Anti-CD79b antibodies and chimeric antigen receptors and their usage

Abstract

This article provides a CD79b antibody and a CD79b-specific chimeric antigen receptor (CAR). Further, this article provides immune cells expressing the CD79b-specific CAR and a method for treating cancer by administering the CD79b-specific CAR immune cells.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 018,266, filed April 30, 2020 (which is incorporated herein by reference in its entirety). Background of the Invention

[0003] 1. Field of Invention

[0004] In general, this disclosure relates to the fields of immunology, cell biology, molecular biology, and medicine. More specifically, it relates to CD79b antibodies and related compositions (including at least chimeric antigen receptors) and methods of use thereof.

[0005] 2. Description of related technologies

[0006] Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 is highly effective in B-cell malignancies. Recently, two anti-CD19 CAR T-cell therapy products were approved by the US FDA for relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) and / or large B-cell lymphoma. In pivotal trials, durable remission lasting more than one year has been observed in ~40-50% of these patients. However, relapse or progression occurs in ~50-60% of cases, and a major cause of resistance appears to be due to CD19 antigen loss. Therefore, there is an urgent need to develop CAR T-cell therapies targeting novel targets to further improve outcomes in these patients. Invention Overview

[0008] This disclosure relates to methods and compositions associated with specific antibodies. The antibodies can be used in any type of immunotherapy and for any medical application where the targeting of CD79b is therapeutic. In some embodiments, this disclosure provides isolated monoclonal antibodies that specifically bind to CD79b and comprise:

[0009] (I):

[0010] (a) The first V containing SEQ ID NO:1 H CDR;

[0011] (b) The second V containing SEQ ID NO:2 H CDR;

[0012] (c) The third V containing SEQ ID NO:3 H CDR;

[0013] (d) The first V containing SEQ ID NO:4 L CDR;

[0014] (e) The second V containing SEQ ID NO:5L CDR; and

[0015] (f) The third V containing SEQ ID NO:6 L CDR;

[0016] (II):

[0017] (a) The first V containing SEQ ID NO:11 H CDR;

[0018] (b) The second V containing SEQ ID NO:12 H CDR;

[0019] (c) The third V containing SEQ ID NO:13 H CDR;

[0020] (d) The first V containing SEQ ID NO:14 L CDR;

[0021] (e) The second V containing SEQ ID NO:15 L CDR; and

[0022] (f) The third V containing SEQ ID NO:16 L CDR; or

[0023] (III):

[0024] (a) The first V containing SEQ ID NO:21 H CDR;

[0025] (b) The second V containing SEQ ID NO:22 H CDR;

[0026] (c) The third V containing SEQ ID NO:23 H CDR;

[0027] (d) The first V containing SEQ ID NO:24 L CDR;

[0028] (e) The second V containing SEQ ID NO:25 L CDR; and

[0029] (f) The third V containing SEQ ID NO:26 L CDR.

[0030] In some respects, the antibody comprises:

[0031] (a) The first V containing SEQ ID NO:1 H CDR;

[0032] (b) The second V containing SEQ ID NO:2 H CDR;

[0033] (c) The third V containing SEQ ID NO:3 H CDR;

[0034] (d) The first V containing SEQ ID NO:4 L CDR;

[0035] (e) The second V containing SEQ ID NO:5 L CDR; and

[0036] (f) The third V containing SEQ ID NO:6 L CDR.

[0037] In a further aspect, the antibody comprises V with SEQ ID NO:7 H The structural domain is at least approximately 80% identical to the V. H The structural domain and V with SEQ ID NO:9 L The structural domain is at least approximately 80% identical to the V. L Domain. In other aspects, the antibody comprises the V domain of SEQ ID NO:7. H V with the same structural domain H The structural domain and V with SEQ ID NO:9 L V with the same structural domain L Structural domain.

[0038] In some respects, the antibody comprises:

[0039] (a) The first V containing SEQ ID NO:11 H CDR;

[0040] (b) The second V containing SEQ ID NO:12 H CDR;

[0041] (c) The third V containing SEQ ID NO:13 H CDR;

[0042] (d) The first V containing SEQ ID NO:14 L CDR;

[0043] (e) The second V containing SEQ ID NO:15 L CDR; and

[0044] (f) The third V containing SEQ ID NO:16 L CDR.

[0045] In a further aspect, the antibody comprises V with SEQ ID NO:17 H The structural domain is at least approximately 80% identical to the V. H The structural domain and V of SEQ ID NO:19 L The structural domain is at least approximately 80% identical to the V. L Domain. In other aspects, the antibody comprises V, which is consistent with SEQ ID NO:17. H V with the same structural domain H The structural domain and V of SEQ ID NO:19 L V with the same structural domain L Structural domain.

[0046] In some respects, the antibody comprises:

[0047] (a) The first V containing SEQ ID NO:21 H CDR;

[0048] (b) The second V containing SEQ ID NO:22 H CDR;

[0049] (c) The third V containing SEQ ID NO:23 H CDR;

[0050] (d) The first V containing SEQ ID NO:24 L CDR;

[0051] (e) The second V containing SEQ ID NO:25 L CDR; and

[0052] (f) The third V containing SEQ ID NO:26 L CDR.

[0053] In a further aspect, the antibody comprises V with SEQ ID NO:27 H The structural domain is at least approximately 80% identical to the V. H The structural domain and V of SEQ ID NO:29 L The structural domain is at least approximately 80% identical to the V. L Domain. In other aspects, the antibody comprises V, which is consistent with SEQ ID NO:27. H V with the same structural domain HThe structural domain and V of SEQ ID NO:29 L V with the same structural domain L Structural domain.

[0054] In some aspects, the antibody is recombinant. In some aspects, the antibody is IgG, IgM, IgA, or an antigen-binding fragment thereof. In some aspects, the antibody is Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or a single-domain antibody. In some aspects, the antibody is a human antibody, a humanized antibody, or a de-immunized antibody. In some aspects, the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

[0055] In other embodiments, this disclosure provides compositions which contain the antibody of this disclosure in a pharmaceutically acceptable carrier.

[0056] In other embodiments, this disclosure provides isolated polynucleotide molecules containing nucleic acid sequences encoding antibodies of this disclosure.

[0057] In other embodiments, this disclosure provides a recombinant polypeptide comprising antibody V. H The structural domain, the antibody V H The domain contains the V of clone T26 H CDRs 1-3 (SEQ ID NO: 1, 2 and 3) of the structural domain; and V of clone T26 L CDRs 1-3 of the structural domain (SEQ ID NO: 4, 5 and 6).

[0058] In other embodiments, this disclosure provides recombinant polypeptides comprising antibody V. H The structural domain, the antibody V H The domain contains V of clone 5B. H CDRs 1-3 of the domain (SEQ ID NO: 11, 12 and 13); and V of clone 5B L CDRs 1-3 of the structural domain (SEQ ID NO: 14, 15 and 16).

[0059] In other embodiments, this disclosure provides a recombinant polypeptide comprising antibody V. H The structural domain, the antibody V H The domain contains V of clone 28B. H CDRs 1-3 (SEQ ID NO: 21, 22 and 23) of the structural domain; and V of clone 28B LCDRs 1-3 of the structural domain (SEQ ID NO: 24, 25 and 26).

[0060] In other embodiments, this disclosure provides isolated polynucleotide molecules comprising nucleic acid sequences encoding polypeptides of this disclosure.

[0061] In other embodiments, this disclosure provides a host cell comprising one or more polynucleotide molecules encoding an antibody or a recombinant polypeptide of this disclosure. In some aspects, the host cell is a mammalian cell, yeast cell, bacterial cell, ciliate cell, or insect cell.

[0062] In some other embodiments, this disclosure provides a method for treating a subject with cancer, comprising administering an effective amount of an antibody of this disclosure to the subject. In some aspects, the cancer is a B-cell malignancy. In some aspects, the antibody is in a pharmaceutically acceptable composition. In some aspects, the antibody is administered systemically. In some aspects, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally. In some aspects, the method further comprises administering at least a second anticancer therapy to the subject. In a further aspect, the second anticancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In some aspects, the second anticancer therapy includes adoptive T-cell therapy.

[0063] In other embodiments, this disclosure provides a modified CD79b CAR or TCR having an antigen-binding domain comprising:

[0064] (I):

[0065] (a) The first V containing SEQ ID NO:1 H CDR;

[0066] (b) The second V containing SEQ ID NO:2 H CDR;

[0067] (c) The third V containing SEQ ID NO:3 H CDR;

[0068] (d) The first V containing SEQ ID NO:4 L CDR;

[0069] (e) The second V containing SEQ ID NO:5 L CDR; and

[0070] (f) The third V containing SEQ ID NO:6 L CDR;

[0071] (II):

[0072] (a) The first V containing SEQ ID NO:11 H CDR;

[0073] (b) The second V containing SEQ ID NO:12 H CDR;

[0074] (c) The third V containing SEQ ID NO:13 H CDR;

[0075] (d) The first V containing SEQ ID NO:14 L CDR;

[0076] (e) The second V containing SEQ ID NO:15 L CDR; and

[0077] (f) The third V containing SEQ ID NO:16 L CDR; or

[0078] (III):

[0079] (a) The first V containing SEQ ID NO:21 H CDR;

[0080] (b) The second V containing SEQ ID NO:22 H CDR;

[0081] (c) The third V containing SEQ ID NO:23 H CDR;

[0082] (d) The first V containing SEQ ID NO:24 L CDR;

[0083] (e) The second V containing SEQ ID NO:25 L CDR; and

[0084] (f) The third V containing SEQ ID NO:26 L CDR.

[0085] In some respects, the antigen-binding domain includes:

[0086] (a) The first V containing SEQ ID NO:1H CDR;

[0087] (b) The second V containing SEQ ID NO:2 H CDR;

[0088] (c) The third V containing SEQ ID NO:3 H CDR;

[0089] (d) The first V containing SEQ ID NO:4 L CDR;

[0090] (e) The second V containing SEQ ID NO:5 L CDR; and

[0091] (f) The third V containing SEQ ID NO:6 L CDR.

[0092] In a further aspect, the antigen-binding domain includes the V of SEQ ID NO:7. H The structural domain is at least approximately 80% identical to the V. H The structural domain and V with SEQ ID NO:9 L The structural domain is at least approximately 80% identical to the V. L Domain. In some respects, the antigen-binding domain is associated with V of SEQ ID NO:7. H V with the same structural domain H The structural domain and V with SEQ ID NO:9 L V with the same structural domain L Structural domain.

[0093] In some respects, the antibody comprises:

[0094] (a) The first V containing SEQ ID NO:11 H CDR;

[0095] (b) The second V containing SEQ ID NO:12 H CDR;

[0096] (c) The third V containing SEQ ID NO:13 H CDR;

[0097] (d) The first V containing SEQ ID NO:14 L CDR;

[0098] (e) The second V containing SEQ ID NO:15 L CDR; and

[0099] (f) The third V containing SEQ ID NO:16 L CDR.

[0100] In a further aspect, the antigen-binding domain includes the V of SEQ ID NO:17. H The structural domain is at least approximately 80% identical to the V. H The structural domain and V of SEQ ID NO:19 L The structural domain is at least approximately 80% identical to the V. L Domain. In some aspects, the antigen-binding domain comprises the V of SEQ ID NO:17. H V with the same structural domain H The structural domain and V of SEQ ID NO:19 L V with the same structural domain L Structural domain.

[0101] In some respects, the antigen-binding domain includes:

[0102] (a) The first V containing SEQ ID NO:21 H CDR;

[0103] (b) The second V containing SEQ ID NO:22 H CDR;

[0104] (c) The third V containing SEQ ID NO:23 H CDR;

[0105] (d) The first V containing SEQ ID NO:24 L CDR;

[0106] (e) The second V containing SEQ ID NO:25 L CDR; and

[0107] (f) The third V containing SEQ ID NO:26 L CDR.

[0108] In a further aspect, the antigen-binding domain includes the V of SEQ ID NO:27. H The structural domain is at least approximately 80% identical to the V. H The structural domain and V of SEQ ID NO:29 L The structural domain is at least approximately 80% identical to the V. L Domain. In some aspects, the antigen-binding domain comprises the V of SEQ ID NO:27. H V with the same structural domain HThe structural domain and V of SEQ ID NO:29 L V with the same structural domain L Structural domains. In some aspects, the CAR includes one or more signal transduction domains CD3ξ, CD28, OX40 / CD134, 4-1BB / CD137, and combinations thereof. In some aspects, the CAR includes CD3ζ and CD28 signal transduction domains. In some aspects, the CAR includes CD3ζ and 4-1BB signal transduction domains. In some aspects, the CAR includes CD3ζ and OX-40 signal transduction domains. In some aspects, the CAR or TCR is encoded by a viral vector. In a further aspect, the viral vector is a lentiviral vector.

[0109] In some aspects, the antigen-binding domain includes a linker connected to V L V connected by structural domains H Structural domain. In a further aspect, the connector is connector 1 (SEQ ID NO: 44 or 45), connector 2 (SEQ ID NO: 46 or 47), connector 3 (SEQ ID NO: 48 or 49), or connector 4 (SEQ ID NO: 50 or 51). In some aspects, the CAR includes V. L -Connector 1-V H V L -Connector 2-V H V L -Connector 3-V H V L -Connector 4-V H V H -Connector 1-V L V H -Connector 2-V L V H -Connector 3-V L or V H -Connector 4-V LIn some aspects, the CAR or TCR includes a hinge. In a further aspect, the hinge is CD8 hinge 1 (SEQ ID NO: 52 or 53), CD8 hinge 2 (SEQ ID NO: 54 or 55), CD8 hinge 3 (SEQ ID NO: 56 or 57), CD28 hinge (SEQ ID NO: 58 or 59), IgG4 hinge (SEQ ID NO: 60 or 61), IgG4CH2 (SEQ ID NO: 62 or 63), IgG4 CH2CH3 (SEQ ID NO: 64 or 65), or IgG4 CH1CH2CH3 (SEQ ID NO: 66 or 67). In some aspects, the CAR includes a transmembrane domain. In a further aspect, the transmembrane domain is CD8TM1 (SEQ ID NO: 68 or 69), CD8TM2 (SEQ ID NO: 70 or 71), or CD28TM (SEQ ID NO: 72 or 73).

[0110] In some aspects, the method further includes a transduction marker and / or a safety switch. In a further aspect, the transduction marker is enhanced green fluorescent protein (eGFP). In a further further aspect, the eGFP has the amino acid sequence of SEQ ID NO:83. In some aspects, the transduction marker and / or safety switch is a truncated epidermal growth factor (EGFR). In a further aspect, the EGFR has the amino acid sequence of SEQ ID NO:41. In some aspects, the transduction marker and / or safety switch is linked to the CAR via a cleavage peptide. In a further aspect, the cleavage peptide is a 2A peptide. In a further further aspect, the 2A peptide is a T2A peptide. In an even further aspect, the T2A peptide has the amino acid sequence of SEQ ID NO:85. In some aspects, the CAR further includes a second antigen-binding domain. In a further aspect, the second antigen-binding domain is a CD19, CD20, or CD22 antigen-binding domain.

[0111] In other embodiments, this disclosure provides an expression carrier that encodes the CAR or TCR of this disclosure.

[0112] In other embodiments, this disclosure provides host cells modified to express CD79b CAR or CD79b TCR. In some aspects, the cells are modified to express the CAR of this disclosure. In some aspects, the host cells are immune cells. In a further aspect, the immune cells are T cells. In an even further aspect, the T cells are primary human T cells or TILs. In other aspects, the T cells are CD4+ T cells or CD8+ T cells. In some aspects, the primary human T cells are obtained from a healthy donor. In some aspects, the T cells are autologous. In some aspects, the T cells are allogeneic. In some aspects, the cells are modified using a CRISPR or transposase system.

