <211> 258 <212> DNA <213> Artificial Sequence <220> <223> Linker KIRS2 <400> 18 ggtggcggag gttctggagg tgggggttcc tcacccactg aaccaagctc caaaaccggt 60 aaccccagac acctgcatgt tctgattggg acctcagtgg tcaaaatccc tttcaccatc 120 ctcctcttct ttctccttca tcgctggtgc tccaacaaaa aaaatgctgc tgtaatggac 180 caagagcctg cagggaacag aacagtgaac agcgaggatt ctgatgaaca agaccatcag 240 gaggtgtcat acgcataa 258 <210> 19 <211> 1464 <212> DNA <213> Artificial Sequence <220> <223> 806 BBZ CAR <400> 19 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgggatccg atgtccagct gcaagagtct ggccctagcc tggtcaagcc tagccagagc 120 ctgagcctga catgtaccgt gaccggctac agcatcacca gcgacttcgc ctggaactgg 180 atcagacagt tccccggcaa caagctggaa tggatgggct acatcagcta cagcggcaac 240 acccggtaca accccagcct gaagtcccgg atctccatca ccagagacac cagcaagaac 300 cagttcttcc tgcagctgaa cagcgtgacc atcgaggaca ccgccaccta ctactgtgtg 360 acagccggca gaggcttccc ttattgggga cagggaaccc tggtcacagt gtctgctggt 420 ggcggaggat ctggcggagg cggatcttct ggcggtggct ctgatatcct gatgacacag 480 agccccagca gcatgtctgt gtccctgggc gataccgtgt ccatcacctg tcacagcagc 540 caggacatca acagcaacat cggctggctg cagcagaggc ctggcaagtc ttttaagggc 600 ctgatctacc acggcaccaa cctggatgat gaggtgccca gcagattttc cggctctgga 660 agcggagccg actactccct gacaatcagc agcctggaaa gcgaggactt cgccgattac 720 tactgcgtgc agtacgccca gtttccttgg acctttggag gcggcacaaa gctggaaatc 780 aagcgggcta gcaccactac cccagcaccg aggccaccca ccccggctcc taccatcgcc 840 tcccagcctc tgtccctgcg tccggaggca tgtagacccg cagctggtgg ggccgtgcat 900 acccggggtc ttgacttcgc ctgcgatatc tacatttggg cccctctggc tggtacttgc 960 ggggtcctgc tgctttcact cgtgatcact ctttactgta agcgcggtcg gaagaagctg 1020 ctgtacatct ttaagcaacc cttcatgagg cctgtgcaga ctactcaaga ggaggacggc 1080 tgttcatgcc ggttcccaga ggaggaggaa ggcggctgcg aactgcgcgt gaaattcagc 1140 cgcagcgcag atgctccagc ctacaagcag gggcagaacc agctctacaa cgaactcaat 1200 cttggtcgga gagaggagta cgacgtgctg gacaagcgga gaggacggga cccagaaatg 1260 ggcgggaagc cgcgcagaaa gaatccccaa gagggcctgt acaacgagct ccaaaaggat 1320 aagatggcag aagcctatag cgagattggt atgaaagggg aacgcagaag aggcaaaggc 1380 cacgacggac tgtaccaggg actcagcacc gccaccaagg acacctatga cgctcttcac 1440 atgcaggccc tgccgcctcg gtga 1464 <210> 20 <211> 487 <212> PRT <213> Artificial Sequence <220> <223> 806 BBZ CAR <400> 20 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Asp Val Gln Leu Gln Glu Ser Gly Pro 20 25 30 Ser Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr 35 40 45 Gly Tyr Ser Ile Thr Ser Asp Phe Ala Trp Asn Trp Ile Arg Gln Phe 50 55 60 Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Asn 65 70 75 80 Thr Arg Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp 85 90 95 Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Ile Glu 100 105 110 Asp Thr Ala Thr Tyr Tyr Cys Val Thr Ala Gly Arg Gly Phe Pro Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Leu Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Met Ser Val Ser Leu Gly Asp Thr Val Ser Ile Thr 165 170 175 Cys His Ser Ser Gln Asp Ile Asn Ser Asn Ile Gly Trp Leu Gln Gln 180 185 190 Arg Pro Gly Lys Ser Phe Lys Gly Leu Ile Tyr His Gly Thr Asn Leu 195 200 205 Asp Asp Glu Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp 210 215 220 Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr 225 230 235 240 Tyr Cys Val Gln Tyr Ala Gln Phe Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255 Lys Leu Glu Ile Lys Arg Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg 485 <210> 21 <211> 1461 <212> DNA <213> Artificial Sequence <220> <223> 806 KIR CAR <400> 21 atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60 ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120 ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180 gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240 aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300 gtctacagcg acctcaacac acagaggccg tattacaaag tcgagggcgg cggagagggc 360 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gatggcctta 420 ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc acgccgccag gccgggatcc 480 gatgtccagc tgcaagagtc tggccctagc ctggtcaagc ctagccagag cctgagcctg 540 acatgtaccg tgaccggcta cagcatcacc agcgacttcg cctggaactg gatcagacag 600 ttccccggca acaagctgga atggatgggc tacatcagct acagcggcaa cacccggtac 660 aaccccagcc tgaagtcccg gatctccatc accagagaca ccagcaagaa ccagttcttc 720 ctgcagctga acagcgtgac catcgaggac accgccacct actactgtgt gacagccggc 780 agaggcttcc cttattgggg acagggaacc ctggtcacag tgtctgctgg tggcggagga 840 tctggcggag gcggatcttc tggcggtggc tctgatatcc tgatgacaca gagccccagc 900 agcatgtctg tgtccctggg cgataccgtg tccatcacct gtcacagcag ccaggacatc 960 aacagcaaca tcggctggct gcagcagagg cctggcaagt cttttaaggg cctgatctac 1020 cacggcacca acctggatga tgaggtgccc agcagatttt ccggctctgg aagcggagcc 1080 gactactccc tgacaatcag cagcctggaa agcgaggact tcgccgatta ctactgcgtg 1140 cagtacgccc agtttccttg gacctttgga ggcggcacaa agctggaaat caagcgggct 1200 agcggtggcg gaggttctgg aggtgggggt tcctcaccca ctgaaccaag ctccaaaacc 1260 ggtaacccca gacacctgca tgttctgatt gggacctcag tggtcaaaat ccctttcacc 1320 atcctcctct tctttctcct tcatcgctgg tgctccaaca aaaaaaatgc tgctgtaatg 1380 gaccaagagc ctgcagggaa cagaacagtg aacagcgagg attctgatga acaagaccat 1440 caggaggtgt catacgcata a 1461 <210> 22 <211> 486 <212> PRT <213> Artificial Sequence <220> <223> 806 KIR CAR <400> 22 Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp 20 25 30 Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met 35 40 45 Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu 50 55 60 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 65 70 75 80 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 85 90 95 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 100 105 110 Lys Val Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly 115 120 125 Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala 130 135 140 Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser 145 150 155 160 Asp Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 165 170 175 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 180 185 190 Phe Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 195 200 205 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg Tyr Asn Pro Ser Leu 210 215 220 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 225 230 235 240 Leu Gln Leu Asn Ser Val Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys 245 250 255 Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 260 265 270 Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly 275 280 285 Gly Gly Ser Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val 290 295 300 Ser Leu Gly Asp Thr Val Ser Ile Thr Cys His Ser Ser Gln Asp Ile 305 310 315 320 Asn Ser Asn Ile Gly Trp Leu Gln Gln Arg Pro Gly Lys Ser Phe Lys 325 330 335 Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp Glu Val Pro Ser Arg 340 345 350 Phe Ser Gly Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser 355 360 365 Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln 370 375 380 Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 385 390 395 400 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro Thr Glu Pro 405 410 415 Ser Ser Lys Thr Gly Asn Pro Arg His Leu His Val Leu Ile Gly Thr 420 425 430 Ser Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe Phe Leu Leu His 435 440 445 Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu Pro 450 455 460 Ala Gly Asn Arg Thr Val Asn Ser Glu Asp Ser Asp Glu Gln Asp His 465 470 475 480 Gln Glu Val Ser Tyr Ala 485 <210> 23 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Humanized 806 VH <400> 23 caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60 acctgtaccg tgtccggcta cagcatcagc agcgacttcg cctggaactg gatcagacag 120 cctcctggca aaggactgga atggatgggc tacatcagct acagcggcaa caccagatac 180 cagcctagcc tgaagtcccg gatcaccatc agcagagaca ccagcaagaa ccagttcttc 240 ctgaagctga acagcgtgac agccgccgat accgccacct actattgtgt gacagctggc 300 agaggcttcc cctattgggg acagggaaca ctggtcaccg ttagctct 348 <210> 24 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Humanized 806 VL <400> 24 gatatccaga tgacacagag ccccagcagc atgtccgtgt ccgtgggaga cagagtgacc 60 atcacctgtc acagcagcca ggacatcaac agcaacatcg gctggctgca gcagaagccc 120 ggcaagtctt ttaagggcct gatctaccac ggcaccaacc tggatgatgg cgtgcccagc 180 agattttctg gcagcggctc tggcaccgac tacaccctga ccatatctag cctgcagcct 240 gaggacttcg ccacctatta ctgcgtgcag tacgcccagt ttccttggac ctttggaggc 300 ggcacaaagc tggaaatcaa gcgg 324 <210> 25 <211> 672 <212> DNA <213> Artificial Sequence <220> <223> Humanized 806 scFv <400> 25 caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60 acctgtaccg tgtccggcta cagcatcagc agcgacttcg cctggaactg gatcagacag 120 cctcctggca aaggactgga atggatgggc tacatcagct acagcggcaa caccagatac 180 cagcctagcc tgaagtcccg gatcaccatc agcagagaca ccagcaagaa ccagttcttc 240 ctgaagctga acagcgtgac agccgccgat accgccacct actattgtgt gacagctggc 300 agaggcttcc cctattgggg acagggaaca ctggtcaccg ttagctctga tatccagatg 360 acacagagcc ccagcagcat gtccgtgtcc gtgggagaca gagtgaccat cacctgtcac 420 agcagccagg acatcaacag caacatcggc tggctgcagc agaagcccgg caagtctttt 480 aagggcctga tctaccacgg caccaacctg gatgatggcg tgcccagcag attttctggc 540 agcggctctg gcaccgacta caccctgacc atatctagcc tgcagcctga ggacttcgcc 600 acctattact gcgtgcagta cgcccagttt ccttggacct ttggaggcgg cacaaagctg 660 gaaatcaagc gg 672 <210> 26 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Humanized 806 VH <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg Tyr Gln Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 27 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanized 806 VL <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys His Ser Ser Gln Asp Ile Asn Ser Asn 20 25 30 Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly Thr Asn Leu Asp Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 28 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Humanized 806 scFv <400> 28 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg Tyr Gln Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser 115 120 125 Val Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ser Ser Gln Asp 130 135 140 Ile Asn Ser Asn Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe 145 150 155 160 Lys Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp Gly Val Pro Ser 165 170 175 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 180 185 190 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala 195 200 205 Gln Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 210 215 220 <210> 29 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Affinity maturized 806 VH <400> 29 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Arg Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asn Gly Asn Thr Arg Tyr Gln Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Ser Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Affinity maturized 806 VL <400> 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys His Ser Ser Gln Asp Ile Asn Ser Asn 20 25 30 Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly Thr Asn Leu Asp Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 31 <211> 717 <212> PRT <213> Artificial Sequence <220> <223> 806 scFv <400> 31 Gly Ala Thr Ala Thr Thr Cys Thr Gly Ala Thr Gly Ala Cys Thr Cys 1 5 10 15 Ala Ala Thr Cys Thr Cys Cys Gly Thr Cys Thr Thr Cys Thr Ala Thr 20 25 30 Gly Ala Gly Cys Gly Thr Gly Ala Gly Cys Thr Thr Gly Gly Gly Thr 35 40 45 Gly Ala Cys Ala Cys Cys Gly Thr Cys Ala Gly Cys Ala Thr Cys Ala 50 55 60 Cys Cys Thr Gly Thr Cys Ala Thr Thr Cys Cys Ala Gly Cys Cys Ala 65 70 75 80 Gly Gly Ala Thr Ala Thr Ala Ala Ala Cys Thr Cys Ala Ala Ala Thr 85 90 95 Ala Thr Cys Gly Gly Cys Thr Gly Gly Cys Thr Cys Cys Ala Gly Cys 100 105 110 Ala Ala Cys Gly Cys Cys Cys Ala Gly Gly Cys Ala Ala Gly Thr Cys 115 120 125 Ala Thr Thr Cys Ala Ala Gly Gly Gly Gly Cys Thr Thr Ala Thr Thr 130 135 140 Thr Ala Thr Cys Ala Thr Gly Gly Cys Ala Cys Cys Ala Ala Thr Cys 145 150 155 160 Thr Thr Gly Ala Cys Gly Ala Thr Gly Ala Ala Gly Thr Cys Cys Cys 165 170 175 Ala Thr Cys Ala Cys Gly Cys Thr Thr Cys Ala Gly Cys Gly Gly Ala 180 185 190 Thr Cys Ala Gly Gly Cys Thr Cys Ala Gly Gly Thr Gly Cys Gly Gly 195 200 205 Ala Cys Thr Ala Thr Thr Cys Cys Thr Thr Gly Ala Cys Thr Ala Thr 210 215 220 Ala Ala Gly Thr Thr Cys Cys Cys Thr Cys Gly Ala Ala Thr Cys Thr 225 230 235 240 Gly Ala Gly Gly Ala Thr Thr Thr Cys Gly Cys Cys Gly Ala Cys Thr 245 250 255 Ala Thr Thr Ala Thr Thr Gly Cys Gly Thr Ala Cys Ala Ala Thr Ala 260 265 270 Cys Gly Cys Cys Cys Ala Gly Thr Thr Thr Cys Cys Cys Thr Gly Gly 275 280 285 Ala Cys Cys Thr Thr Cys Gly Gly Ala Gly Gly Cys Gly Gly Cys Ala 290 295 300 Cys Cys Ala Ala Ala Thr Thr Gly Gly Ala Gly Ala Thr Ala Ala Ala 305 310 315 320 Ala Ala Gly Gly Gly Gly Thr Gly Gly Ala Gly Gly Ala Gly Gly Ala 325 330 335 Thr Cys Ala Gly Gly Cys Gly Gly Gly Gly Gly Thr Gly Gly Ala Ala 340 345 350 Gly Cys Gly Gly Cys Gly Gly Ala Gly Gly Ala Gly Gly Cys Ala Gly 355 360 365 Cys Gly Ala Cys Gly Thr Ala Cys Ala Ala Cys Thr Gly Cys Ala Ala 370 375 380 Gly Ala Ala Thr Cys Cys Gly Gly Gly Cys Cys Gly Ala Gly Thr Thr 385 390 395 400 Thr Gly Gly Thr Cys Ala Ala Gly Cys Cys Cys Thr Cys Thr Cys Ala 405 410 415 Ala Thr Cys Thr Cys Thr Thr Thr Cys Thr Cys Thr Cys Ala Cys Thr 420 425 430 Thr Gly Cys Ala Cys Gly Gly Thr Cys Ala Cys Cys Gly Gly Ala Thr 435 440 445 Ala Cys Thr Cys Cys Ala Thr Ala Ala Cys Cys Ala Gly Cys Gly Ala 450 455 460 Thr Thr Thr Thr Gly Cys Gly Thr Gly Gly Ala Ala Thr Thr Gly Gly 465 470 475 480 Ala Thr Thr Cys Gly Ala Cys Ala Ala Thr Thr Thr Cys Cys Ala Gly 485 490 495 Gly Gly Ala Ala Thr Ala Ala Ala Thr Thr Gly Gly Ala Ala Thr Gly 500 505 510 Gly Ala Thr Gly Gly Gly Ala Thr Ala Thr Ala Thr Cys Ala Gly Thr 515 520 525 Thr Ala Thr Thr Cys Thr Gly Gly Thr Ala Ala Thr Ala Cys Cys Ala 530 535 540 Gly Ala Thr Ala Cys Ala Ala Cys Cys Cys Gly Thr Cys Ala Thr Thr 545 550 555 560 Gly Ala Ala Ala Ala Gly Thr Cys Gly Cys Ala Thr Cys Thr Cys Thr 565 570 575 Ala Thr Ala Ala Cys Ala Cys Gly Ala Gly Ala Cys Ala Cys Thr Thr 580 585 590 Cys Ala Ala Ala Gly Ala Ala Thr Cys Ala Gly Thr Thr Cys Thr Thr 595 600 605 Cys Cys Thr Thr Cys Ala Gly Cys Thr Cys Ala Ala Thr Thr Cys Thr 610 615 620 Gly Thr Ala Ala