[0113] In other embodiments, this disclosure provides pharmaceutical compositions comprising CD79b-targeting T cells and a pharmaceutical carrier, wherein the CD79b-targeting T cells are modified to express the CAR or TCR of this disclosure.

[0114] In other embodiments, this disclosure provides compositions comprising an effective amount of CD79b-targeting T cells for treating cancer in subjects, wherein the CD79b-targeting T cells are modified to express the CAR or TCR of this disclosure.

[0115] In other embodiments, this disclosure provides the use of a composition comprising an effective amount of CD79b-targeting T cells for the treatment of cancer in a subject, wherein the CD79b-targeting T cells are modified to express the CAR or TCR of this disclosure.

[0116] In other embodiments, this disclosure provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of CD79b-targeting T cells, wherein the CD79b-targeting T cells are modified to express a CAR or TCR of this disclosure. In a further aspect, the cancer is a B-cell malignancy. In an even further aspect, the B-cell malignancy is B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt lymphoma, or chronic lymphocytic leukemia. In some aspects, the subject has previously received CD19 CAR therapy. In some aspects, the subject is resistant to CD19 CAR therapy. In a further aspect, the subject has CD19 antigen loss. In an even further aspect, the subject has relapsed with a CD19-negative tumor. In some aspects, the CD79b-targeting T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally. In some aspects, the CD79b-targeting T cells are administered intravenously. In some aspects, the method further includes administering at least a second anticancer therapy to the subject. In further aspects, the second anticancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In some aspects, the cancer is a CD79b-expressing cancer.

[0117] In some aspects, the CAR further includes a second antigen-binding domain. In some aspects, the second antigen-binding domain is a CD19, CD20, or CD22 antigen-binding domain.

[0118] In another embodiment, an expression vector is provided that encodes the CD79b CAR of the present invention embodiment.

[0119] This document further provides host cells modified to express CD79b CAR (e.g., CD79b according to embodiments of the present invention). In some aspects, the host cells are immune cells, such as T cells. In some aspects, the T cells are primary human T cells. In some aspects, the T cells are CD4+ T cells or CD8+ T cells. In some aspects, the primary human T cells are obtained from a healthy donor. The T cells can be autologous or allogeneic.

[0120] Pharmaceutical compositions comprising CD79b CAR T cells (e.g., CAR T cells according to embodiments of the present invention) and a pharmaceutical carrier are also provided herein. Compositions comprising an effective amount of CD79b CAR T cells (e.g., CAR T cells according to embodiments of the present invention) for treating cancer in a subject are further provided herein. In other embodiments, the use of compositions comprising an effective amount of CD79b CAR T cells (e.g., CAR T cells according to embodiments of the present invention) for treating cancer in a subject is provided.

[0121] In a further embodiment, a method for treating cancer in a subject is provided, comprising administering to the subject an effective amount of CD79b CAR T cells, such as the CAR T cells of the embodiments of the present invention. In some aspects, the cancer is a B-cell malignancy, such as B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt lymphoma, or chronic lymphocytic leukemia. In some aspects, the cancer is a CD79b-expressing cancer.

[0122] In some respects, the subject had previously received CD19 CAR therapy. In some respects, the subject was resistant to CD19 CAR therapy, for example, due to loss of the CD19 antigen. In some respects, the subject had a recurrence of CD19-negative tumors.

[0123] In some aspects, the CD79b CAR T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally. In other aspects, the method further includes administering at least a second anticancer therapy to the subject. In some aspects, the second anticancer therapy is surgical therapy, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0124] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while preferred embodiments of the invention have been indicated, the detailed description and specific embodiments are given by way of example only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Brief description of the attached diagram

[0126] The accompanying drawings, which form part of this specification, are included to further illustrate certain aspects of the invention. A better understanding of the invention will be achieved by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0127] Figure 1A-1D :( Figure 1A CD79b expression in cell lines. Figure 1B CD79b expression in human tissues. Figure 1C CD79b expression in leukemia. Figure 1D CD79b expression in lymphoma.

[0128] Figures 2A-2D :( Figure 2A Flow cytometry analysis was performed on CD79b-transduced cells. Figure 2B Binding affinity of CD79b monoclonal antibody. Figure 2C Characterization of CD79b monoclonal antibody. Figure 2D Clone 14 staining of lymphoma cell lines.

[0129] Figures 3A-3D :( Figure 3A A schematic depiction of the construct for the CD79b CAR. Figure 3B Flow cytometry analysis of CD79b CAR and CD19 CAR. Figure 3C The percentage of cytotoxicity of CD79b CAR and CD19 CAR was compared with untransduced T cells as a control. Figure 3D Flow cytometry of CD79b CAR and CD19 CAR, with untransduced T cells as a control.

[0130] Figures 4A-4D :( Figure 4A T cells co-cultured with CD79b CAR and CD19 exon 2Δ splicing variants. Figure 4B Flow cytometry analysis of the efficacy of CAR incubated for 4 days at an effector:target ratio of 5:1. Figure 4C Absolute cell counts of Daudi cells using CD79b CAR. Figure 4D The absolute cell count of cells was knocked down using CD19 knockout of CD79b CAR.

[0131] Figures 5A-5C :( Figure 5A A schematic diagram of preclinical research. Figure 5B Bioluminescent images of mice during the study. Figure 5C The percentage of mice that survived during the study period.

[0132] Figures 6A-6E :( Figure 6A CD79b CAR constructs for certain implementations. Figures 6B-6CLine plots and frequency distribution maps showed that anti-CD79b CAR T cells exhibited in vitro cytotoxicity against Daudi lymphoma cells. Figure 6D-6E Linear graphs and imaging showed that anti-CD79b CAR T cells exhibited in vivo efficacy against Daudi lymphoma xenografts.

[0133] Figure 7 : The binding of anti-CD79b antibodies (clones 5B and 28B) to human CD79b.

[0134] Figures 8A-8B :( Figure 8A The structural domain map of the anti-CD79b CAR used in the embodiments described herein. Figure 8B A map of CAR constructs in a lentiviral vector (pLVEG) containing the EF1α promoter.

[0135] Figures 9A-9D :( Figure 9A CAR was transduced into NFAT recipient cells. Figure 9B Luciferase activity in transduced NFAT reporter cells co-cultured with antibodies or Dadui Burkitt cells. Figure 9C-9D Luciferase activity in transduced NFAT reporter cells co-cultured with SUDHL6.

[0136] Figures 10A-10B :( Figure 10A The representative transduction efficiency of CAR in T cells. Figure 10B Phosphorylation of CD3ζ and ERK1 / 2 in T cells that express or do not express CAR.

[0137] Figure 11A-11B :( Figure 11A-11B Proliferation of T cells that express or do not express CAR.

[0138] Figure 12 Cytokine expression in T cells that express or do not express CAR.

[0139] Figures 13A-13B :( Figures 13A-13B In response to lymphoma cells, T cells expressing or not expressing CAR undergo degranulation.

[0140] Figures 14A-14B :( Figure 14A The cytotoxic activity of T cells expressing or not expressing CAR against lymphoma cells. Figure 14B ) via the lysis of SUDHL6 cells by CAR T cells.

[0141] Figures 15A-15B :( Figure 15ABioluminescent imaging of tumor burden in mice treated with T cells expressing or not expressing CAR. Figure 15B Survival probability in mouse cancer models treated with cells expressing or not expressing CAR.

[0142] Description of illustrative implementation schemes

[0143] CD79b is a pan-B-cell lineage marker and an important component of the B-cell receptor complex. CD79b is widely expressed in normal B-cells and B-cell malignancies, and its expression is typically maintained in CD19-negative tumors that relapse after CD19-specific CAR T-cell therapy. Therefore, in some embodiments, this disclosure provides CD79b monoclonal antibodies and CD79b-specific CARs, for example, for CD79b-CAR T cells.

[0144] This study demonstrated the efficacy of the CD79b-specific CAR T cell product of this invention in in vitro and in vivo models. Three murine monoclonal antibodies against human CD79b were developed using hybridoma technology, and their specific binding to recombinant human CD79b with high affinity (Kd range of 1.44-17.8 nM) was demonstrated, along with staining of various lymphoma cell lines. Subsequently, the variable regions of the heavy and light chains of the CD79b antibodies were cloned, and a lentiviral construct for an anti-CD79b CAR with CD3ζ and CD28 / 4-1BB co-stimulatory domains was developed. It was demonstrated that the anti-CD79b CAR construct could be transduced into primary CD4+ and CD8+ T cells from healthy donors using lentivirus, achieving a transduction efficiency exceeding 70%.

[0145] Anti-CD79b CAR T cells (rather than untransduced T cells) were observed to exhibit significant cytotoxic activity against Daudi Burkitt lymphoma and Mino mantle cell lymphoma cell lines, comparable to control anti-CD19 CAR T cells. More importantly, anti-CD79b, but not anti-CD19 CAR T cells, lysed CD19-CD79b+ lymphoma cells. Also on CD4... + and CD8 + Significant degranulation was observed in all anti-CD79b CAR T cells when they were co-cultured with lymphoma cells. The efficacy of anti-CD79b CAR T cells was also examined in vivo in an NSG mouse model of Mino lymphoma xenograft. Luciferase-labeled Mino mantle cell lymphoma cells were cultured at 2 × 10⁻⁶ cells / year. 6 One tumor cell / mouse IV was injected into NSG mice. Eighteen days later, the cells were administered via the tail vein at a dose of 10 × 10⁻⁶. 6Mice were treated with untransduced primary T cells, anti-CD19 CAR T cells, or anti-CD79b CAR T cells. Tumor burden was assessed using bioluminescence imaging. Results showed progressive tumor growth in mice treated with untransduced T cells. In mice treated with anti-CD19 and anti-CD79b CAR T cells, tumor growth was suppressed and survival was improved. Therefore, these results demonstrate the efficacy of this novel anti-CD79b CAR T cell therapy in patients with B-cell malignancies, which could be a novel strategy for overcoming resistance caused by CD19 loss following CD19-specific CAR T cell therapy.

[0146] In some aspects, the anti-CD79b CAR construct of the present invention is encoded by a lentiviral vector. The vector can be transduced into immune cells (e.g., T cells). The construct may contain CD28, CD3ζ, and / or 4-1BB signaling domains. The construct may contain a transduction marker, such as eGFP or a truncated EGFR domain. The transduction marker can be linked to the CAR via a cleavage peptide (e.g., 2A peptide).

[0147] This document further provides a method for treating cancer by administering CD79b-specific CAR immune cells (e.g., T cells) provided herein. The cancer can be a CD79b-expressing B-cell malignancy, such as B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt lymphoma, or chronic lymphocytic leukemia. The therapy of this invention can be used to treat subjects with CD19-negative B-cell malignancies who have relapsed after anti-CD19-CAR T-cell therapy.

[0148] II. Definition

[0149] As used herein, "substantially free" with respect to a particular component means that none of the particular component was intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the particular component due to any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, a composition is one in which the amount of the particular component is undetectable by standard analytical methods.

[0150] As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claims, when combined with the word "comprising (including)," the word "a" or "an" may mean one or more.

[0151] The use of the term "or" in the claims is intended to mean "and / or" unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive, although this disclosure supports the definition of referring only to alternatives and "and / or". As used herein, "another" may mean at least a second or more. The terms "about", "substantially", and "approximately" generally mean the stated value ±5%.

[0152] "Treatment" of a disease or condition refers to the implementation of a program of treatment that may include administering one or more medications to a patient in an effort to reduce the signs or symptoms of the disease. Desired therapeutic effects include slowing the rate of disease progression, improving or alleviating the disease state, and achieving a reduced or improved prognosis. Relief may occur before or after the signs or symptoms of the disease or condition appear. Therefore, "treatment" may include "prevention" of a disease or undesirable condition. Furthermore, "treatment" does not require complete reduction of signs or symptoms, does not require a cure, and particularly includes programs that have only a marginal effect on the patient.

[0153] As used throughout this application, the terms "therapeutic benefit" or "therapeutic effectiveness" mean anything that promotes or enhances the well-being of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, cancer treatment may involve, for instance, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in the rate of cancer growth, or prevention of metastasis. Cancer treatment may also refer to prolonging the survival of a subject with cancer.

[0154] "Subject" and "patient" refer to humans or non-human animals, such as primates, mammals, and vertebrates. In a particular embodiment, the subject is a human.

[0155] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that, when administered to animals such as humans (where appropriate), do not produce adverse, allergic, or other troublesome reactions. In light of this disclosure, the preparation of pharmaceutical compositions comprising antibodies or other active ingredients will be known to those skilled in the art. Furthermore, for animal (e.g., human) administration, it will be understood that the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Biostandards Agency.

[0156] As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral carriers such as sodium chloride, Ringer's glucose solution, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption-delaying agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, liquids, and nutrient supplements, as well as similar materials and combinations thereof, as will be known to those skilled in the art. The pH and precise concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0157] III. CD79b antibody

[0158] In some embodiments, an antibody or fragment thereof is considered that binds to at least a portion of CD79b and inhibits any type of CD79b activity (including at least signaling). As used herein, the term "antibody" is intended to refer broadly to any immunobinding agent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG, as well as polypeptides containing an antibody CDR domain that retains antigen-binding activity. The antibody may be selected from the group consisting of chimeric antibodies, affinity-matured antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, or antigen-binding antibody fragments or natural or synthetic ligands. In particular, the anti-CD79b antibody is a monoclonal antibody or a humanized antibody.

[0159] Therefore, polyclonal or monoclonal antibodies, antibody fragments and binding domains and CDRs (including modified forms of any of the above), or conjugates of any of the above, specific for CD79b, one or more of their respective epitopes, can be generated by known methods and as described herein, regardless of whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of natural compounds.

[0160] Examples of antibody fragments suitable for embodiments of the present invention include, but are not limited to: (i) Fab fragments, which are composed of V L V H C L and C H1 (ii) The “Fd” segment, which is composed of V H and C H1 (iii) The “Fv” fragment, which consists of a single antibody’s V domain. L and V H(iv) The “dAb” segment, which is composed of V H Domain composition; (v) separated CDR regions; (vi) F(ab')2 fragments, which are bivalent fragments containing two connected Fab fragments; (vii) single-chain Fv molecules (“scFv”), where V H Domain and V L The domains are linked by allowing the two domains to combine to form a peptide linker that binds the domains; (viii) a bispecific single-chain Fv dimer (see U.S. Patent No. 5,091,513); and (ix) a diabody, which is a multivalent or multispecific fragment constructed by gene fusion (U.S. Patent Application Publication 20050214860). Fv, scFv, or diabody molecules can be linked by incorporation of V... H and V L Stabilization is achieved through disulfide bridges in the CH3 domain. Minibody antibodies containing scFv linked to the CH3 domain can also be prepared.

[0161] Antibody-like binding peptide mimics are also considered in the implementation scheme. Liu et al. (2003) described “antibody-like binding peptide mimics” (ABiPs), which are peptides that can act as reduced antibodies and have certain advantages such as a longer serum half-life and a less cumbersome synthetic method.

[0162] Animals can be inoculated with antigens, such as the CD79b extracellular domain (ECD) protein, to generate antibodies specific to CD79b. Often, antigens are bound to or conjugated to another molecule to enhance the immune response. As used herein, a conjugate is any peptide, polypeptide, protein, or non-protein substance that binds to an antigen used to elicit an immune response in an animal. Antibodies generated in animals in response to antigen inoculation comprise a wide variety of dissimilar molecules (polyclonal antibodies), prepared from a wide variety of B lymphocytes that produce individual antibodies. Polyclonal antibodies are a mixed population of antibody classes, each capable of recognizing different epitopes on the same antigen. If the correct conditions are given for the production of polyclonal antibodies in an animal, the majority of antibodies in the animal's serum will recognize a collective epitope on the antigen compound to which the animal has been immunized. This specificity is further enhanced by affinity purification (to select only those antibodies that recognize the target antigen or epitope).

[0163] Monoclonal antibodies are a single class of antibodies in which each antibody molecule recognizes the same epitope because all antibody-producing cells originate from a single B-lymphocyte cell line. Methods for generating monoclonal antibodies (MAbs) generally begin along the same routes as those used to prepare polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to generate monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used to generate monoclonal antibodies. The use of rats is well-known and can offer certain advantages. Mice (e.g., BALB / c mice) are routinely used, and they typically give a high percentage of stable fusions.

[0164] Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with the CD79b antigen with an uncontrolled proliferation of myeloma cells (typically mouse myeloma). This technology provides a method for propagating a single antibody-producing cell to an unlimited number of generations, thereby generating an unlimited quantity of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

[0165] Plasma B cells (CD45) + CD5 - CD19 + CD79b binding molecules can be isolated from freshly prepared rabbit peripheral blood mononuclear cells from immunized rabbits, and further selected for CD79b binding. After enriching antibody-producing B cells, total RNA can be isolated and cDNA synthesized. DNA sequences from the antibody variable regions of both the heavy and light chains can be amplified, constructed into phage display Fab expression vectors, and transformed into *E. coli*. CD79b-specific binding Fabs can be selected through multiple rounds of enrichment panning and sequenced. Selected CD79b binding molecules can be expressed as full-length IgG in rabbit and rabbit / human chimeric forms in human embryonic kidney (HEK293) cells (Invitrogen) using a mammalian expression vector system, and purified using a rapid protein liquid chromatography (FPLC) separation unit with G protein resin.

[0166] In one embodiment, the antibody is a chimeric antibody, such as an antibody comprising an antigen-binding sequence from a non-human donor grafted with a heterologous non-human, human, or humanized sequence (e.g., framework and / or constant domain sequence). Methods have been developed to replace the light and heavy chain constant domains of a monoclonal antibody with similar domains derived from humans, while keeping the variable region of the exogenous antibody intact. Alternatively, “fully human” monoclonal antibodies are generated in transgenic mice relating to human immunoglobulin genes. Methods have also been developed to convert the variable domains of monoclonal antibodies into a more human form by recombinantly constructing antibody variable domains having both rodent (e.g., mouse) and human amino acid sequences. In “humanized” monoclonal antibodies, only the hypervariable CDR is derived from a mouse monoclonal antibody, and the framework and constant regions are derived from human amino acid sequences (see U.S. Patent Nos. 5,091,513 and 6,881,557). It is believed that replacing the characteristic amino acid sequence in antibodies for rodents with the amino acid sequence found at the corresponding positions in human antibodies would reduce the likelihood of adverse immune responses during therapeutic use. Hybridomas or other antibody-producing cells may also undergo gene mutations or other changes that may or may not alter the binding specificity of antibodies produced by that hybridoma.

[0167] Methods for generating polyclonal antibodies in various animal species and for generating various types of monoclonal antibodies (including humanized, chimeric, and fully human) are well known in the art and are highly predictable. For example, the following U.S. patents and patent applications provide enabling descriptions of such methods: U.S. Patent Application Nos. 2004 / 0126828 and 2002 / 0172677; and U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,196,265, 4,275,149, and 4,277,437. 4,366,241, 4,469,797, 4,472,509, 4,606,855, 4,703,003, 4,742,159, 4,767,720, 4,816,567, 4,867,973, 4,938,948, 4,946,778, 5,021,236, 5,164,296, 5,196 066、5,223,409、5,403,484、5,420,253、5,565,332、5,571,698、5,627,052、5,656,434、5,770,376、5,789,208、5,821,337、5,844,091、5,858,657、5,861,155、5,8 71,907, 5,969,108, 6,054,297, 6,165,464, 6,365,157, 6,406,867, 6,709,659, 6,709,873, 6,753,407, 6,814,965, 6,849,259, 6,861,572, 6,875,434, and 6,891,024. All patents, patent application publications, and other publications cited herein and therein are incorporated herein by reference.

[0168] Antibodies can be produced from any animal source, including birds and mammals. Preferably, the antibodies are from sheep, rodents (e.g., mice and rats), rabbits, goats, guinea pigs, camels, horses, or chickens. Additionally, newer technologies allow for the development and screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression technology allows for the generation of specific antibodies in the absence of animal immunization, as described in U.S. Patent No. 6,946,546 (which is incorporated herein by reference).

[0169] It is fully anticipated that antibodies against CD79b will have the ability to neutralize or counteract the effects of CD79b, regardless of the animal species, monoclonal cell line, or other source of the antibody. Certain animal species may be less preferred for generating therapeutic antibodies because they may be more likely to elicit an allergic response due to activation of the complement system via the antibody's "Fc" portion. However, intact antibodies can be enzymatically digested into an "Fc" (complement-binding) fragment, as well as an antibody fragment with a binding domain or CDR. Removal of the Fc portion reduces the likelihood of the antigen-antibody fragment triggering an undesirable immune response, and therefore antibodies without the Fc may be preferentially used for prophylactic or therapeutic treatment. As described above, antibodies can also be constructed as chimeric or partially or fully human to reduce or eliminate adverse immunological consequences resulting from the administration of antibodies produced in other species or having sequences from other species to animals.

[0170] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein and can be programmed to modulate one or more properties of the polypeptide, with or without loss of other functions or properties. Substitutions can be conservative, meaning one amino acid is replaced by an amino acid with a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, the substitution can be non-conservative, thereby affecting the function or activity of the polypeptide. Non-conservative changes typically involve substituting residues with chemically different residues, such as replacing a polar or charged amino acid with a nonpolar or uncharged amino acid, or vice versa.

[0171] The protein can be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also contemplated that bacteria containing such variants can be incorporated into the composition and method. Therefore, protein isolation is not required.

[0172] Consideration has been given to the presence of approximately 0.001 mg to approximately 10 mg of total polypeptides, peptides, and / or proteins per ml in the composition. Therefore, the protein concentration in the composition can be approximately, at least approximately, or at most approximately 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derived therefrom). Among these, approximately, at least approximately, or at most approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% can be antibodies that bind to CD79b.

[0173] Antibodies, or preferably the immunological portion of antibodies, may be chemically conjugated to other proteins or expressed as fusion proteins with other proteins. For the purposes of this specification and the appended claims, all such fused proteins are included in the definition of antibodies or the immunological portion of antibodies.

[0174] The implementation provides antibodies and antibody-like molecules, peptides, and polypeptides targeting CD79b, which are linked to at least one reagent to form antibody conjugates or payloads. To enhance the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link, covalently bind, or conjugate at least one desired molecule or moiety. Such molecules or moiety can be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules having the desired activity (e.g., cytotoxic activity). Non-limiting examples of effector molecules attached to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, etc. Conversely, reporter molecules are defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabeled molecules, haptens, fluorescently labeled molecules, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands (e.g., biotin).

[0175] Several methods for attaching or conjugating antibodies to their conjugate portions are known in the art. Some attachment methods involve the use of metal chelate complexes, employing, for example, organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3,6-diphenylglyuron-3, to attach the antibody. Monoclonal antibodies can also be reacted with enzymes in the presence of conjugating agents such as glutaraldehyde or periodate. Conjugates with fluorescein labels are prepared in the presence of these conjugating agents or by reaction with isothiocyanates.

[0176] IV. Cell Therapy

[0177] Some embodiments of this disclosure involve obtaining cells and administering the cells to a subject (as an immunotherapy) to target cancer cells. The cells may deliver antibody compositions covered herein, but they may or may not be immune cells. In particular embodiments, the cells are immune cells. Examples of cells include T cells (including αβT cells or γδT cells), natural killer (NK) cells, invariant NKT (iNKT) cells, B cells, macrophages, any type of stem cell (including MSCs or induced pluripotent stem cells), or dendritic cells.

[0178] Several fundamental approaches for the derivation, activation, and expansion of functional anti-tumor effector T cells have been described over the past two decades. These include: autologous cells, such as tumor-infiltrating lymphocytes (TILs); in vitro activated T cells, obtained by using autologous dendritic cells (DCs), lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T-cell ligands and activating antibodies, or by relying on the capture of target cell membranes; allogeneic cells that naturally express anti-host tumor T-cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells that have been genetically reprogrammed or “modified” to express tumor-reactive TCRs or chimeric TCR molecules exhibiting antibody-like tumor recognition capabilities, known as “T-bodies.” These approaches have led to numerous protocols for T-cell preparation and immunization that can be incorporated into the methods described in this disclosure.

[0179] AT cell preparation

[0180] In some embodiments, the T cells are derived from blood, bone marrow, lymph, or lymphatic organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as those directly isolated from a subject and / or isolated from a subject and frozen. In some embodiments, the cells comprise one or more T cell subclasses or other cell types, such as the entire T cell population, CD4+, etc. + Cells, CD8 + Cells and their subpopulations, for example, those defined by the following aspects: function, activation state, maturity, potential for differentiation, expansion, recycling, localization, and / or persistence, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, biomarker or cytokine secretion profile, and / or degree of differentiation. Regarding the subject to be treated, the cells may be allogeneic and / or autologous. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and / or specifically pluripotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from the subject, preparing, processing, culturing, and / or modifying them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0181] In T cell subtypes and subsets (e.g., CD4) + and / or CD8 + T cells include naive T cells (T2). N ) cells, effector T cells (T cells) EFF ), memory T cells and their subtypes, such as stem cell memory T cells (TSCs). M ), central memory T cells (T CM ), effector memory T cells (T EM) or terminal differentiation effect memory T cells (T TEMRA Tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells.

[0182] In some embodiments, one or more cells in the T cell population are enriched or depleted that are positive for a specific marker (e.g., a surface marker) or negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels in some T cell populations (e.g., non-memory cells), but present or expressed at relatively higher levels in some other T cell populations (e.g., memory cells). In one embodiment, the cells (e.g., CD8+) are enriched or depleted. + Cells or CD3 + Cells are enriched (i.e., positively selected) for CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, or expressing high surface levels of these cells, and / or depleted (e.g., negatively selected) for CD45RA, or expressing high surface levels of CD45RA. In some embodiments, the cells are enriched or depleted for CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127), or expressing high surface levels of these cells. In some examples, CD8+ cells are enriched or depleted for CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). + T cells are enriched in cells that are negative for CD45RO (or CD45RA) and positive for CD62L.

[0183] In some implementations, T cells are separated from PBMC samples by negative selection of markers (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes, or other leukocytes). In some aspects, CD4 is used. + or CD8 + Choose the steps to separate CD4 + Helper T cells and CD8 + Cytotoxic T cells. These CD4+ cells can be classified by positive or negative selection for markers expressed or expressed at relatively high levels on one or more naive, memory, and / or effector T cell subsets.+ and CD8 + The group was further subdivided into subgroups.

[0184] In some implementations, CD8 is made + Cells are further enriched or depleted of naive, central memory, effector memory, and / or central memory stem cells, for example, through positive or negative selection based on surface antigens associated with their respective subsets. In some embodiments, selection is performed on central memory T(T) cells. CM Enrichment of cells to increase efficacy, such as to improve long-term survival, expansion, and / or translocation after application, is particularly robust in some respects in this subpopulation. In some embodiments, the cells will be enriched via T... CM Enriched CD8 + T cells and CD4 + The combination of T-cells further enhances efficacy.

[0185] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from the patient, and a single-cell suspension is obtained. The single-cell suspension can be obtained in any suitable manner, such as mechanically (by depolymerizing the tumor, for example using gentleMACS). TM Dissociator, Miltenyi Biotec, Auburn, Calif., or enzymatically (e.g., collagenase or DNase). A single-cell suspension of the tumor enzymatic digest is cultured in interleukin-2 (IL-2). The cells are cultured until confluence (e.g., approximately 2 × 10⁻⁶). 6 (e.g., about 5 to about 21 days, preferably about 10 to about 14 days). For example, the cells can be cultured for 5, 5.5 or 5.8 days to 21, 21.5 or 21.8 days, such as 10, 10.5 or 10.8 days to 14, 14.5 or 14.8 days.

[0186] Cultured T cells can be pooled and rapidly expanded. Rapid expansion provides at least a 50-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or greater) increase in the number of antigen-specific T cells over a period of approximately 10 to approximately 14 days (preferably approximately 14 days). More preferably, rapid expansion provides at least a 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900, or greater) increase over a period of approximately 10 to approximately 14 days (preferably approximately 14 days).

[0187] Expansion can be accomplished by any of the many methods known in the art. For example, T cells can be rapidly expanded by using nonspecific T-cell receptor stimulation in the presence of fed lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15) (wherein IL-2 is preferred). The nonspecific T-cell receptor stimulation may comprise approximately 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (from Ortho- Raritan, NJ (available). Alternatively, T cells can be rapidly expanded by stimulating peripheral blood mononuclear cells (PBMCs) in vitro with one or more antigens of the cancer (including its antigenic portions, such as epitopes or cells) (which may optionally be expressed from a vector) (e.g., human leukocyte antigen A2 (HLA-A2) binding peptide) in the presence of T-cell growth factors such as 300 IU / ml IL-2 or IL-15 (wherein IL-2 is preferred). The in vitro induced T cells are then rapidly expanded by restimulation with the same antigen of the cancer pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, the T cells can be restimulated, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0188] The autologous T cells can be modified to express T-cell growth factors that promote the growth and activation of the autologous T cells. Suitable T-cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable modification methods are known in the art, see, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In particular, the modified T cells express T-cell growth factors at high levels. T-cell growth factor coding sequences (e.g., the coding sequence for IL-12) are readily available in the art, and operatively linked to T-cell growth factor coding sequences, as with promoters, promote high-level expression.

[0189] B. Genetically modified antigen receptors

[0190] The cells can be genetically modified to express a modified antigen receptor, such as a modified TCR or a chimeric antigen receptor (CAR). For example, autologous T cells can be modified to express a T-cell receptor (TCR) with antigen specificity for a cancer antigen (e.g., CD79b). Suitable TCRs include, for example, those with antigen specificity for melanoma antigens (e.g., gp100 or MART-1). Suitable modification methods are known in the art. See, for example, Sambrook and Ausubel, above. For example, the T cells can be transduced to express a TCR with antigen specificity for a cancer antigen using the transduction techniques described in Heemskerk et al., Hum Gene Ther. 19:496-510 (2008) and Johnson et al., Blood 114:535-46 (2009).

[0191] In some embodiments, the T cell comprises one or more genetically engineered nucleic acids encoding one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or a sample obtained from the cell, such as from another organism or cell, and is not typically found in the cell being engineered and / or the organism from which such cells are derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).

[0192] In some embodiments, the CAR includes an extracellular antigen recognition domain that specifically binds to CD79b. In some embodiments, the antigen is a protein expressed on the cell surface. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of major histocompatibility complex (MHC) molecules.

[0193] Exemplary antigen receptors (including CARs and recombinant TCRs) and methods for modifying said receptors and introducing said receptors into cells, including, for example, those described in International Patent Application Publications Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publications Nos. US2002131960, US2013287748, US20130149337, and U.S. Patent No. 6,450. 1,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and those described in European Patent Application No. EP2537416; and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some aspects, the genetically modified antigen receptor includes the CAR described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668 A1.