Cys Cys Ala Thr Cys Gly Ala Ala Gly Ala Thr Ala 625 630 635 640 Cys Thr Gly Cys Thr Ala Cys Thr Thr Ala Thr Thr Ala Cys Thr Gly 645 650 655 Thr Gly Thr Ala Ala Cys Gly Gly Cys Gly Gly Gly Thr Cys Gly Ala 660 665 670 Gly Gly Ala Thr Thr Cys Cys Cys Cys Thr Ala Cys Thr Gly Gly Gly 675 680 685 Gly Cys Cys Ala Gly Gly Gly Thr Ala Cys Ala Cys Thr Gly Gly Thr 690 695 700 Thr Ala Cys Thr Gly Thr Thr Thr Cys Cys Gly Cys Cys 705 710 715 <210> 32 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> 806 scFv <400> 32 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly 1 5 10 15 Asp Thr Val Ser Ile Thr Cys His Ser Ser Gln Asp Ile Asn Ser Asn 20 25 30 Ile Gly Trp Leu Gln Gln Arg Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly Thr Asn Leu Asp Asp Glu Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Gln 115 120 125 Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr 130 135 140 Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp Phe Ala Trp Asn Trp 145 150 155 160 Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser 165 170 175 Tyr Ser Gly Asn Thr Arg Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser 180 185 190 Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser 195 200 205 Val Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys Val Thr Ala Gly Arg 210 215 220 Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 225 230 235 <210> 33 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> 806 VL <400> 33 gatatcctga tgacacagag ccccagcagc atgtctgtgt ccctgggcga taccgtgtcc 60 atcacctgtc acagcagcca ggacatcaac agcaacatcg gctggctgca gcagaggcct 120 ggcaagtctt ttaagggcct gatctaccac ggcaccaacc tggatgatga ggtgcccagc 180 agattttccg gctctggaag cggagccgac tactccctga caatcagcag cctggaaagc 240 gaggacttcg ccgattacta ctgcgtgcag tacgcccagt ttccttggac ctttggaggc 300 ggcacaaagc tggaaatcaa gcgg 324 <210> 34 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> 806 VH <400> 34 gatgtccagc tgcaagagtc tggccctagc ctggtcaagc ctagccagag cctgagcctg 60 acatgtaccg tgaccggcta cagcatcacc agcgacttcg cctggaactg gatcagacag 120 ttccccggca acaagctgga atggatgggc tacatcagct acagcggcaa cacccggtac 180 aaccccagcc tgaagtcccg gatctccatc accagagaca ccagcaagaa ccagttcttc 240 ctgcagctga acagcgtgac catcgaggac accgccacct actactgtgt gacagccggc 300 agaggcttcc cttattgggg acagggaacc ctggtcacag tgtctgct 348 <210> 35 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 35 His Gly Thr Asn Leu Asp Asp 1 5 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <220> <221> REPEAT <222> (1)..(5) <223> repeat n times, where n represents an integer of at least 1 <400> 36 Gly Ser Gly Gly Ser 1 5 <210> 37 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker <220> <221> REPEAT <222> (1)..(4) <223> repeat n times, where n represents an integer of at least 1 <400> 37 Gly Gly Gly Ser 1 <210> 38 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <220> <221> REPEAT <222> (1)..(5) <223> repeat n times, where n represents an integer of at least 1 <400> 38 Gly Gly Gly Gly Ser 1 5 <210> 39 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 39 Gly Gly Ser Gly 1 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 40 Gly Gly Ser Gly Gly 1 5 <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 41 Gly Ser Gly Ser Gly 1 5 <210> 42 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 42 Gly Ser Gly Gly Gly 1 5 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 43 Gly Gly Gly Ser Gly 1 5 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 44 Gly Ser Ser Ser Gly 1 5 <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 45 Gly Gly Gly Gly Ser 1 5 <210> 46 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 46 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 47 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Linker <400> 47 ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45 <210> 48 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 48 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser 1 5 10 15 <210> 49 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Linker <400> 49 ggtggcggag gatctggcgg aggcggatct tctggcggtg gctct 45 <210> 50 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 50 Asp Lys Thr His Thr 1 5 <210> 51 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 51 Cys Pro Pro Cys 1 <210> 52 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 52 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 1 5 10 15 <210> 53 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 53 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr 1 5 10 <210> 54 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 54 Lys Ser Cys Asp Lys Thr His Thr Cys Pro 1 5 10 <210> 55 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 55 Lys Cys Cys Val Asp Cys Pro 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 56 Lys Tyr Gly Pro Pro Cys Pro 1 5 <210> 57 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 57 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 58 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 1 5 10 <210> 59 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 59 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 60 Ser Pro Asn Met Val Pro His Ala His His Ala Gln 1 5 10 <210> 61 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Hinge <400> 61 Glu Pro Lys Ser Cys Asp Lys Thr Tyr Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 62 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Affinity maturated humanized 806 VH <400> 62 gaggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60 acctgtaccg tgtccggcta cagcatcagc agagacttcg cctggaactg gatcagacag 120 cctcctggca aaggactgga atggatgggc tacatcagct acaacggcaa caccagatac 180 cagcctagcc tgaagtcccg gatcaccatc tccagagaca ccagcaagaa ccagttcttc 240 ctgaagctga acagcgtgac agccgccgat accgccacct actattgtgt gacagccagc 300 agaggcttcc cctattgggg acagggaacc ctggtcacag ttagctct 348 <210> 63 <211> 321 <212> PRT <213> Artificial Sequence <220> <223> Affinity maturated humanized 806 VL <400> 63 Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Ala Cys 1 5 10 15 Ala Gly Ala Gly Cys Cys Cys Cys Ala Gly Cys Ala Gly Cys Ala Thr 20 25 30 Gly Thr Cys Cys Gly Thr Gly Thr Cys Cys Gly Thr Gly Gly Gly Ala 35 40 45 Gly Ala Cys Ala Gly Ala Gly Thr Gly Ala Cys Cys Ala Thr Cys Ala 50 55 60 Cys Cys Thr Gly Thr Cys Ala Cys Ala Gly Cys Ala Gly Cys Cys Ala 65 70 75 80 Gly Gly Ala Cys Ala Thr Cys Ala Ala Cys Ala Gly Cys Ala Ala Cys 85 90 95 Ala Thr Cys Gly Gly Cys Thr Gly Gly Cys Thr Gly Cys Ala Gly Cys 100 105 110 Ala Gly Ala Ala Gly Cys Cys Cys Gly Gly Cys Ala Ala Gly Thr Cys 115 120 125 Thr Thr Thr Thr Ala Ala Gly Gly Gly Cys Cys Thr Gly Ala Thr Cys 130 135 140 Thr Ala Cys Cys Ala Cys Gly Gly Cys Ala Cys Cys Ala Ala Cys Cys 145 150 155 160 Thr Gly Gly Ala Thr Gly Ala Thr Gly Gly Cys Gly Thr Gly Cys Cys 165 170 175 Cys Ala Gly Cys Ala Gly Ala Thr Thr Thr Thr Cys Thr Gly Gly Cys 180 185 190 Ala Gly Cys Gly Gly Cys Thr Cys Thr Gly Gly Cys Ala Cys Cys Gly 195 200 205 Ala Cys Thr Ala Cys Ala Cys Cys Cys Thr Gly Ala Cys Cys Ala Thr 210 215 220 Ala Thr Cys Thr Ala Gly Cys Cys