[0194] 1. Chimeric antigen receptor

[0195] In some embodiments, the CAR includes: a) an intracellular signal transduction domain; b) a transmembrane domain; and c) an extracellular domain containing an antigen-binding region.

[0196] In some embodiments, the modified antigen receptor comprises a CAR, including activating or stimulatory CARs, co-stimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). The CAR typically contains an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some respects via a linker and / or a transmembrane domain. Such molecules typically mimic or approximate signaling via natural antigen receptors, signaling via such receptors and co-stimulatory receptors in combination therewith, and / or signaling via individual co-stimulatory receptors.

[0197] Some embodiments of this disclosure relate to the use of nucleic acids, including nucleic acids encoding polypeptides such as antigen-specific CAR polypeptides, including humanized CARs (hCARs) to reduce immunogenicity, which comprise intracellular signal transduction domains, transmembrane domains, and extracellular domains (containing one or more signal transduction motifs). In some embodiments, the CAR can recognize an epitope contained in a shared space between one or more antigens. In some embodiments, the binding region may comprise a complementarity-determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, that specificity derives from a peptide (e.g., a cytokine) that binds to a receptor.

[0198] The human CAR nucleic acid is considered to be a human gene for enhancing cell immunotherapy for human patients. In a particular embodiment, this disclosure includes a full-length CAR cDNA or coding region. The antigen-binding region or domain may comprise a V-shaped variable fragment (scFv) derived from a specific human monoclonal antibody (e.g., those described in U.S. Patent 7,109,304, which is incorporated herein by reference). H and V L The fragment is a segment of the chain. The fragment can also be any number of different antigen-binding domains of human antigen-specific antibodies. In a more particular embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage in order to be expressed in human cells.

[0199] The arrangement can be multimeric, such as a double-chain antibody or a multimer. The multimer is most likely formed by cross-pairing variable portions of the light and heavy chains into a double-chain antibody. The hinge portion of the construct can have multiple options, from complete deletion to retaining the first cysteine, to proline instead of serine substitution, to truncation down to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerized protein can be used for this purpose. Only one of the Fc domains can be used, such as the CH2 or CH3 domains from human immunoglobulins. The hinge, CH2, and CH3 regions of human immunoglobulins modified to improve dimerization can also be used. Only the hinge portion of the immunoglobulin can be used. A portion of CD8α can also be used.

[0200] In some embodiments, the CAR nucleic acid includes sequences encoding other co-stimulatory receptors, such as transmembrane domains and modified CD28 intracellular signaling domains. Other co-stimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), and 4-1BB (CD137).

[0201] In some embodiments, a CAR is constructed that is specific for a particular antigen (or biomarker or ligand), such as an antigen expressed in a specific cell type to be targeted by adoptive therapy, such as a cancer biomarker, and / or an antigen intended to induce a mitigating response, such as an antigen expressed in normal or disease-free cell types. Therefore, the CAR typically contains one or more antigen-binding molecules in its extracellular portion, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR contains the antigen-binding portion of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0202] The sequence encoding the open reading frame of the chimeric receptor can be obtained from genomic DNA, cDNA, or can be synthetic (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns are found to stabilize the mRNA. Furthermore, it may be even more advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0203] It is considered that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods for stably transfecting cells using naked DNA via electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA that encodes a chimeric receptor contained in a plasmid expression vector with appropriate orientation for expression.

[0204] Alternatively, a viral vector (e.g., a retroviral vector, adenovirus vector, adeno-associated virus vector, or lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors used in accordance with the methods of this disclosure are non-replicative in the immune cells. A large number of virus-based vectors are known in which the viral copy number in the cells is maintained sufficiently low to preserve the viability of the cells, such as vectors based on HIV, SV40, EBV, HSV, or BPV.

[0205] In some aspects, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to an extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0206] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., transmembrane regions containing at least) the following molecules: the α, β, or ζ chain of the T-cell receptor, CD28, CD3ζ, CD3ε, CD3γ, and CD3δ. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain primarily comprises hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0207] In a particular embodiment, the CAR construct of the present invention comprises a light chain-connector-heavy chain-hinge-transmembrane structural domain-signal transduction structural domain. The connector may comprise, consist of, or substantially consist of the following: connector 1 (SEQ ID NO: 44 or 45), connector 2 (SEQ ID NO: 46 or 47), connector 3 (SEQ ID NO: 48 or 49), or connector 4 (SEQ ID NO: 50 or 51). The hinge may comprise, consist of, or substantially consist of the following: CD8 hinge 1 (SEQ ID NO: 52 or 53), CD8 hinge 2 (SEQ ID NO: 54 or 55), CD8 hinge 3 (SEQ ID NO: 56 or 57), CD28 hinge (SEQ ID NO: 58 or 59), IgG4 hinge (SEQ ID NO: 60 or 61), IgG4 CH2 (SEQ ID NO: 62 or 63), IgG4 CH2CH3 (SEQ ID NO: 64 or 65), or IgG4CH1CH2CH3 (SEQ ID NO: 66 or 67). The transmembrane domain may comprise, consist of, or substantially consist of the following: CD8TM1 (SEQ ID NO: 68 or 69), CD8TM2 (SEQ ID NO: 70 or 71), CD28TM (SEQ ID NO: 72 or 73), or CD8αTM (SEQ ID NO: 87). In special cases, CD8αTM with an amino acid sequence lacking LYC and / or NHRN (including successive sequences) (e.g., at its C-terminus) is used. The signal transduction domain may comprise, consist of, or substantially comprise the following: CD28 (SEQ ID NO: 74 or 75), 4-1BB (SEQ ID NO: 76 or 77), OX-40 (SEQ ID NO: 78 or 79), and / or intracellular CD3 (SEQ ID NO: 80 or 81). The CAR construct may further comprise, consist of, or substantially comprise the following: GFP (SEQ ID NO: 82 or 83), T2A (SEQ ID NO: 84 or 85), and / or EGFR (SEQ ID NO: 40 or 41). Exemplary combinations of heavy chains (HC), linkers, and light chains (LC) may include, but are not limited to: LC-linker 1-HC; LC-linker 2-HC; LC-linker 3-HC; LC-linker 4-HC; HC-linker 1-LC; HC-linker 2-LC; HC-linker 3-LC; or HC-linker 4-LC.

[0208] 2. T cell receptor (TCR)

[0209] In some embodiments, the genetically modified antigen receptor comprises a recombinant TCR and / or a TCR cloned from naturally occurring T cells. A “T cell receptor” or “TCR” refers to a molecule containing variable α and β chains (also referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (also referred to as TCRγ and TCRδ, respectively) and capable of specifically binding to an antigen that binds to an MHC receptor. In some embodiments, the TCR is in αβ form.

[0210] Typically, TCRs existing in αβ and γδ forms are structurally similar, but T cells expressing them can have different anatomical locations or functions. TCRs can be found on the cell surface or in a soluble form. Typically, TCRs are found on the surface of T cells (or T lymphocytes), where they are usually responsible for recognizing antigens that bind to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et al., 1997). For example, in some aspects, each chain of a TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs are associated with the invariant protein of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term “TCR” should be understood to encompass its functional TCR fragments. The term also encompasses complete or full-length TCRs, including TCRs in αβ or γδ form.

[0211] Therefore, for the purposes of this document, reference to TCR includes any TCR or functional fragment, such as the antigen-binding moiety of a TCR, that binds to a specific antigenic peptide (i.e., an MHC-peptide complex) bound to an MHC molecule. The term "antigen-binding moiety" or "antigen-binding fragment" of a TCR (these terms are used interchangeably) refers to a molecule that contains a portion of the structural domains of a TCR but binds to the antigen (e.g., an MHC-peptide complex) that the full TCR binds to. In some cases, the antigen-binding moiety contains variable domains of the TCR, such as variable α-chains and variable β-chains of the TCR, sufficient to form binding sites for binding to a specific MHC-peptide complex, typically where each chain contains three complementarity-determining regions.

[0212] In some embodiments, the variable domains of the TCR chain associate to form loops or complementarity-determining regions (CDRs) similar to those of immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the major CDR responsible for recognizing the processed antigen, although CDR1 of the α chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β chain may include a further highly variable (HV4) region.

[0213] In some embodiments, the TCR chain comprises constant domains. For example, like immunoglobulins, the extracellular portion of the TCR chain (e.g., α-chain, β-chain) may contain two immunoglobulin domains and a variable domain at the N-terminus (e.g., V...). a Or Vp; typically, based on amino acid 1 to 116 numbered by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," US Dept. Health and Human Services, Public Health Service, National Institutes of Health, 1991, 5th edition), and a constant domain adjacent to the cell membrane (e.g., α-chain constant domain or C). a Typically, the β-chain constant domain or Cp is based on Kabat amino acids 117 to 259; typically, it is based on Kabat amino acids 117 to 295. For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two proximal membrane constant domains and two distal membrane variable domains containing CDRs. The constant domains of the TCR contain short linker sequences in which cysteine ​​residues form disulfide bonds, thereby constituting the link between the two chains. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α and β chains, so that the TCR contains two disulfide bonds in the constant domain.

[0214] In some embodiments, the TCR chain may include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail region. In some cases, the structure allows the TCR to associate with other molecules such as CD3. For example, a TCR containing a constant domain with a transmembrane region can anchor to proteins in the cell membrane and associate with invariant subunits of CD3 signaling transducers or complexes.

[0215] Typically, CD3 is a multi-protein complex that can have three distinct chains (γ, δ, and ε) (in mammals) and a ζ-chain. For example, in mammals, the complex can consist of a homodimer of one CD3γ chain, one CD3δ chain, two CD3ε chains, and a CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly associated cell surface proteins of the immunoglobulin superfamily, each containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to associate with positively charged T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif (called an immunoreceptor tyrosine-based activation motif, or ITAM), while each CD3ζ chain has three. Typically, ITAMs are involved in the signal transduction capabilities of the TCR complex. These associated molecules have negatively charged transmembrane regions and play a role in transmitting signals from the TCR into the cell. The CD3- and ζ- chains together with the TCR form the so-called T-cell receptor complex.

[0216] In some embodiments, the TCR may be a heterodimer of two strands, α and β (or optionally γ and δ), or it may be a single-stranded TCR construct. In some embodiments, the TCR is a heterodimer comprising two linked (e.g., by disulfide bonds) separate strands (α and β chains or γ and δ chains). In some embodiments, a TCR is identified with respect to a target antigen (e.g., a cancer antigen) and introduced into cells. In some embodiments, the nucleic acid encoding the TCR may be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as cells like T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, the T cells may be cells isolated from within the body. In some embodiments, high-affinity T cell clones may be isolated from a patient, and the TCR may be isolated. In some embodiments, the T cells may be cultured T cell hybridomas or clones. In some embodiments, a TCR clone targeting the target antigen is generated in a transgenic mouse modified with human immune system genes (e.g., the human leukocyte antigen system or HLA). In some embodiments, phage display is used to isolate the TCR targeting the target antigen. In some embodiments, the TCR or its antigen-binding portion can be generated synthetically from knowledge of the TCR's sequence.

[0217] C. Delivery method

[0218] Those skilled in the art will be able to construct vectors for the expression of any antigen receptor of this disclosure using standard recombination techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference). Vectors include, but are not limited to: plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses) and artificial chromosomes (e.g., YAC), such as retroviral vectors (e.g., derived from Moloney mouse leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors (including their replicative, replication-defective, and content-free forms), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey mouse sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maraba virus vectors, and group B adenovirus enadenotucirev vectors.

[0219] 1. Viral vector

[0220] In certain aspects of this disclosure, viral vectors encoding antigen receptors may be provided. In the generation of recombinant viral vectors, non-essential genes are typically replaced by genes or coding sequences concerning foreign (or non-natural) proteins. Viral vectors are a class of expression constructs that utilize viral sequences to introduce nucleic acids and, possibly proteins, into cells. The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis and integrate into the host cell genome, and to stably and efficiently express viral genes, makes them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids from certain aspects of this disclosure are described below.

[0221] Lentivirals are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, for example, U.S. Patents 6,013,516 and 5,994,136).

[0222] Recombinant lentiviruses can infect non-dividing cells and can be used for in vivo and in vitro gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells—in which suitable host cells are transfected with two or more vectors carrying packaging functions, namely gag, pol, and env, and rev and tat—are described in U.S. Patent 5,994,136 (which is incorporated herein by reference).

[0223] 2. Control element

[0224] The expression cassettes contained in vectors useful in this disclosure specifically include (in a 5' to -3' orientation) eukaryotic transcription promoters, splicing signals (including intercalation sequences), and transcription termination / polyadenylation sequences operatively linked to protein-coding sequences. In eukaryotic cells, promoters and enhancers controlling transcription of protein-coding genes consist of multiple genetic elements. Cellular machinery can collect and integrate the regulatory information conveyed by each element, thereby allowing different genes to develop different, often complex, transcriptional regulatory patterns. Promoters used in the context of this disclosure include constitutive, inducible, and tissue-specific promoters.

[0225] a. Promoters / enhancers

[0226] The expression constructs presented herein contain promoters to drive the expression of antigen receptors. Promoters typically contain sequences that function to place the initiation site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters lacking the TATA box, such as those for mammalian terminal deoxynucleotidyl transferase genes and SV40 late-stage genes, discrete elements superimposed on the initiation site itself help to fix the initiation position. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in a region 30-110 bp upstream of the initiation site, although many promoters have been shown to also contain functional elements downstream of the initiation site. To bring the coding sequence under the “control” of the promoter, the 5′ end of the transcription start site of the transcription reading frame is placed “downstream” (i.e., 3′) of the selected promoter. The “upstream” promoter stimulates transcription of the DNA and promotes the expression of the encoded RNA.

[0227] The spacing between promoter elements is often flexible, thus preserving promoter function when an element is inverted or moved relative to another element. In the tk promoter, the spacing between promoter elements can increase to 50 bp, after which activity begins to decline. Depending on the promoter, it appears that individual elements can function both cooperatively and independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which are cis-regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0228] Promoters can be promoters naturally associated with a nucleic acid sequence, such as those obtained by isolating a 5′-noncoding sequence located upstream of a coding region and / or exon. Such promoters can be called “endogenous.” Similarly, enhancers can be enhancers naturally associated with a nucleic acid sequence, located downstream or upstream of that sequence. Alternatively, certain advantages may be gained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter (which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment). Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with a nucleic acid sequence in their natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not “naturally occurring,” i.e., those containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, the most frequently used promoters in recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to generating promoter and enhancer nucleic acid sequences synthetically, the compositions disclosed herein can also be used by recombinant cloning and / or nucleic acid amplification techniques (including PCR). TMSequences can be generated using [a specific method / mechanism]. Furthermore, it has been considered that control sequences could also be used to guide the transcription and / or expression of sequences within non-nuclear organelles (e.g., mitochondria, chloroplasts, etc.).

[0229] Naturally, it will be important to employ promoters and / or enhancers that effectively direct the expression of DNA segments in the organelles, cell types, tissues, organs, or organisms selected for expression. Those skilled in the art of molecular biology are generally familiar with the use of combinations of promoters, enhancers, and cell types for protein expression (see, for example, Sambrook et al., 1989, which are incorporated herein by reference). The promoters employed can be constitutive, tissue-specific, inducible, and / or, under suitable conditions, useful for directing high-level expression of the introduced DNA segment, for example, advantageous in the large-scale production of recombinant proteins and / or peptides. The promoters can be heterologous or endogenous.

[0230] Alternatively, expression can be driven using any promoter / enhancer combination (according to, for example, the eukaryotic promoter database EPDB, available at epd.isb-sib.ch / via the World Wide Web). The use of T3, T7, or SP6 cytoplasmic expression systems is another possible implementation. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters, provided a suitable bacterial polymerase is available, either as part of a delivery complex or as an additional genetic expression construct.

[0231] Non-limiting examples of promoters include: early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus (RSV) early promoter; eukaryotic cell promoters, such as the β-actin promoter, the GADPH promoter, and the metallothionein promoter; and cascaded response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the response element promoter (tre) near the minimal TATA box. It is also possible to use human growth hormone promoter sequences (e.g., the minimal human growth hormone promoter described in Genbank, accession number X05244, nucleotides 283-341) or mouse mammary tumor promoters (available from ATCC, catalog number ATCC 45007). In some embodiments, the promoter is CMV IE, dectin-1, dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, β-actin, a class I MHC or class II MHC promoter, but any other promoter useful for driving the expression of therapeutic genes may also be applicable to the practice of this disclosure.

[0232] In some respects, the methods of this disclosure also involve enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have the potential to function cis- and regardless of orientation, even at relatively long distances (up to several thousand bases away from the target promoter). However, enhancer function is not necessarily limited to such long distances, as they can also function very close to a given promoter.

[0233] b. Initial signals and linkage expressions

[0234] Specific start signals may also be used in the expression constructs provided in this disclosure for efficient translation of the coding sequence. These signals include the ATG start codon or a neighboring sequence. Exogenous translation control signals, including the ATG start codon, may need to be provided. Those skilled in the art will readily be able to determine this and provide the necessary signals. It is well known that the start codon must "frame-match" with the desired coding sequence's reading frame to ensure translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be enhanced by including suitable transcriptional enhancer elements.

[0235] In some implementations, internal ribosome entry site (IRES) elements are used to create multi-gene or polycistronic information. IRES elements bypass the cap-dependent ribosome scanning pattern of 5' methylation translation and initiate translation at an internal site. IRES elements from two members of the small RNA virus family (poliovirus and encephalomyovirus), as well as IRES from mammalian information, have been described. IRES elements can be linked to heterologous open reading frames (OPFs). Multiple OPFs can be transcribed together, each separated by an IRES, thus producing polycistronic information. With IRES elements, each OPF is accessible to the ribosome for efficient translation. Multiple genes can be efficiently expressed by transcribing a single message using a single promoter / enhancer.

[0236] Additionally, certain 2A sequence elements can be used to generate synergistic or co-expression of genes within the constructs provided in this disclosure. For example, a cleavage sequence can be used to co-express genes by connecting open reading frames to form a single cistron. An exemplary cleavage sequence is F2A (foot-and-mouth disease virus 2A) or a “2A-like” sequence (e.g., Thoseea asigna virus 2A; T2A).

[0237] 3. Copy the starting point

[0238] To propagate the vector in host cells, it may contain one or more origin of replication sites (commonly referred to as "ori"), for example, a nucleic acid sequence corresponding to the oriP of EBV described above, or a genetically modified oriP with similar or enhanced programmed function, which is the specific nucleic acid sequence at which replication begins. Alternatively, origin of replication or autonomous replication sequence (ARS) of other extrachromosomal replicating viruses described above may be used.

[0239] 4. Selection and Filterable Tags

[0240] In some embodiments, cells containing constructs of this disclosure can be identified in vitro or in vivo by including markers in the expression vector. Such markers confer identifiable variations on the cells, thereby allowing easy identification of cells containing the expression vector. Typically, a selection marker is a marker that imparts a property that allows selection. A positive selection marker is one whose presence allows selection, while a negative selection marker is one whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0241] Typically, drug-selective markers are helpful for the cloning and identification of transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histamine are useful selectable markers. In addition to markers that allow for condition-based differentiation of transformant phenotypes, other types of markers are considered, including selectable markers such as GFP, which are based on colorimetric analysis. Alternatively, selectable enzymes, such as herpes simplex virus thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT), can be used as negative selectable markers. Those skilled in the art will also know how to employ immunomarkers, possibly in conjunction with FACS analysis. The marker used is not considered important as long as it can be co-expressed with the nucleic acid encoding the gene product. Further examples of selectable and selectable markers are well known to those skilled in the art.

[0242] 5. Other methods for nucleic acid delivery

[0243] In addition to viral delivery of nucleic acids encoding the antigen receptor, the following are other methods of delivering recombinant genes to a given host cell and are therefore considered in this disclosure.

[0244] The introduction of nucleic acids, such as DNA or RNA, into the immune cells of this disclosure can be achieved using any suitable method for nucleic acid delivery to transform cells, which is described herein or will be known to those skilled in the art. Such methods include, but are not limited to: direct delivery of DNA, such as by in vitro transfection, by injection (including microinjection); by electroporation; by calcium phosphate precipitation; by the use of DEAE-glucan followed by polyethylene glycol; by direct sonic loading; by liposome-mediated and receptor-mediated transfection; by microparticle bombardment; by shaking with silicon carbide fibers; by Agrobacterium-mediated transformation; by drying / inhibition-mediated DNA uptake; and any combination of such methods. By applying techniques such as these, organelles, cells, tissues, or organisms can be transformed stably or transiently.

[0245] V. Treatment Methods

[0246] Certain aspects of embodiments of the present invention can be used to prevent or treat diseases or conditions associated with CD79b signaling, including those in which the killing of CD79b-positive cells would improve at least one symptom of said disease or condition. CD79b signaling can be reduced by any suitable composition used to prevent the proliferation of cancer cells. In particular, such a substance would be an anti-CD79b antibody or cells expressing anti-CD79b CAR.

[0247] In some embodiments, this disclosure provides methods for immunotherapy comprising administering an effective amount of a composition containing an antibody (at least including CAR T cells) of this disclosure. In one embodiment, a medical disease or condition is treated by administering a population of CAR-expressing cells that elicit an immune response. In some embodiments of this disclosure, cancer is treated by administering a population of CAR-expressing immune cells that elicit an immune response. Methods for treating cancer or delaying cancer progression in an individual are provided herein, comprising administering an effective amount of antigen-specific cell therapy to the individual. This method can be applied to treat, for example, immune disorders, solid tumors, and hematologic cancers. In particular, the cancer can be a B-cell malignancy, such as B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia.

[0248] Tumors for which this treatment method is useful include any malignant cell type, such as those found in solid tumors or hematologic malignancies. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of: pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematologic malignancies include tumors of the bone marrow, T- or B-cell malignancies, leukemia, lymphoma, germ cell tumor, myeloma, etc. Further examples of cancers that can be treated using the methods presented herein include, but are not limited to: lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, stomach or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0249] The cancer may specifically be, but is not limited to, the following histological types: growths, malignant; carcinoma; undifferentiated carcinoma; giant and fusiform cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilonidal stromal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous polypoid adenocarcinoma; adenocarcinoma, familial adenomatous polyposis; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic cell carcinoma; eosinophilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granulocytic carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-capsulated Sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Skin adnexal carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous gland carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma w / squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant; Granulosarcoma, malignant; Androcytoma, malignant; Setori cell carcinoma; Ledich cell tumor, malignant; Lipiocytoma, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Hemangiosarcoma; Malignant melanoma Tumors; amelanoma; superficial diffuse melanoma; malignant lentigines melanoma; acral lentigines melanoma; nodular melanoma; malignant melanoma within a giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; small vesicular rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Miller mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner's tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroscleroma, Malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; subcortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primary neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma;Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granulocytoma, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's disease; Granulomatoid; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specialized non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate / follicular NHL; Intermediate diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cleaved cell NHL; Massive Diseases include: NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0250] Some implementation schemes involve methods for treating leukemia. Leukemia is a cancer of the blood or bone marrow and is characterized by the abnormal proliferation (through doubling) of blood cells (usually white blood cells). It is part of a large group of diseases known as hematologic vegetations. Leukemia is a broad term encompassing a range of diseases. Leukemia is clinically and pathologically classified into its acute and chronic forms.

[0251] In some embodiments of the methods disclosed herein, activated CD4 and / or CD8 T cells in an individual are characterized by CD4 and / or CD8 T cells producing γ-IFN and / or enhanced cytolytic activity relative to prior to the administration of the combination. γ-IFN can be measured by any means known in the art, including, for example, intracellular cytokine staining (ICS), which involves cell fixation, permeabilization, and staining with an antibody against γ-IFN. Cytolytic activity can be measured by any means known in the art, such as using a cell-killing assay with a mixture of effector cells and target cells.

[0252] In some implementations, the subject may be given non-myeloablative lymphocyte depletion chemotherapy prior to T-cell therapy. The non-myeloablative lymphocyte depletion chemotherapy can be any suitable such therapy, which can be administered via any suitable route. The non-myeloablative lymphocyte depletion chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which may be metastatic. An exemplary route of administration of cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine can be administered. In a particular aspect, approximately 60 mg / kg of cyclophosphamide is administered for two days, followed by approximately 25 mg / kg of cyclophosphamide. 2 Fludarabine for five days.

[0253] In some embodiments, a T-cell growth factor that promotes the growth and activation of the autologous T cells is administered to the subject, either concurrently with or after the autologous T cells. The T-cell growth factor can be any suitable growth factor that promotes the growth and activation of the autologous T cells. Examples of suitable T-cell growth factors include interleukin (IL)-2, IL-7, IL-15, and / or IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2. IL-12 is a particular T-cell growth factor.

[0254] Therapeutic amounts of immune cells can be administered via a variety of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intra-articular injection or infusion.

[0255] For dispersed, solid, and accessible tumors, intratumoral injection or injection into the tumor vascular system should be considered. Local, regional, or systemic administration may also be appropriate. For tumors >4 cm, the volume to be administered will be approximately 4–10 ml (especially 10 ml), while for tumors <4 cm, a volume of approximately 1–3 ml (especially 3 ml) will be used. Multiple injections delivered as a single dose contain a volume of approximately 0.1 to approximately 0.5 ml.

[0256] The T-cell population can be administered according to a treatment regimen consistent with the disease, such as single or several doses over one to several days to improve the disease status, or regular doses over an extended period to suppress disease progression and prevent relapse. The precise dosage used in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be determined based on the physician's judgment and the individual patient's situation. The therapeutically effective dose of T cells will depend on the subject undergoing treatment, the severity and type of the disease, and the method of administration. In some embodiments, the dose that can be used in the treatment of human subjects starts from at least 3.8 × 10⁻⁶. 4 At least 3.8 × 10 5 At least 3.8 × 10 6 At least 3.8 × 10 7 At least 3.8 × 10 8 At least 3.8 × 10 9 Or at least 3.8 × 10 10 T cells / m 2 Variations are made. In some implementations, the dose used in the treatment of human subjects varies from approximately 3.8 × 10⁻⁶. 9 Up to approximately 3.8 × 10 10 T cells / m 2 The dosage can be modified. In another implementation, the therapeutically effective dose of T cells can be increased from approximately 5 × 10⁻⁶. 6 Cells / kg body weight up to approximately 7.5 × 10⁻⁶ 8 Cells / kg body weight, for example, approximately 2 × 10⁻⁶ 7 1 cell to approximately 5 × 10 8 Cells / kg body weight, or approximately 5 × 10 7 1 cell to approximately 2 × 10 8 The number of T cells varies per kg of body weight. The accurate number of T cells can be readily determined by those skilled in the art based on the subject's age, weight, sex, and physiological condition. The effective amount can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0257] In some embodiments of this disclosure, an effective amount of immune cells expressing CD79b CAR is delivered to an individual in need, such as an individual with cancer. The cells then enhance the individual's immune system to attack the cancer cells. In some cases, the individual is provided with one or more doses of the immune cells. In cases where the individual is provided with two or more doses of the immune cells, the duration between administrations should be sufficient to allow time for proliferation within the individual, and in particular embodiments, the duration between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0258] In a particular implementation, cells modified to express CD79b CAR are provided to an individual to improve at least one therapeutically effective dose (in 10) of cancer cell-related symptoms in that individual. 3 Up to 10 10 (within the range). The effective therapeutic dose can be 10. 3 Up to 10 10 10 3 Up to 10 9 10 3 Up to 10 8 10 3 Up to 10 7 10 3 Up to 10 6 10 3 Up to 10 5 10 3 Up to 10 4 10 4 Up to 10 10 10 4 Up to 10 9 10 4 Up to 10 8 10 4 Up to 10 7 10 4 Up to 10 6 10 4 Up to 10 5 10 5 Up to 10 10 10 5 Up to 10 9 10 5 Up to 10 8 10 5 Up to 10 7 10 5 Up to 10 6 10 6 Up to 10 10 10 6 Up to 10 9 10 6 Up to 10 8 10 6 Up to 10 7 10 7 Up to 10 10 10 7 Up to 10 9 10 7 Up to 10 8 10 8 Up to 10 10 10 8 Up to 10 9 Or 10 9Up to 10 10 Individual cells. Therefore, in a particular implementation, an individual with a certain type of cancer is provided with a therapeutically effective amount of cells expressing CD79b CAR once or multiple times.

[0259] A. Pharmaceutical Compositions

[0260] Pharmaceutical compositions and formulations comprising CAR-expressing cells and pharmaceutically acceptable carriers are also provided herein.

[0261] The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (e.g., an antibody or peptide) with the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd edition, 2012), in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids). Examples of pharmaceutically acceptable carriers include glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (…). Baxter International, Inc.). In one aspect, sHASEGP is combined with one or more other glycosaminoglycans, such as chondroitinase.

[0262] B. Combination Therapy

[0263] In some embodiments, the compositions and methods of the present invention involve a population of T cells in combination with at least one additional therapy. The additional therapy may be radiotherapy, surgery (e.g., tumor resection and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0264] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-transfer agent. In some embodiments, the additional therapy is the administration of a side-effect limiting agent (e.g., an agent designed to reduce the occurrence and / or severity of treatment side effects, such as an anti-nausea agent). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma radiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0265] Immunotherapy can be administered before, during, after, or in various combinations with other cancer therapies (e.g., immune checkpoint therapy). The administration can be at intervals ranging from simultaneous to minutes to days or weeks. In embodiments where immunotherapy is administered to the patient separately from other therapeutic agents, it is generally ensured that there is no significant time gap between each delivery so that the two compounds will still be able to exert a beneficial combined effect on the patient. In such cases, it is considered that the antibody therapy and anticancer therapy can be administered to the patient within approximately 12 to 24 or 72 hours of each other, and more particularly within approximately 6 to 12 hours of each other. In some cases, it may be desirable to significantly extend the duration of treatment, with intervals from several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) between administrations.

[0266] Various combinations can be used. In the example below, immune cell therapy is "A," while anti-cancer treatment is "B":

[0267] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / BB / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A.

[0268] Administration of any compound or therapy according to embodiments of the present invention to a patient will follow a general protocol for the administration of such compounds, taking into account the toxicity of the agent, if any. Therefore, in some embodiments, there is a step to monitor for toxicity attributable to the combination therapy.