Thr Gly Cys Ala Gly Cys Cys Thr 225 230 235 240 Gly Ala Gly Gly Ala Cys Thr Thr Cys Gly Cys Cys Ala Cys Cys Thr 245 250 255 Ala Thr Thr Ala Cys Thr Gly Cys Gly Thr Gly Cys Ala Gly Thr Ala 260 265 270 Cys Gly Cys Cys Cys Ala Gly Thr Thr Thr Cys Cys Thr Thr Gly Gly 275 280 285 Ala Cys Cys Thr Thr Thr Gly Gly Ala Gly Gly Cys Gly Gly Cys Ala 290 295 300 Cys Ala Ala Ala Gly Cys Thr Gly Gly Ala Ala Ala Thr Cys Ala Ala 305 310 315 320 Gly <210> 64 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> 806 CAR <400> 64 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggatgtcc agctgcaaga gtctggccct agcctggtca agcctagcca gagcctgagc 120 ctgacatgta ccgtgaccgg ctacagcatc accagcgact tcgcctggaa ctggatcaga 180 cagttccccg gcaacaagct ggaatggatg ggctacatca gctacagcgg caacacccgg 240 tacaacccca gcctgaagtc ccggatctcc atcaccagag acaccagcaa gaaccagttc 300 ttcctgcagc tgaacagcgt gaccatcgag gacaccgcca cctactactg tgtgacagcc 360 ggcagaggct tcccttattg gggacaggga accctggtca cagtgtctgc tggtggcgga 420 ggatctggcg gaggcggatc ttctggcggt ggctctgata tcctgatgac acagagcccc 480 agcagcatgt ctgtgtccct gggcgatacc gtgtccatca cctgtcacag cagccaggac 540 atcaacagca acatcggctg gctgcagcag aggcctggca agtcttttaa gggcctgatc 600 taccacggca ccaacctgga tgatgaggtg cccagcagat tttccggctc tggaagcgga 660 gccgactact ccctgacaat cagcagcctg gaaagcgagg acttcgccga ttactactgc 720 gtgcagtacg cccagtttcc ttggaccttt ggaggcggca caaagctgga aatcaagcgg 780 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 840 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 900 gacttcgcct gtgatatcta catctgggcc cctctggccg gcacctgtgg cgtgctgctg 960 ctgtccctgg tcatcaccct gtactgcaag cggggcagaa agaagctgct gtacatcttc 1020 aagcagccct tcatgcggcc tgtgcagacc acacaggaag aggacggctg tagctgtaga 1080 ttccccgagg aagaggaagg cggctgcgag ctgagagtga agttcagcag aagcgccgac 1140 gcccctgcct atcagcaggg ccagaaccag ctgtacaacg agctgaacct gggcagacgg 1200 gaggaatacg acgtgctgga caagagaaga ggccgggacc ctgagatggg cggcaagccc 1260 agacggaaga acccccagga aggcctgtat aacgaactgc agaaagacaa gatggccgag 1320 gcctacagcg agatcggcat gaagggcgag cggagaagag gcaagggcca tgacggcctg 1380 taccagggcc tgagcaccgc caccaaggac acctacgacg ccctgcacat gcaggccctg 1440 cctccaaga 1449 <210> 65 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> 806 CAR <400> 65 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Val Gln Leu Gln Glu Ser Gly Pro Ser Leu 20 25 30 Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr 35 40 45 Ser Ile Thr Ser Asp Phe Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly 50 55 60 Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg 65 70 75 80 Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser 85 90 95 Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Ile Glu Asp Thr 100 105 110 Ala Thr Tyr Tyr Cys Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Leu Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Met Ser Val Ser Leu Gly Asp Thr Val Ser Ile Thr Cys His 165 170 175 Ser Ser Gln Asp Ile Asn Ser Asn Ile Gly Trp Leu Gln Gln Arg Pro 180 185 190 Gly Lys Ser Phe Lys Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp 195 200 205 Glu Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Tyr Ser 210 215 220 Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys 225 230 235 240 Val Gln Tyr Ala Gln Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 245 250 255 Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 260 265 270 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 275 280 285 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 290 295 300 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 305 310 315 320 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 325 330 335 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 340 345 350 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 355 360 365 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 370 375 380 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 385 390 395 400 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 405 410 415 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 420 425 430 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 435 440 445 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 450 455 460 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 465 470 475 480 Pro Pro Arg <210> 66 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Humanized 806 CAR <400> 66 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcaggttc agctgcaaga gtctggccct ggcctggtca agcctagcca aacactgagc 120 ctgacctgta ccgtgtccgg ctacagcatc agcagcgact tcgcctggaa ctggatcaga 180 cagcctcctg gcaaaggact ggaatggatg ggctacatca gctacagcgg caacaccaga 240 taccagccta gcctgaagtc ccggatcacc atcagcagag acaccagcaa gaaccagttc 300 ttcctgaagc tgaacagcgt gacagccgcc gataccgcca cctactattg tgtgacagct 360 ggcagaggct tcccctattg gggacaggga acactggtca ccgttagctc tggtggcgga 420 ggatctggcg gaggcggatc ttctggcggt ggctctgata tccagatgac acagagcccc 480 agcagcatgt ccgtgtccgt gggagacaga gtgaccatca cctgtcacag cagccaggac 540 atcaacagca acatcggctg gctgcagcag aagcccggca agtcttttaa gggcctgatc 600 taccacggca ccaacctgga tgatggcgtg cccagcagat tttctggcag cggctctggc 660 accgactaca ccctgaccat atctagcctg cagcctgagg acttcgccac ctattactgc 720 gtgcagtacg cccagtttcc ttggaccttt ggaggcggca caaagctgga aatcaagcgg 780 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 840 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 900 gacttcgcct gtgatatcta catctgggcc cctctggccg gcacctgtgg cgtgctgctg 960 ctgtccctgg tcatcaccct gtactgcaag cggggcagaa agaagctgct gtacatcttc 1020 aagcagccct tcatgcggcc tgtgcagacc acacaggaag aggacggctg tagctgtaga 1080 ttccccgagg aagaggaagg cggctgcgag ctgagagtga agttcagcag aagcgccgac 1140 gcccctgcct atcagcaggg ccagaaccag ctgtacaacg agctgaacct gggcagacgg 1200 gaggaatacg acgtgctgga caagagaaga ggccgggacc ctgagatggg cggcaagccc 1260 agacggaaga acccccagga aggcctgtat aacgaactgc agaaagacaa gatggccgag 1320 gcctacagcg agatcggcat gaagggcgag cggagaagag gcaagggcca tgacggcctg 1380 taccagggcc tgagcaccgc caccaaggac acctacgacg ccctgcacat gcaggccctg 1440 cctccaaga 1449 <210> 67 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Humanized 806 CAR <400> 67 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr 35 40 45 Ser Ile Ser Ser Asp Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg 65 70 75 80 Tyr Gln Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser 85 90 95 Lys Asn Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr 100 105 110 Ala Thr Tyr Tyr Cys Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Met Ser Val Ser Val Gly Asp Arg Val Thr Ile Thr Cys His 165 170 175 Ser Ser Gln Asp Ile Asn Ser Asn Ile Gly Trp Leu Gln Gln Lys Pro 180 185 190 Gly Lys Ser Phe Lys Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp 195 200 205 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 210 215 220 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 Val Gln Tyr Ala Gln Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 245 250 255 Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 260 265 270 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 275 280 