[0269] 1. Chemotherapy

[0270] A wide variety of chemotherapeutic agents can be used according to embodiments of the present invention. Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, indomethacin, and piperazine; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; azacyclopropanes and methylamelamines, including hexamethylmelamine, triethylmelamine, triethylphosphamide, triethylthiophosphamide, and trihydroxymethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including... Synthetic analogs (topotecan); lichenin; callystatin; CC-1065 (including its synthetic analogs adolaxine, calcetin, and pyrazine); cryptophytes (especially cryptophyte 1 and cryptophyte 8); dolastatin; pyruvicin (including synthetic analogs KW-2189 and CB1-TM1); arugula; pancratistatin; sarcodictyin; spongiform septicemia; nitrogen mustards, such as chlorambucil, naphthylmustine, cyclophosphamide, estradiol, ifosfamide, nitrogen mustard, oxynitric acid, melphalan, neonitric acid, benzylmustine, prednimustine, trazophosphatamide, and uracil mustard; nitrosourea Classes such as carmustine, chloramphenicol, formustine, lomustine, nimustine, and ramustine; antibiotics such as enemidyne antibiotics (e.g., calcipomycin, especially calcipomycin γ1I and calcipomycin ωI1); enemidyne anthracycline antibiotics, including enemidyne anthracycline antibiotics A; bisphosphonates such as clophosphonates; esporomycin; and new carcinogen chromophores and related chromogens, enemidyne antibiotic chromophores, aclarubicin, actinomycin, autramycin, diazoserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinogen, chromomycin, actinomycin D, daunorubicin, detoxin, 6-diazo-5- Oxy-L-leucine, doxorubicin (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolinyl doxorubicin and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, maceralomycin, mitomycin C, mycophenolic acid, nogamycin, olivomycin, pepromycin, puromycin, triamcinolone acetonide, rhodorubicin, streptozotocin, streptozotocin, tuberculin, ubenmex, fenestrated statin and zolrubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, pteroxate and trimethyltraxa; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazoline and thioguanine;Pyrimidine analogues, such as ancitabine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fluorouridine; androgens, such as calotestosterone, drotaloferrin propionate, cyclothionol, meandrolone, and testrolide; antiadrenergic drugs, such as mitotane and tralostertan; folic acid supplements, such as folinic acid; aceglucuronolactone; aldehydephosphoramide glycosides; 5-aminolevulinic acid; enuracil; acridine; bestrabucil; Bismuth subsalicylate; Idatraxa; Desphosphonamide; Colchicine; Ditraquinone; Elformithine; Elimethicone; Epomycin; Etoglobulin; Gallium nitrate; Hydroxyurea; Lentinan; Clonidamine; Mestansens, such as Maytansine and Ansomidine; Mitoguanidine hydrazone; Mitoantrone; Mopiperol; Nitratriazine; Pentostatin; Pheanamet; Pirarubicin; Loxoantrone; Podophyllotoxin; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex Compounds; Rezosen; Lisol; Cizonan; Germonospiramine; Alternaria solanilic acid; Triaminoquinone; 2,2′,2″-Trichlorotriethylamine; Trichothecene compounds (especially T-2 toxin, verracurin A, baculosporin A, and serpentin); Urethane; Vinpocetine; Dacarbazine; Mannomustine; Dibromomannitol; Dibromoeugenol; Piperbromobromide; Gacytosine; Arabinoside (“Ara-C”); Cyclohexane Phosphoramides; taxanes, such as paclitaxel and docetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novotoxin; teniposide; edaraxacum; donomycin; aminopterin; capecitabine; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin; procarbazine; purcamycin; gemcitabine; novoben; farnesyl-protein transferase inhibitors; antiplatinum; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0271] 2. Radiation therapy

[0272] Other factors that cause DNA damage and have been widely used include the targeted delivery of gamma rays, X-rays, and / or radioactive isotopes to tumor cells, commonly referred to as gamma rays, X-rays, and / or radioactive isotopes. Other forms of DNA damage have also been considered, such as microwaves, proton beam radiation, and UV radiation. Most likely, all of these factors cause a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dosage ranges for X-rays vary widely, from daily doses of 50 to 200 roentgens over extended periods (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dosage ranges for radioactive isotopes vary extensively and depend on the isotope's half-life, the intensity and type of radiation emitted, and the uptake by the proliferating cells.

[0273] 3. Immunotherapy

[0274] Those skilled in the art will understand that immunotherapy can be used in combination with or in conjunction with the methods described in the implementation scheme. In the context of cancer treatment, immunotherapeutic agents typically rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab This is one such example. Immune effectors can be, for instance, antibodies specific to certain markers on the surface of tumor cells. The antibody alone can act as an effector of a therapy, or it can recruit other cells to actually influence cell killing. The antibody can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and act as a targeting agent. Alternatively, the effector can be a lymphocyte carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0275] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) covalently linked to a cytotoxic drug and can be used in combination therapies. This approach combines the high specificity of the MAb targeting its antigenic target with a highly potent cytotoxic drug, resulting in a “armed” MAb that delivers its payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, leading to reduced toxicity and an improved therapeutic index. Exemplary ADC drugs include… (brentuximabvedotin) and (trastuzumab emtansine or T-DM1).

[0276] In one aspect of immunotherapy, tumor cells must carry markers that are easily targeted, i.e., not present on most other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of the embodiments of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, erb B2, and p155. An alternative aspect of immunotherapy is the combination of anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γ-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0277] Examples of immunotherapy include: immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapies, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapies, such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is considered that one or more anticancer therapies may be used in conjunction with the antibody therapies described herein.

[0278] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either upregulate (e.g., co-stimulatory molecules) or downregulate. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and T-cell activation V-domain Ig inhibitor (VISTA). In particular, the immune checkpoint inhibitor targets the PD-1 axis and / or CTLA-4.

[0279] The immune checkpoint inhibitors may be drugs, such as small molecules, recombinant forms of ligands or receptors, or particularly antibodies, such as human antibodies. Known inhibitors of immune checkpoint proteins or their analogues may be used, particularly chimeric, humanized, or human forms of antibodies. As those skilled in the art will appreciate, alternative and / or equivalent names may be used for certain antibodies mentioned in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0280] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In one particular aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In one particular aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In one particular aspect, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0281] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab (also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and...) Pembrolizumab is a usable anti-PD-1 antibody. (Also known as MK-3475, Merck 3475, Lanlolizumab, etc.) SCH-900475 is an example of an anti-PD-1 antibody. CT-011 (also known as hBAT or hBAT-1) is also an anti-PD-1 antibody. AMP-224 (also known as B7-DCIg) is a PD-L2-Fc fusion soluble receptor.

[0282] Another immune checkpoint that can be targeted using the methods presented in this paper is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T-cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and is likely important for their function. T cell activation via T cell receptors and CD28 leads to increased expression of CTLA-4 (an inhibitory receptor for the B7 molecule).

[0283] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0284] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in this method can be generated using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and...). Or its antigen-binding fragments and variants. In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes with the antibodies mentioned above for binding to CTLA-4 and / or binds to the same epitopes on CTLA-4 as the antibodies mentioned above. In yet another embodiment, the antibody has at least approximately 90% variable region amino acid sequence identity with the antibodies mentioned above (e.g., at least approximately 90%, 95%, or 99% variable region identity with ipilimumab).

[0285] 4. Surgical procedures

[0286] Approximately 60% of people with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgeries. Curative surgeries include resections in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can be used in combination with other therapies (e.g., treatments according to embodiments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies). Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical procedures (Moss surgery).

[0287] After the removal of some or all cancerous cells, tissue, or tumors, a cavity may form in the body. Treatment can be performed by perfusion, direct injection, or local application of additional anticancer therapies to the area. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also have varying dosages.

[0288] 5. Other reagents

[0289] Other agents can be used in combination with certain aspects of embodiments of the present invention to improve the therapeutic efficacy. These additional agents include: agents that affect the upregulation of cell surface receptors and GAP connections, cell inhibition and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of highly proliferating cells to apoptosis-inducing agents, or other biological agents. Increased intercellular signaling resulting from increased GAP connections will increase the anti-hyperproliferative effect on adjacent highly proliferating cell populations. In other embodiments, cell inhibition or differentiation agents can be used in combination with certain aspects of embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are considered for improving the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin.

[0290] VI. Manufactured products or reagent kits

[0291] This document also provides manufactured articles or kits comprising immune cells, antibodies, reagents, buffers, or combinations thereof. The manufactured articles or kits may further include a packaging insert containing instructions on using the immune cells to treat cancer in an individual or delay cancer progression or enhance the immune function of an individual with cancer. Any antigen-specific immune cells described herein may be included in the manufactured articles or kits. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a wide variety of materials such as glass, plastics (e.g., polyvinyl chloride or polyolefins), or metal alloys (e.g., stainless steel or Hastelloy corrosion-resistant nickel-based alloys). In some embodiments, the container contains the formulation and a label (which may indicate instructions for use) on or attached to the container. The manufactured articles or kits may further include other materials desired from a commercial or user standpoint, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some embodiments, the manufactured articles further include one or more additional reagents (e.g., chemotherapy reagents and antitumor reagents). Suitable containers for said one or more reagents include, for example, bottles, vials, bags, and syringes.

[0292] VII. Sequences used in some implementation schemes

[0293] Cloned T26 VH zone:

[0294] nucleotide sequence

[0295] GAGGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACCAGGCCTTGAGTGGATCGGAGCAATTGATCCTTCAGATAGTTATACTGGCTACAATC AAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTACAAGAAGCTACTATGGTAACTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(357nt)(SEQ IDNO:8)

[0296] amino acid sequence

[0297] EVQLQESGAELVKPGASVKMSCKASGYTFTSYWMHWVKQRPGPGLEWIGAIDPSDSYTGYNQKFKGKATLTVDTSSSTAYMHLSSLTSEDSAVYFCTRSYYGNSWFAYWGQGTLVTVSA(SEQ ID NO:7)(119aa)

[0298] T26 VH CDR1: GYTFTSYW(SEQ ID NO:1)

[0299] T26 VH CDR2: IDPSDSYT(SEQ ID NO:2)

[0300] T26 VH CDR3: NSWFAYWGQGTLV(SEQ ID NO:3)

[0301] Cloned T26 VL zone:

[0302] Nucleotide sequence

[0303] ACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATAAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGAATCCCAGCCAGGTTTAGTGCCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGTTGCAGCCTATTACTGTCAGCAAAGTAATGAGGACCCATTCACGTTCGGCTCGGGGACAAGGTTGGAAATAAAAC(330nt)(SEQ ID NO:10)

[0304] Amino acid sequence

[0305] IVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYINWYQQKPGQPPKLLIYAASNLESGIPARFSASGSGTDFTLNIHPVEEEDVAAYYCQQSNEDPFTFGSGTRLEIK(110aa)(SEQ ID NO:9)

[0306] T26 VL CDR1: QSVDYDGDSY (SEQ ID NO:4)

[0307] T26 VL CDR2: AAS (SEQ ID NO:5)

[0308] T26 VL CDR3: QQSNEDPFT (SEQ ID NO:6)

[0309] Clones 5B VH zone:

[0310] Nucleotide sequence

[0311] GAGGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGCAATTGATCCTTCAGATAGTTATACTGGCTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTACAAGAAGCTACTATGGTAACTCCTGGTTTGATTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(357nt)(SEQ IDNO:18)

[0312] Amino acid sequence

[0313] EVQLQESGAELVKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIDPSDSYTGYNQKFKGKATLTVDTSSSTAYMHLSSLTSEDSAVYFCTRSYYGNSWFDYWGQGTLVTVSA(119aa)(SEQ ID NO:17)

[0314] 5B VH CDR1: GYTFTSYW (SEQ ID NO:11)

[0315] 5B VH CDR2: IDPSDSYT (SEQ ID NO:12)

[0316] 5B VH CDR3: NSWFDYWGQGTLV (SEQ ID NO:13)

[0317] Clones 5B VL zone:

[0318] Nucleotide sequence

[0319] GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGAAGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGAATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATCACTGTCAGCAAAGTAATGAGGACCCGTTCACGTTCGGAGGGGGGACCAAGTTGGAAATAAAA(333nt)(SEQ ID NO:20)

[0320] Amino acid sequence

[0321] DIVLTQSPASLAVSLGQRATISCKASQSVDYEGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYHCQQSNEDPFTFGGGTKLEIK(111aa)(SEQ ID NO:19)

[0322] 5B VL CDR1: QSVDYEGDSY (SEQ ID NO:14)

[0323] 5B VL CDR2: AAS (SEQ ID NO:15)

[0324] 5B VL CDR3: QQSNEDPFT (SEQ ID NO:16)

[0325] Cloned 28B VH zone:

[0326] Nucleotide sequence

[0327] GAGGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGCAATTGATCCTTCAGATAGTTATACTGGCTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTACAAGAAGCTACTATGGTAACTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(357nt)(SEQ IDNO:28)

[0328] Amino acid sequence

[0329] EVQLQESGAELVKPGASVKMSCKASGYTFTSYWMHWVVKQRPGQGLEWIGAIDPSDSYTGYNQKFKGKATLTVDTSSSTAYMHLSSLTSEDSAVYFCTRSYYGNSWFAYWGQGTLVTVSA(119aa)(SEQ ID NO:27)

[0330] 28B VH CDR1: GYTFTSYW(SEQ ID NO:21)

[0331] 28B VH CDR2: DPSDSYT(SEQ ID NO:22)

[0332] 28B VH CDR3: SWFAYWGQGTLV(SEQ ID NO:23)

[0333] Cloned 28B VL zone:

[0334] Nucleotide sequence

[0335] GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATTTATGTTGCATCCAATCTAGAATCTGGAATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAC(333nt)(SEQ ID NO:30)

[0336] Amino acid sequence

[0337] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYVASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIN(111aa)(SEQ ID NO:29)

[0338] 28B VL CDR1: QSVDYDGDSY(SEQ ID NO:24)

[0339] 28B VL CDR2: VAS(SEQ ID NO:25)

[0340] 28B VL CDR3: QQSNEDPFT(SEQ ID NO:26)

[0341] SEQ ID NO:40

[0342] Truncated human EGFR

[0343]

[0344] SEQ ID NO:41

[0345] EGFRIII-IV

[0346] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0347] SEQ ID NO:42

[0348] CD8 leader

[0349] ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCC

[0350] SEQ ID NO:43

[0351] CD8 leader

[0352] MALPVTALLLPLALLLHAARP

[0353] SEQ ID NO:44

[0354] Linker 1

[0355] GGTGGCGGAGGTTCT

[0356] SEQ ID NO:45

[0357] Linker 1

[0358] GGGGS

[0359] SEQ ID NO:46

[0360] Linker 2

[0361] GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC

[0362] SEQ ID NO:47

[0363] Linker 2

[0364] GGGGSGGGGS

[0365] SEQ ID NO:48

[0366] Linker 3

[0367] GGAGGAGGTGGTAGTGGTGGAGGAGGAAGTGGAGGAGGAGGAAGT

[0368] SEQ ID NO:49

[0369] Linker 3

[0370] GGGGSGGGGSGGGGS

[0371] SEQ ID NO:50

[0372] Linker 4

[0373] GGAGGAGGTGGTAGTGGTGGAGGAGGAAGTGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC

[0374] SEQ ID NO:51

[0375] Linker 4

[0376] GGGGSGGGGSGGGGSGGGGS

[0377] SEQ ID NO:52

[0378] CD8 hinge 1

[0379] ACAACTACTCCAGCACCACGACCACCAACACCTGCTCCAACTATCGCATCTCAACCACTTTCTCTACGTCCAGAAGCATGCCGACCAGCTGCAGGAGGTGCAGTTCATACGAGAGGTCTAGATTTCGCATGTGAT

[0380] SEQ ID NO:53

[0381] CD8 hinge 1

[0382] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0383] SEQ ID NO:54

[0384] CD8 hinge 2

[0385] AAGCCCACAACTACTCCAGCACCACGACCACCAACACCTGCTCCAACTATCGCATCTCAACCACTTTCTCTACGTCCAGAAGCATGCCGACCAGCTGCAGGAGGTGCAGTTCATACGAGAGGTCTAGATTTCGCATGTGAT