285 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 290 295 300 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 305 310 315 320 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 325 330 335 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 340 345 350 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 355 360 365 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 370 375 380 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 385 390 395 400 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 405 410 415 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 420 425 430 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 435 440 445 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 450 455 460 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 465 470 475 480 Pro Pro Arg <210> 68 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> Affinity maturated humanized 806 CAR <400> 68 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggaggttc agctgcaaga gtctggccct ggcctggtca agcctagcca aacactgagc 120 ctgacctgta ccgtgtccgg ctacagcatc agcagagact tcgcctggaa ctggatcaga 180 cagcctcctg gcaaaggact ggaatggatg ggctacatca gctacaacgg caacaccaga 240 taccagccta gcctgaagtc ccggatcacc atctccagag acaccagcaa gaaccagttc 300 ttcctgaagc tgaacagcgt gacagccgcc gataccgcca cctactattg tgtgacagcc 360 agcagaggct tcccctattg gggacaggga accctggtca cagttagctc tggtggcgga 420 ggatctggcg gaggcggatc ttctggcggt ggctctgata tccagatgac acagagcccc 480 agcagcatgt ccgtgtccgt gggagacaga gtgaccatca cctgtcacag cagccaggac 540 atcaacagca acatcggctg gctgcagcag aagcccggca agtcttttaa gggcctgatc 600 taccacggca ccaacctgga tgatggcgtg cccagcagat tttctggcag cggctctggc 660 accgactaca ccctgaccat atctagcctg cagcctgagg acttcgccac ctattactgc 720 gtgcagtacg cccagtttcc ttggaccttt ggaggcggca caaagctgga aatcaagacc 780 acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 840 ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 900 ttcgcctgtg atatctacat ctgggcccct ctggccggca cctgtggcgt gctgctgctg 960 tccctggtca tcaccctgta ctgcaagcgg ggcagaaaga agctgctgta catcttcaag 1020 cagcccttca tgcggcctgt gcagaccaca caggaagagg acggctgtag ctgtagattc 1080 cccgaggaag aggaaggcgg ctgcgagctg agagtgaagt tcagcagaag cgccgacgcc 1140 cctgcctatc agcagggcca gaaccagctg tacaacgagc tgaacctggg cagacgggag 1200 gaatacgacg tgctggacaa gagaagaggc cgggaccctg agatgggcgg caagcccaga 1260 cggaagaacc cccaggaagg cctgtataac gaactgcaga aagacaagat ggccgaggcc 1320 tacagcgaga tcggcatgaa gggcgagcgg agaagaggca agggccatga cggcctgtac 1380 cagggcctga gcaccgccac caaggacacc tacgacgccc tgcacatgca ggccctgcct 1440 ccaaga 1446 <210> 69 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> Affinity maturated humanized 806 CAR <400> 69 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr 35 40 45 Ser Ile Ser Arg Asp Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly 50 55 60 Lys Gly Leu Glu Trp Met Gly Tyr Ile Ser Tyr Asn Gly Asn Thr Arg 65 70 75 80 Tyr Gln Pro Ser Leu Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser 85 90 95 Lys Asn Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr 100 105 110 Ala Thr Tyr Tyr Cys Val Thr Ala Ser Arg Gly Phe Pro Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Met Ser Val Ser Val Gly Asp Arg Val Thr Ile Thr Cys His 165 170 175 Ser Ser Gln Asp Ile Asn Ser Asn Ile Gly Trp Leu Gln Gln Lys Pro 180 185 190 Gly Lys Ser Phe Lys Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp 195 200 205 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 210 215 220 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 Val Gln Tyr Ala Gln Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 245 250 255 Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 260 265 270 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 275 280 285 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 290 295 300 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 305 310 315 320 Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu 325 330 335 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 340 345 350 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 355 360 365 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 370 375 380 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 385 390 395 400 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 405 410 415 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 420 425 430 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 435 440 445 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 450 455 460 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 465 470 475 480 Pro Arg <210> 70 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> SRP <400> 70 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 71 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> CD8 hinge <400> 71 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 72 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> CD8 hinge <400> 72 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 73 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> CD8 transmembrane <400> 73 atctacatct gggcccctct ggccggcacc tgtggcgtgc tgctgctgtc cctggtcatc 60 accctgtact gc 72 <210> 74 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> CD8 transmembrane <400> 74 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 75 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 41BB <400> 75 aagcggggca gaaagaagct gctgtacatc ttcaagcagc ccttcatgcg gcctgtgcag 60 accacacagg aagaggacgg ctgtagctgt agattccccg aggaagagga aggcggctgc 120 gagctg 126 <210> 76 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> 41BB <400> 76 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 77 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CD3zeta <400> 77 agagtgaagt tcagcagaag cgccgacgcc cctgcctatc agcagggcca gaaccagctg 60 tacaacgagc tgaacctggg cagacgggag gaatacgacg tgctggacaa gagaagaggc 120 cgggaccctg agatgggcgg caagcccaga cggaagaacc cccaggaagg cctgtataac 180 gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240 agaagaggca agggccatga cggcctgtac cagggcctga gcaccgccac caaggacacc 300 tacgacgccc tgcacatgca ggccctgcct ccaaga 336 <210> 78 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3zeta <400> 78 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 79 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Humanized 806 scFv <400> 79 caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60 acctgtaccg tgtccggcta cagcatcagc agcgacttcg cctggaactg gatcagacag 120 cctcctggca aaggactgga atggatgggc tacatcagct acagcggcaa caccagatac 180 cagcctagcc tgaagtcccg gatcaccatc agcagagaca ccagcaagaa ccagttcttc 240 ctgaagctga acagcgtgac agccgccgat accgccacct actattgtgt gacagctggc 300 agaggcttcc cctattgggg acagggaaca ctggtcaccg ttagctctgg tggcggagga 360 tctggcggag gcggatcttc tggcggtggc tctgatatcc agatgacaca gagccccagc 420 agcatgtccg tgtccgtggg agacagagtg accatcacct gtcacagcag ccaggacatc 480 aacagcaaca tcggctggct gcagcagaag cccggcaagt cttttaaggg cctgatctac 540 cacggcacca acctggatga tggcgtgccc agcagatttt ctggcagcgg ctctggcacc 600 gactacaccc tgaccatatc tagcctgcag cctgaggact tcgccaccta