[0386] SEQ ID NO:55

[0387] CD8 hinge 2

[0388] KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0389] SEQ ID NO:56

[0390] CD8 hinge 3

[0391] TTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACAACTACTCCAGCACCACGACCACCAACACCTGCTCCAACTATCGCATCTCAACCACTTTCTCTACGTCCAGAAGCATGCCGACCAGCTGCAGGAGGTGCAGTTCATACGAGAGGTCTAGATTTCGCATGTGAT

[0392] SEQ ID NO:57

[0393] CD8 hinge 3

[0394] FSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0395] SEQ ID NO:58

[0396] CD28 hinge

[0397] ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGG

[0398] SEQ ID NO:59

[0399] CD28 hinge

[0400] IEVMYPPPYLDNEKSNGTIIHVKG

[0401] SEQ ID NO:60

[0402] IgG4 hinge

[0403] GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCA

[0404] SEQ ID NO:61

[0405] IgG4 hinge

[0406] ESKYGPPCPSCP

[0407] SEQ ID NO:62

[0408] IgG4 CH2

[0409] GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGG

[0410] SEQ ID NO:63

[0411] IgG4 CH2

[0412] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG

[0413] SEQ ID NO:64

[0414] IgG4 CH2CH3

[0415] GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

[0416] SEQ ID NO:65

[0417] IgG4 CH2CH3

[0418] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0419] SEQ ID NO:66

[0420] IgG4 CH1CH2CH3

[0421] GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

[0422] SEQ ID NO:67

[0423] IgG4 CH1CH2CH3

[0424] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0425] SEQ ID NO:68

[0426] CD8 TM 1

[0427] ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCCTTCTCCTATCACTGGTTATCACCCTTTACTGC

[0428] SEQ ID NO:69

[0429] CD8 TM 1

[0430] IYIWAPLAGTCGVLLLSLVITLYC

[0431] SEQ ID NO:70

[0432] CD8 TM 2

[0433] ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCCTTCTCCTATCACTGGTTATCACCCTTTACTGCAACCACAGGAAC

[0434] SEQ ID NO:71

[0435] CD8 TM 2

[0436] IYIWAPLAGTCGVLLLSLVITLYCNHRN

[0437] SEQ ID NO:72

[0438] CD28 TM

[0439] AAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC

[0440] SEQ ID NO:73

[0441] CD28 TM

[0442] KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF

[0443] SEQ ID NO:74

[0444] CD28

[0445] AGAAGTAAAAGAAGTAGGCTACTTCATAGTGATTACATGAATATGACTCCTCGACGACCTGGTCCCACCCGTAAGCATTATCAGCCCTATGCACCACCACGAGATTTCGCAGCCTATCGCTCC

[0446] SEQ ID NO:75

[0447] CD28

[0448] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0449] SEQ ID NO:76

[0450] 4-1BB

[0451] AAACGAGGTAGAAAAAAACTTCTTTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGATGTAGTTGTCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0452] SEQ ID NO:77

[0453] 4-1BB

[0454] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0455] SEQ ID NO:78

[0456] OX-40

[0457] AGGCGCGACCAGCGGCTGCCACCTGATGCACACAAGCCACCAGGAGGAGGCTCTTTCCGGACCCCAATCCAGGAGGAGCAGGCAGACGCACACAGCACACTGGCCAAGATC

[0458] SEQ ID NO:79

[0459] OX-40

[0460] RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0461] SEQ ID NO:80

[0462] CD3ζ intracellular

[0463] AGAGTTAAATTTAGCAGAAGTGCAGATGCTCCTGCGTATAAACAGGGTCAAAACCAACTATATAATGAACTAAATCTAGGACGAAGAGAAGAATATGATGTTTTAGATAAAAGACGTGGTCGAGATCCTGAAATGGGAGGAAAACCTAGAAGAAAAAATCCTCAAGAAGGCCTATATAATGAACTACAAAAAGATAAGATGGCAGAAGCTTATAGTGAAATTGGAATGAAAGGAGAACGTCGTAGAGGTAAAGGTCATGATGGTCTTTATCAAGGTCTTAGTACAGCAACAAAAGATACATATGATGCACTTCATATGCAAGCACTTCCACCTCGT

[0464] SEQ ID NO:81

[0465] CD3ζ intracellular

[0466] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0467] SEQ ID NO:82

[0468] Enhanced GFP

[0469] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

[0470] SEQ ID NO:83

[0471] Enhanced GFP

[0472] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0473] SEQ ID NO:84

[0474] T2A

[0475] GAGGGCAGAGGCAGTCTGCTGACATGCGGTGACGTGGAAGAGAATCCCGGCCCT

[0476] SEQ ID NO:85

[0477] T2A

[0478] EGRGSLLTCGDVEENPGP

[0479] SEQ ID NO:86

[0480] CD8αTM (Nucleotide sequence)

[0481] ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCCTTCTCCTATCACTGGTTATCACC

[0482] SEQ ID NO:87

[0483] CD8αTM (Amino acid sequence)

[0484] IYIWAPLAGTCGVLLLSLVIT

[0485] VIII. Examples

[0486] The following embodiments are included to illustrate particular implementations of this disclosure. Those skilled in the art will recognize that the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in the practice of the invention, and therefore can be considered to constitute preferred embodiments of the invention. However, those skilled in the art will recognize that many variations can be made in the disclosed particular embodiments, and that they still yield the same or similar results without departing from the spirit and scope of the invention.

[0487] Example 1 – Development of CD79b antibody and CAR

[0488] CD79b expression is limited to the B-cell lineage. Using real-time PCR, CD79b was found to be expressed in a wide range of B-cell lymphoma cell lines (including Mino, Daudi, HBC-1, Jeko, SUDHL6, SUDHL4, and U2932), but not in the Jurkat and J76 T-cell lymphoma / leukemia cell lines. Figure 1A To determine whether CD79b is expressed in normal tissues, the FirstChoice Human Total RNA Survey Panel, containing total RNA from 20 normal human tissues, was obtained from Applied Biosystems. Total RNA was extracted from purified B and T cells from human tonsil samples and used as positive and negative controls, respectively. CD79b transcripts were found to be present only in lymphoid tissues such as the spleen and lymph nodes, but not in any non-lymphoid normal tissues. Figure 1B ).

[0489] Using publicly available gene expression datasets (Oncomine), CD79b was found to be present in ALL and chronic lymphocytic leukemia. Figure 1C ) and various subtypes of B-cell lymphoma, such as Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma. Figure 1D High expression in (). The number of samples for each tumor type is shown in parentheses.

[0490] Generation of various monoclonal antibodies against human CD79b and identification of the heavy and light chain sequences of the antibodies. Anti-human CD79b monoclonal antibodies are generated using hybridoma technology, in which mice are immunized with L cells of mouse fibroblasts expressing human CD79b. Figure 2A Three clones with high binding capacity to recombinant human CD79b protein were identified by ELISA. Figure 2BThe affinities of these three monoclonal antibodies were further determined using the Octet assay, and three clones with Kd values ​​at 1.44, 17.8, and 2.0 nM, respectively—namely 14 (IgG1), 16A (IgG2), and 45 (IgG2)—were selected for further development. Figure 2C Monoclonal antibody clone #14 was conjugated with fluorescent dye and showed staining for B-cell lymphoma cell lines comparable to that of the commercial anti-CD79b antibody from BDBiosciences. Figure 2D Total RNA was extracted from the hybridomas containing the monoclonal antibody, and cDNA was synthesized. The V-genes for the heavy and light chains were cloned using 5'-RACE PCR (rapid amplification of cDNA ends). Protein sequences were predicted from the DNA sequences. The hybridoma culture supernatant was purified, and the heavy and light chain protein sequences were confirmed by mass spectrometry from MD Anderson Proteomics Core Facility.

[0491] Generation of anti-CD79b CAR T cells Several anti-CD79b CAR constructs were generated using a specific sequence of the variable region single-stranded fragment (scFv). To detect CAR expression in transduced T cells, CAR-enhanced green fluorescent protein (eGFP) fusion constructs or truncated human epidermal growth factor receptor (huEGFRt) were used. Figure 3A The latter can also act as a safety switch to eliminate CAR T cells in cases of severe toxicity. Incorporation of the CD3-ζ (CD3z) chain provides signal 1 regarding T cell activation, and incorporation of the co-stimulatory domain CD28 or 4-1BB provides signal 2. Figure 3A ).

[0492] These constructs were cloned into the lentiviral vector pHR_SFFV and then used to transduce primary healthy donor T cells. Figure 3B The image shows the use of clone 45-CD79b-CD28-CAR via CD4 + and CD8 + The representative transduction efficiency (>70%) was determined by eGFP expression in T cells. Anti-CD19-CAR T cells were used as a control.

[0493] The cytotoxic activity of CAR T cells against Daudi cells labeled with CellTrace Far Red was determined by Aqua staining in a 16-hour flow cytometry assay with the effector:target (E:T) ratio as shown. Figure 3C ).exist Figure 3DThe image shows a representative dot plot with the percentage of dead cells at an E:T ratio of 20:1 for various culture conditions (top right quadrant). The data show that both anti-CD19-CAR T cells and anti-CD79b-CAR T cells are highly cytotoxic to Daudi Burkitt lymphoma cells compared to untransduced control T cells.

[0494] Anti-CD79b CAR T cells against CD19 + and CD19 - Lymphoma cells are all cytotoxic. To determine the effectiveness of anti-CD79b CAR T against CD19-negative (CD19) antibodies. - The efficacy of lymphoma cells was assessed using a diffuse large B-cell lymphoma cell line, SUDHL6 (lacking CD19), through degranulation and cytotoxicity assays. First, CD19 was knocked out using CRISPR-Cas9 (CD19KOSUDHL6), and these lymphoma cells were then transduced with a CD19 splice variant lacking exon 2 (CD19Dex2), which relates to the binding site of the anti-CD19 antibody clone FMC63 used in the anti-CD19 CAR construct.

[0495] Untransduced primary T cells (Ctrl T), clone-14-CD79b-CD28 CAR, 14-CD79b-4-1BB CAR, and FMC63-CD19-CD28 CAR were co-cultured with Daudi or the aforementioned SUDHL6 cells (CD19KOSUDHL6-CD19Dex2) at a 5:1 E:T ratio. T cells were labeled with CellTrace Far Red, and target cells were labeled with CellTrace Violet. After 2 hours, a Golgi inhibitor and a degranulation marker (CD107a / b) were added to the culture to measure T cell degranulation. Cytotoxic activity against tumor cells was measured after 4 days of co-culture. 14-CD79b-CD28 and 14-CD79b-4-1BB CAR T cells, instead of control T cells, showed significantly increased degranulation and cytotoxic activity against both cell lines. Figure 4A And B). Conversely, FMC63-CD19-CD28 CAR for CD19 + Daudi is cytotoxic, but not cytotoxic to CD19KOSUDHL6-CD19Dex2 tumor cells.

[0496] The absolute number of viable tumor cells was also determined using CountBright absolute counting beads for flow cytometry after 4 days of co-culture. Figure 4C The results were consistent with the observed percentage of viable tumor cells. Figure 4B The map shows representative dot plots of target cells and effector T cells. Figure 4A (and B). The experiment was repeated at least three times, with similar results.

[0497] Anti-CD79b CAR T cells demonstrate in vivo efficacy against lymphoma xenografts. To test the in vivo efficacy of anti-CD79b CAR T cells, Mino mantle cell lymphoma cell lines expressing the firefly luciferase gene were administered via intravenous infusion at a dose of 2 × 10⁻⁶ cells / mL. 6 One tumor cell / mouse was injected into NSG mice. Eighteen days later, the tumor cells were administered via tail vein at a dose of 10 × 10⁻⁶. 6 CAR + T cells or untransduced T cells / mice were used to treat mice with untransduced primary T cells, anti-CD19-CD28 CAR T cells, or clone 45 anti-CD79b-CD28 CAR T cells. Tumor burden was assessed using bioluminescence imaging. Figure 5A The results showed progressive tumor growth in mice treated with untransduced T cells. Conversely, good tumor control and significantly improved survival were observed in mice treated with both anti-CD19- and anti-CD79b CAR T cells (p<0.05). Figure 5B and 5C For each individual experiment, in vitro results have been validated at least three times, and in vivo results have been validated twice. Therefore, anti-CD79b CAR therapy can be used to treat B-cell malignancies with or without CD19 expression.

[0498] Example 2 – Development of additional CD79b antibodies and CARs

[0499] Using novel anti-CD79b antibody clones to generate anti-CD79b CAR T cells Several anti-CD79b CAR constructs were generated using specific sequences of the variable region single-stranded fragment (scFv) from other antibody clones (T26, 5B, and 28B). To detect CAR expression in transduced T cells, CAR-enhanced green fluorescent protein (eGFP) fusion constructs or truncated human epidermal growth factor receptor (huEGFRt) were used. Figure 6A The latter can also act as a safety switch to eliminate CAR T cells in cases of severe toxicity. Incorporation of the CD3-ζ (CD3z) chain provides signal 1 regarding T cell activation, and incorporation of co-stimulatory domains CD28 or 4-1BB or OX-40 provides signal 2. Figure 6A ).

[0500] Anti-CD79b CAR T cells demonstrated in vitro cell targeting of Daudi lymphoma cells.Toxicity. The above-described CAR constructs (T26 and 28B) were cloned into the lentiviral vector pLVEG and then used to transduce primary healthy donor T cells to generate anti-CD79b-CAR T cells. Anti-CD19-CAR T cells were used as a control. At 1 and 4 days of co-culture, the cytotoxic activity of CAR T cells (labeled with CellTrace Far Red) against Daudi cells (labeled with CellTrace Violet) was determined by flow cytometry at the effector:target (E:T) ratio shown. Figure 6B ).exist Figure 6C The image shows a representative dot plot. Data shows that both anti-CD19-CAR T cells and anti-CD79b-CAR T cells are highly cytotoxic to Daudi Burkitt lymphoma cells.

[0501] Anti-CD79b CAR T cells demonstrated in vivo efficacy against Daudi lymphoma xenografts. To test the in vivo efficacy of anti-CD79b CAR T cells (clone T26), Daudi Burkitt lymphoma cell line expressing the firefly luciferase gene was administered via intravenous infusion at a dose of 2 × 10⁻⁶ cells / mL. 4 One tumor cell / mouse was injected into NSG mice. Ten days later, the mice were either left untreated or administered a 3×10⁻⁶ tumor cell / mouse dose via tail vein injection. 6 Each CAR+ T cell / mouse was treated with either anti-CD19-CAR T cells or cloned T26 anti-CD79b-CAR T cells. Bioluminescence imaging was used to assess tumor burden. Figure 6D The results showed progressive tumor growth in untreated mice. Conversely, good tumor control and significantly improved survival were observed in mice treated with both anti-CD19- and anti-CD79b CAR T cells (p = 0.01). Figure 6E These results demonstrate that anti-CD79b CAR T cells possess strong in vivo anti-tumor activity.

[0502] Example 3 – Further Development and Exploration of CD79b Antibody and CAR

[0503] Binding of anti-CD79b antibody to human CD79b Full-length anti-CD79b monoclonal antibodies (clones 5B and 28B) isolated from hybridoma supernatant were used to test binding to human CD79b expressed on mouse fibroblast L cells by ELISA. Figure 7 ).