ttactgcgtg 660 cagtacgccc agtttccttg gacctttgga ggcggcacaa agctggaaat caagcgg 717 <210> 80 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Humanized 806 scFv <400> 80 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg Tyr Gln Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Gly Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly 115 120 125 Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Val 130 135 140 Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ser Ser Gln Asp Ile 145 150 155 160 Asn Ser Asn Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys 165 170 175 Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp Gly Val Pro Ser Arg 180 185 190 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser 195 200 205 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln 210 215 220 Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 225 230 235 <210> 81 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Humanized 806 scFv <400> 81 ggcgaactaa aggtcgaaac acggcggagg tttccaggtt cctttgaccc gcatgacgtg 60 cgtcattatc caccgcttca ggagtccgac gtccgatcta taccagtccc acatcagcca 120 cggtctcggc gacggtcttt tagacgaccc gtgcggtagt aggtccaacc acggcaccat 180 ctagtccggg aattttctga acggcccgaa gacgacgtcg gtcggctaca acgacaacta 240 caggaccgac gacactgtcc actaccagtg agacagaggg tgcctgtgcc tgtacgacga 300 ccccgagaca cagtagacct atagtctcgg tggcggtctt ctaggcggag gcggtctagg 360 aggcggtggt ctcgattgcc actggtcaca agggacaggg gttatcccct tcggagacgg 420 tcgacagtgt gttatcatcc accgccatag ccgccgacag tgcgacaagt cgaagtcctt 480 cttgaccaag aacgaccaca gagacgacta ccactaggcc ctgaagtccg atccgaccat 540 agaccacaac ggcgacatcg actacatcgg gtaggtaagg tcaggaaacg gtcctccgac 600 agactaggtc aaggtccgct tcagcgacga ctacgacatc ggcctgtgcc atgtccagtc 660 cgagtcacaa accgatccga actggtccgg tcccggtctg agaacgtcga cttggac 717 <210> 82 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Humanized 806 scFv <400> 82 Arg Lys Ile Glu Leu Lys Thr Gly Gly Gly Phe Thr Trp Pro Phe Gln 1 5 10 15 Ala Tyr Gln Val Cys Tyr Tyr Thr Ala Phe Asp Glu Pro Gln Leu Ser 20 25 30 Ser Ile Thr Leu Thr Tyr Asp Thr Gly Ser Gly Ser Gly Ser Phe Arg 35 40 45 Ser Pro Val Gly Asp Asp Leu Asn Thr Gly His Tyr Ile Leu Gly Lys 50 55 60 Phe Ser Lys Gly Pro Lys Gln Gln Leu Trp Gly Ile Asn Ser Asn Ile 65 70 75 80 Asp Gln Ser Ser His Cys Thr Ile Thr Val Arg Asp Gly Val Ser Val 85 90 95 Ser Met Ser Ser Pro Ser Gln Thr Met Gln Ile Asp Ser Gly Gly Gly 100 105 110 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Val Thr Val 115 120 125 Leu Thr Gly Gln Gly Trp Tyr Pro Phe Gly Arg Gly Ala Thr Val Cys 130 135 140 Tyr Tyr Thr Ala Thr Asp Ala Ala Thr Val Ser Asn Leu Lys Leu Phe 145 150 155 160 Phe Gln Asn Lys Ser Thr Asp Arg Ser Ile Thr Ile Arg Ser Lys Leu 165 170 175 Ser Pro Gln Tyr Arg Thr Asn Gly Ser Tyr Ser Ile Tyr Gly Met Trp 180 185 190 Glu Leu Gly Lys Gly Pro Pro Gln Arg Ile Trp Asn Trp Ala Phe Asp 195 200 205 Ser Ser Ile Ser Tyr Gly Ser Val Thr Cys Thr Leu Ser Leu Thr Gln 210 215 220 Ser Pro Lys Val Leu Gly Pro Gly Ser Glu Gln Leu Gln Val Gln 225 230 235 <210> 83 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> Affinity maturated humanized 806 scFv <400> 83 gaggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60 acctgtaccg tgtccggcta cagcatcagc agagacttcg cctggaactg gatcagacag 120 cctcctggca aaggactgga atggatgggc tacatcagct acaacggcaa caccagatac 180 cagcctagcc tgaagtcccg gatcaccatc tccagagaca ccagcaagaa ccagttcttc 240 ctgaagctga acagcgtgac agccgccgat accgccacct actattgtgt gacagccagc 300 agaggcttcc cctattgggg acagggaacc ctggtcacag ttagctctgg tggcggagga 360 tctggcggag gcggatcttc tggcggtggc tctgatatcc agatgacaca gagccccagc 420 agcatgtccg tgtccgtggg agacagagtg accatcacct gtcacagcag ccaggacatc 480 aacagcaaca tcggctggct gcagcagaag cccggcaagt cttttaaggg cctgatctac 540 cacggcacca acctggatga tggcgtgccc agcagatttt ctggcagcgg ctctggcacc 600 gactacaccc tgaccatatc tagcctgcag cctgaggact tcgccaccta ttactgcgtg 660 cagtacgccc agtttccttg gacctttgga ggcggcacaa agctggaaat caag 714 <210> 84 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Affinity maturated humanized 806 scFv <400> 84 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Arg Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Asn Gly Asn Thr Arg Tyr Gln Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Ser Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly 115 120 125 Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Val 130 135 140 Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ser Ser Gln Asp Ile 145 150 155 160 Asn Ser Asn Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys 165 170 175 Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp Gly Val Pro Ser Arg 180 185 190 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser 195 200 205 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln 210 215 220 Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 225 230 235 <210> 85 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> Affinity maturated humanized 806 scFv <400> 85 gaactaaagg tcgaaacacg gcggaggttt ccaggttcct ttgacccgca tgacgtgcgt 60 cattatccac cgcttcagga gtccgacgtc cgatctatac cagtcccaca tcagccacgg 120 tctcggcgac ggtcttttag acgacccgtg cggtagtagg tccaaccacg gcaccatcta 180 gtccgggaat tttctgaacg gcccgaagac gacgtcggtc ggctacaacg acaactacag 240 gaccgacgac actgtccact accagtgaga cagagggtgc ctgtgcctgt acgacgaccc 300 cgagacacag tagacctata gtctcggtgg cggtcttcta ggcggaggcg gtctaggagg 360 cggtggtctc gattgacact ggtcccaagg gacaggggtt atccccttcg gagacgaccg 420 acagtgtgtt atcatccacc gccatagccg ccgacagtgc gacaagtcga agtccttctt 480 gaccaagaac gaccacagag acctctacca ctaggccctg aagtccgatc cgaccataga 540 ccacaacggc aacatcgact acatcgggta ggtaaggtca ggaaacggtc ctccgacaga 600 ctaggtcaag gtccgcttca gagacgacta cgacatcggc ctgtgccatg tccagtccga 660 gtcacaaacc gatccgaact ggtccggtcc cggtctgaga acgtcgactt ggag 714 <210> 86 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Affinity maturated humanized 806 scFv <400> 86 Lys Ile Glu Leu Lys Thr Gly Gly Gly Phe Thr Trp Pro Phe Gln Ala 1 5 10 15 Tyr Gln Val Cys Tyr Tyr Thr Ala Phe Asp Glu Pro Gln Leu Ser Ser 20 25 30 Ile Thr Leu Thr Tyr Asp Thr Gly Ser Gly Ser Gly Ser Phe Arg Ser 35 40 45 Pro Val Gly Asp Asp Leu Asn Thr Gly His Tyr Ile Leu Gly Lys Phe 50 55 60 Ser Lys Gly Pro Lys Gln Gln Leu Trp Gly Ile Asn Ser Asn Ile Asp 65 70 75 80 Gln Ser Ser His Cys Thr Ile Thr Val Arg Asp Gly Val Ser Val Ser 85 90 95 Met Ser Ser Pro Ser Gln Thr Met Gln Ile Asp Ser Gly Gly Gly Ser 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Val Thr Val Leu 115 120 125 Thr Gly Gln Gly Trp Tyr Pro Phe Gly Arg Ser Ala Thr Val Cys Tyr 130 135 140 Tyr Thr Ala Thr Asp Ala Ala Thr Val Ser Asn Leu Lys Leu Phe Phe 145 150 155 160 Gln Asn Lys Ser Thr Asp Arg Ser Ile Thr Ile Arg Ser Lys Leu Ser 165 170 175 Pro Gln Tyr Arg Thr Asn Gly Asn Tyr Ser Ile Tyr Gly Met Trp Glu 180 185 190 Leu Gly Lys Gly Pro Pro Gln Arg Ile Trp Asn Trp Ala Phe Asp Arg 195 200 205 Ser Ile Ser Tyr Gly Ser Val Thr Cys Thr Leu Ser Leu Thr Gln Ser 210 215 220 Pro Lys Val Leu Gly Pro Gly Ser Glu Gln Leu Gln Val Glu 225 230 235

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of the epidermal growth factor receptor (EGFR), a transmembrane domain, and an intracellular domain.

2. The EGFR isoforms are selected from the group consisting of wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR R108K EGFR R108G EGFR G598V EGFR D126Y EGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F and EGFR variant II, the isolated nucleic acid according to claim 1.

3. The isolated nucleic acid according to claim 1, wherein the antigen-binding domain is selected from the group consisting of an antibody, scFv, Fab, or any fragment thereof.

4. The isolated nucleic acid according to claim 1, wherein the antigen-binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 31, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, and SEQ ID NO:

85.

5. The isolated nucleic acid according to claim 1, wherein the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 32, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, and SEQ ID NO:

86.

6. The isolated nucleic acid according to claim 1, wherein the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, and SEQ ID NO:

30.

7. The isolated nucleic acid according to claim 1, wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 26, and SEQ ID NO:

29.

8. The isolated nucleic acid according to claim 1, wherein the antigen-binding domain comprises a light chain complementarity-determining region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, and 7.

9. The isolated nucleic acid according to claim 1, wherein the antigen-binding domain comprises a heavy chain complementarity-determining region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 9, and 10.

10. The isolated nucleic acid according to claim 1, wherein the CAR further comprises a hinge region.

11. The isolated nucleic acid according to claim 10, wherein the hinge region is encoded by the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO:

71.

12. The isolated nucleic acid according to claim 1, wherein the transmembrane domain is encoded by the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO:

73.

13. The isolated nucleic acid according to claim 1, wherein the intracellular domain is encoded by the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO:

75.

14. The isolated nucleic acid according to claim 1, wherein the intracellular domain is encoded by a nucleotide sequence comprising SEQ ID NO: 14 or SEQ ID NO:

77.

15. The isolated nucleic acid according to claim 1, wherein the intracellular domain is encoded by a nucleotide sequence comprising SEQ ID NO: 13 and SEQ ID NO: 14 or a nucleotide sequence comprising SEQ ID NO: 75 and SEQ ID NO:

77.

16. The isolated nucleic acid according to claim 1, wherein the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, 64, 66, or 68.

17. The isolated nucleic acid according to claim 1, wherein the transmembrane domain and / or the intracellular domain comprises a killer cell immunoglobulin-like receptor (KIR).

18. The isolated nucleic acid according to claim 17, further comprising a nucleic acid encoding DAP12.

19. The isolated nucleic acid according to claim 1, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 65, 67, and 69.

20. The isolated nucleic acid according to claim 1, wherein the CAR is capable of binding to an EGFR homodimer, an EGFR heterodimer, an EGFR oligomer, and / or an EGFR / ErbB oligomer.

21. A vector comprising the isolated nucleic acid according to any one of the preceding claims.

22. A modified cell comprising a cross-reactive chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain capable of binding to multiple isoforms of EGFR, a transmembrane domain, and an intracellular domain.

23. The EGFR isoform is selected from the group consisting of wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR R108K EGFR R108G EGFR G598V EGFR D126Y EGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F and EGFR variant II, the modified cell according to claim 22.

24. The modified cell according to claim 22, wherein the antigen-binding domain is selected from the group consisting of an antibody, an scFv, a Fab, or any fragment thereof.

25. The modified cell according to claim 22, wherein the antigen-binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 31, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, and SEQ ID NO:

85.

26. The modified cell according to claim 22, wherein the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 32, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, and SEQ ID NO:

86.

27. The modified cell according to claim 22, wherein the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, and SEQ ID NO:

30.

28. The modified cell according to claim 22, wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 26, and SEQ ID NO:

29.

29. The modified cell according to claim 22, wherein the antigen-binding domain comprises a light chain complementarity determining region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, and 7.

30. The modified cell according to claim 22, wherein the antigen-binding domain comprises a heavy chain complementarity determining region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 9, and 10.

31. The modified cell according to claim 22, wherein the CAR further comprises a hinge region.

32. The modified cell according to claim 31, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:

72.

33. The modified cell according to claim 22, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

74.

34. The modified cell according to claim 22, wherein the intracellular domain comprises the amino acid sequence of SEQ ID NO:

76.

35. The modified cell according to claim 22, wherein the intracellular domain comprises the amino acid sequence of SEQ ID NO:

78.

36. The modified cell according to claim 22, wherein the intracellular domain comprises the amino acid sequences of SEQ ID NO: 76 and SEQ ID NO:

78.

37. The modified cell according to claim 22, wherein the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, 64, 66, or 68.

38. The modified cell according to claim 22, wherein the transmembrane domain and / or the intracellular domain comprises a killer cell immunoglobulin-like receptor (KIR).

39. The modified cell according to claim 38, further comprising a nucleic acid encoding DAP12.

40. The modified cell according to claim 22, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 65, 67, and 69.

41. The modified cell according to claim 22, wherein the CAR is capable of binding to an EGFR homodimer, an EGFR heterodimer, an EGFR oligomer, and / or an EGFR / ErbB oligomer.

42. The modified cell according to claim 22, which is a T cell.

43. The modified cell according to claim 22, which is an autologous cell.

44. The modified cell according to claim 22, which is a human cell.

45. A method for treating cancer in a subject in need thereof, comprising administering to the subject a modified cell according to any one of claims 22 to 44.

46. A method for treating cancer in a subject in need thereof, comprising administering to the subject a modified cell comprising a CAR, wherein the CAR comprises an antigen-binding domain capable of binding to a plurality of isoforms of EGFR, a transmembrane domain, and an intracellular domain. Method.

47. The EGFR isoform is selected from the group consisting of wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR R108K EGFR R108G EGFR G598V EGFR D126Y EGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F and EGFR variant II, the method according to claim 46.

48. The method according to claim 46, wherein the antigen-binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 31, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, and SEQ ID NO:

85.

49. The method according to claim 46, wherein the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 32, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, and SEQ ID NO:

86.

50. The method according to claim 46, wherein the antigen-binding domain comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, and SEQ ID NO:

30.

51. The method according to claim 46, wherein the antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 26, and SEQ ID NO:

29.

52. The method according to claim 46, wherein the antigen-binding domain comprises a light chain complementarity determining region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, and 7.

53. The method according to claim 46, wherein the antigen-binding domain comprises a heavy chain complementarity determining region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 9, and 10.

54. The method according to claim 46, wherein the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, 64, 66, or 68.

55. The method according to claim 46, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 65, 67, and 69.

56. The method according to any one of claims 45 to 55, further comprising the step of administering an additional treatment to the subject.

57. The method according to claim 56, wherein the additional treatment comprises immune checkpoint inhibition (ICB).

58. The method according to claim 57, wherein the ICB is selected from the group consisting of anti-PD-1 treatment, anti-PD-L1 treatment, anti-TIM3 treatment, and anti-CTLA-4 treatment.

59. The method according to any one of claims 45 to 58, wherein the treatment is delivered locally.

60. The method according to claim 45 or 46, wherein the modified cell further comprises a minibody.

61. The method according to claim 60, wherein the minibody comprises an scFv specific for PD-1 and a human IgG CH3 domain.

62. The method according to claim 60, wherein the minibody comprises an scFv specific for CTLA-4 and a human IgG CH3 domain.

63. The method according to claim 60, wherein the minibody comprises an scFv specific for TIM-3 and a human IgG CH3 domain.

64. The method according to claim 60, wherein the minibody comprises an scFv specific for PD-L1 and a human IgG CH3 domain.

65. A method of treating cancer in a subject in need thereof, comprising: culturing a plurality of CAR T cells together with GBM organoids (GBOs) derived from the subject; selecting CAR T cells having the highest potency from the plurality of CAR T cells; and administering the CAR T cells having the highest potency to the subject to thereby treat cancer in the subject. A method comprising the steps of:

66. The method according to claim 65, wherein the plurality of CAR T cells comprises a plurality of modified T cells comprising a plurality of CARs, each CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain.

67. The method according to claim 66, wherein the antigen-binding domain is capable of binding to an antigen selected from the group consisting of CD19, EGFR, multiple isoforms of EGFR (e.g., wild-type EGFR (wtEGFR), mutant EGFR, EGFR A289V EGFR A289D EGFR A289T EGFR R108K EGFR R108G EGFR G598V EGFR D126Y EGFR C628F EGFR R108K / A289V EGFR R108K / D126Y EGFR A289V / G598V EGFR A289V / C628F and EGFR variant II), PSMA, PSCA, and any tumor-associated antigen (TAA).

68. The method according to claim 65, wherein the GBO is generated from a biopsy material derived from the subject.

69. The method according to claim 65, wherein the highest potency is measured as the highest degree of apoptosis and / or tumor cell death.

70. The method according to claim 65, further comprising administering an additional treatment to the subject.

71. The method according to claim 66, wherein the additional treatment comprises immune checkpoint inhibition (ICB).

72. The method according to claim 67, wherein the ICB is selected from the group consisting of anti-PD-1 treatment, anti-PD-L1 treatment, anti-TIM3 treatment, and anti-CTLA-4 treatment.

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