[0504] Generation of CAR constructs : Using specific sequences (shown in) single-stranded fragments (scFv) of the variable region derived from clone 28B of VH and VL Figure 7Several anti-CD79b CAR constructs were generated using CD8α or CD28 hinge / transmembrane domains. To detect CAR expression in transduced T cells, CAR-enhanced green fluorescent protein (eGFP) fusion constructs or truncated human epidermal growth factor receptor (huEGFRt) were used. Figure 8A Anti-CD19-CAR constructs were used as controls. The latter can also act as a safety switch to eliminate CAR T cells in cases of severe toxicity. Incorporation of the CD3-ζ (CD3ζ) chain provides signal 1 regarding T cell activation, and incorporation of co-stimulatory domains CD28 or 4-1BB or OX40 provides signal 2. Figure 8A These constructs were cloned into the lentiviral vector pLVEG with the EF1α promoter. Figure 8B This was then used to transduce T cells and tested in various in vitro and in vivo assays described below.

[0505] Anti-CD79b CAR specifically recognizes human CD79b To determine the signal transduction ability of anti-CD79b CAR, the CAR (clone 28B for CD79b CAR) was transduced into Jurkat-Lucia using lentivirus. TM NFAT reports cells ( Figure 9A In this cell line, the Lucia gene, encoding the coelenterazine-utilizing luciferase, is driven by the ISG54 minimal promoter, which is fused to six copies of the NFAT shared transcriptional response element. These transduced Jurkat cells, co-cultured with anti-CD3 / anti-CD28 antibody or Daudi Burkitt lymphoma cells for 24 hours, induced significantly higher luciferase activity compared to untreated cells. Figure 9B To establish the recognition specificity of the CAR molecule for CD79b, CRISPR / Cas9 was used to generate isogenetic forms of SUDHL6 (a diffuse large B-cell lymphoma cell line expressing CD19 and CD79b): parental or wild-type (WT), CD19 knockout (CD19KO), CD79bKO, and CD19 / CD79b double KO (CD19KO / CD79bKO). Figure 9C Jurkat-Lucia transduced via CAR TMCo-culturing NFAT-reported cells with gene-modified cells such as SUDHL6 showed that CD79b CAR recognized both parental and CD19KO cells, but the responsiveness of CD79bKO cells was significantly reduced when CD79bKO or CD19KO were used. Conversely, CD19 CAR recognized both parental and CD79bKO cells, but not CD19KO or CD19KO / CD79bKO cells. Figure 9D ).

[0506] CAR was transferred into primary human T cells. :exist Figure 10A The image shows the use of the CAR construct described above. Figure 8A Representative transduction efficiency was determined by eGFP expression and / or staining with recombinant human CD79b-Fc protein. Anti-CD19-CAR T cells were used as a control. Anti-CD79b CAR T cells were either untreated or stimulated with anti-CD3 / CD28 antibody or recombinant human CD79b-Fc protein. Phosphorylation of CD3ζ and ERK1 / 2 was assessed by flow cytometry after 15 minutes. The results showed no significant difference in baseline phosphorylation between CAR+ and CAR-T cells, suggesting the absence of robust signaling. Stimulation with anti-CD3 / CD28 antibody induced phosphorylation of CD3ζ in CAR+ but not CAR-T cells. Conversely, stimulation with recombinant human CD79b-Fc protein induced phosphorylation of CD3ζ and ERK1 / 2 in CAR+ but not CAR-T cells, suggesting that the CAR can induce signaling in primary T cells in response to CD79b protein recognition. Figure 10B ).

[0507] Proliferative activity of anti-CD79b CAR T cells : Using CellTrace Far Red to label T cells from healthy donors using the above-described CAR construct ( Figure 8A Anti-CD79b CAR T cells were generated and co-cultured with CD79b-expressing Daudi Burkitt lymphoma tumor cells at a 1:1 effector:target (E:T) ratio. After 4 days, CAR T cell proliferation was assessed by flow cytometry using a dye dilution method. Results showed that both CD4+ and CD8+ anti-CD79b CAR T cells proliferated significantly in response to Daudi tumor cells compared to untransduced T cells. The proliferation of anti-CD79b CAR T cells was comparable to that of anti-CD19 CAR T cells. Figure 11A and 11B ).

[0508] Cytokines produced by anti-CD79b CAR T cells : will be achieved by using the above CAR construct ( Figure 8AAnti-CD79b CAR T cells derived from healthy donor T cells were co-cultured with Daudi Burkitt lymphoma tumor cells at a 1:1 E:T ratio. Cytokine levels in the supernatant were assessed using a multiplex cytokine assay after 24 hours. Results showed that anti-CD79b CAR T cells produced significantly higher levels of IL-2, GM-CSF, IFN-γ, and IL-17A in response to Daudi tumor cells compared to untransduced T cells. Cytokine production by anti-CD79b CAR T cells was comparable to that by anti-CD19 CAR T cells. Figure 12 ).

[0509] Anti-CD79b CAR T cells degranulate in response to lymphoma cells. : will be achieved by using the above CAR construct ( Figure 8A Anti-CD79b CAR T cells derived from healthy donor T cells were co-cultured with Daudi Burkitt lymphoma tumor cells at a 1:1 E:T ratio. Degranulation was assessed by flow cytometry after 6 hours of staining for CD107 a / b. Results showed that both CD4+ and CD8+ anti-CD79b CAR T cells responded to Daudi tumor cells with significant degranulation compared to untransduced T cells. Degranulation by anti-CD79b CAR T cells was comparable to that by anti-CD19 CAR T cells. Degranulation was numerically higher with anti-CD79b CAR T cells containing the CD8α hinge / transmembrane domain compared to anti-CD79b CAR T cells containing the CD28 hinge / transmembrane domain. Figure 13A and 13B ).

[0510] Anti-CD79b CAR T cells are cytotoxic to lymphoma cells. : will be achieved by using the above CAR construct ( Figure 8AAnti-CD79b CAR T cells, derived from healthy donor T cells and labeled with CellTrace Far Red, were co-cultured with Daudi Burkitt lymphoma tumor cells or gene-dependent cell lines such as SUDHL6 (parental, CD19KO, CD79bKO, and CD19 / CD79b double KO) labeled with CellTrace Violet at a 1:1 E:T ratio. Cytotoxicity was assessed by flow cytometry after 4 days by determining the absolute number of residual viable tumor cells and calculating the percentage of lysis. Results showed that anti-CD79b CAR T cells induced significant cytotoxicity against Daudi tumor cells compared to untransduced T cells. The cytotoxic activity of anti-CD79b CAR T cells was comparable to that of anti-CD19 CAR T cells. Compared to anti-CD79b CAR T cells containing a CD28 hinge / transmembrane domain, anti-CD79b CAR T cells containing a CD8α hinge / transmembrane domain exhibit higher cytotoxicity. Figure 14A Furthermore, anti-CD79b CAR T cells induced significant lysis of both parental and CD19KO SUDHL6 cells, but lysis was significantly reduced with CD79bKO or CD19KO / CD79bKO cells. Conversely, anti-CD19 CAR T cells induced significant lysis of both parental and CD79bKO cells, but lysis was significantly reduced with CD19KO or CD19KO / CD79bKO cells. Figure 14B ).

[0511] Anti-CD79b CAR T cells exert anti-tumor effects in vivo To examine the in vivo efficacy of anti-CD79b-CAR T cells, Daudi Burkitt lymphoma cells expressing the firefly luciferase gene were administered via intravenous infusion at a dose of 2 × 10⁻⁶ cells / mL. 4 One tumor cell / mouse was injected into NSG mice. Eleven days later, 5 × 10⁸ tumor cells / mouse were injected via tail vein. 6 CAR+ T cells / mouse using untransduced T cells or anti-CD19-CAR T cells or by using the above-mentioned CAR construct ( Figure 8A The generated anti-CD79b-CAR T cells were used to treat mice. Bioluminescence imaging was used to assess tumor burden. Figure 15AThe results showed rapid tumor growth in mice treated with untransduced T cells. Conversely, good tumor control and significantly improved survival were observed in mice treated with both anti-CD19 CAR and anti-CD79b CAR T cells. Tumor control and survival were significantly higher with anti-CD79b CAR T cells containing the CD8α hinge / transmembrane domain compared to those containing the CD28 hinge / transmembrane domain, with the best survival observed with anti-CD79b CAR T cells containing both the CD8α hinge / transmembrane domain and the OX40 co-stimulatory domain. Figure 15B ).

[0512] ***

[0513] All methods disclosed and claimed herein can be made and performed based on this disclosure without excessive experimentation. While the compositions and methods of the invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be made to the methods described herein and to the steps or sequence of steps thereof without departing from the concept, spirit, and scope of the invention. More particularly, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein with the same or similar results. All such similar substitutions or modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention, as defined by the appended claims.

[0514] References

[0515] The following references are specifically incorporated herein by reference to provide exemplary procedures or other details that are supplementary to those set forth herein.

[0516] Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

[0517] Chothia et al., 1988.

[0518] Davila et al., 2013.

[0519] European patent application number EP2537416

[0520] Heemskerk et al., Hum Gene Ther. 19:496-510, 2008.

[0521] International Patent Publication No. WO / 2014055668 A1.

[0522] International Patent Publication No. WO200014257

[0523] International Patent Publication No. WO2012 / 129514

[0524] International Patent Publication No. WO2013 / 071154

[0525] International Patent Publication No. WO2013 / 123061

[0526] International Patent Publication No. WO2013 / 166321

[0527] International Patent Publication No. WO2013126726

[0528] International Patent Publication No. WO2014031687

[0529] Johnson et al., Blood 114:535-46, 2009.

[0530] Jores et al., 1990.

[0531] Kabat et al., "Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.

[0532] Lefranc et al., 2003.

[0533] Liu et al., 2003.

[0534] Remington's Pharmaceutical Sciences 22 nd edition, 2012.

[0535] Sadelain et al., 2013.

[0536] Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rded., Cold Spring Harbor Press, Cold Spring Harbor, NY2001.

[0537] Turtle et al., 2012.

[0538] US Patent No. 5,091,513

[0539] US Patent No. 5,091,513

[0540] US Patent No. 5,994,136

[0541] US Patent No. 6,013,516

[0542] US Patent No. 6,410,319

[0543] US Patent No. 6,410,319

[0544] US Patent No. 6,451,995

[0545] US Patent No. 6,881,557

[0546] US Patent No. 6,946,546

[0547] US Patent No. 7,070,995

[0548] US Patent No. 7,265,209

[0549] US Patent No. 7,354,762

[0550] US Patent No. 7,446,179

[0551] US Patent No. 7,446,190

[0552] US Patent No. 7,446,191

[0553] US Patent No. 8,252,592

[0554] US Patent No. 8,324,353

[0555] US Patent No. 8,339,645

[0556] US Patent No. 8,398,282

[0557] US Patent No. 8,479,118

[0558] US Patent No. 7,446,190

[0559] US Patent Publication No. US2002131960

[0560] US Patent Publication No. US20050214860

[0561] US Patent Publication No. US20130149337

[0562] US Patent Publication No. US2013287748

[0563] Wu et al., 2012

Claims

1. An isolated monoclonal antibody, wherein the antibody specifically binds to CD79b and comprises: (a) a first V H CDR; (b) a second V H CDR; (c) a third V H CDR; (d) a first V consisting of SEQ ID NO: 24 L CDR; (e) a second V consisting of SEQ ID NO: 25 L CDR; and (f) a third V L CDR.

2. The antibody of claim 1, wherein the antibody comprises V with SEQ ID NO: 27 H At least 80% of the structural domains are identical V H The structural domain and V of SEQ ID NO: 29 L At least 80% of the structural domains are identical V L Structural domain.

3. The antibody of claim 1, wherein the antibody comprises V with SEQ ID NO: 27 H V with the same structural domain H The structural domain and V of SEQ ID NO: 29 L V with the same structural domain L Structural domain.

4. The antibody of any one of claims 1-3, wherein the antibody is recombinant.

5. The antibody of claim 1, wherein the antibody is IgG, IgM, IgA or an antigen-binding fragment thereof.

6. The antibody of any one of claims 1-3, wherein the antibody is Fab', F(ab')2, F(ab')3, monovalent scFv or bivalent scFv.

7. The antibody of any one of claims 1-3, wherein the antibody is a human antibody, a humanized antibody, or a deimmunized antibody.

8. The antibody of any one of claims 1-3, wherein the antibody is conjugated with an imaging agent or a radionuclide.

9. A composition comprising, in a pharmaceutically acceptable carrier, the antibody of any one of claims 1-8.

10. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody of any one of claims 1-7.

11. Recombinant polypeptide, comprising: V H Structural domain, the V H The structural domain contains the V shown in SEQ ID NO: 21, 22 and 23. H CDR 1-3; and V L Structural domain, the V L The structural domain contains the V shown in SEQ ID NO: 24, 25 and 26. L CDR 1-3.

12. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the polypeptide of claim 11.

13. A host cell comprising one or more polynucleotide molecules encoding an antibody of any one of claims 1-7 or a recombinant polypeptide of claim 11.

14. The host cell of claim 13, wherein the host cell is a mammalian cell, yeast cell, bacterial cell, ciliate cell or insect cell.

15. Use of the antibody of any one of claims 1-6 in the preparation of a medicament for treating a subject with cancer, wherein the cancer is diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, or mantle cell lymphoma.

16. The use of claim 15, wherein the antibody is in a pharmaceutically acceptable composition.

17. The use of claim 15, wherein the antibody is administered systemically.

18. The use of claim 15, wherein the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

19. A modified CD79b CAR or TCR having an antigen-binding domain, said antigen-binding domain comprising: (a) The first V consisting of SEQ ID NO: 21 H CDR; (b) The second V consisting of SEQ ID NO: 22 H CDR; (c) The third V consisting of SEQ ID NO: 23 H CDR; (d) The first V consisting of SEQ ID NO: 24 L CDR; (e) The second V consisting of SEQ ID NO: 25 L CDR; and (f) The third V consisting of SEQ ID NO: 26 L CDR.

20. The CAR or TCR of claim 19, wherein the antigen-binding domain comprises the V of SEQ ID NO: 27 H At least 80% of the structural domains are identical V H The structural domain and V of SEQ ID NO: 29 L At least 80% of the structural domains are identical V L Structural domain.

21. The CAR or TCR of claim 19, wherein the antigen-binding domain comprises the V of SEQ ID NO:

27. H V with the same structural domain H The structural domain and V of SEQ ID NO: 29 L V with the same structural domain L Structural domain.

22. The CAR or TCR of claim 19, wherein the CAR comprises one or more signal transduction domains selected from the group consisting of: CD3ξ, CD28, OX40 / CD134 and 4-1BB / CD137.

23. The CAR or TCR of claim 19, wherein the CAR comprises CD3ζ and CD28 signal transduction domains.

24. The CAR or TCR of claim 19, wherein the CAR comprises CD3ζ and 4-1BB signal transduction domains.

25. The CAR or TCR of claim 19, wherein the CAR comprises CD3ζ and OX-40 signal transduction domains.

26. The CAR or TCR of claim 19, wherein the CAR or TCR is encoded by a viral vector.

27. The CAR or TCR of claim 26, wherein the viral vector is a lentiviral vector.

28. The CAR or TCR of claim 19, wherein the antigen-binding domain comprises a linker connected to V L V connected by structural domains H Structural domain.

29. The CAR or TCR of claim 28, wherein the linker comprises linker 1 of SEQ ID NO: 45 or a linker encoded by a polynucleotide of SEQ ID NO: 44, linker 2 of SEQ ID NO: 47 or a linker encoded by a polynucleotide of SEQ ID NO: 46, linker 3 of SEQ ID NO: 49 or a linker encoded by a polynucleotide of SEQ ID NO: 48, or linker 4 of SEQ ID NO: 51 or a linker encoded by a polynucleotide of SEQ ID NO:

50.

30. The CAR or TCR of claim 29, wherein the CAR comprises V L -Connector 1-V H V L -Connector 2-V H V L -Connector 3-V H V L -Connector 4-V H V H -Connector 1-V L V H -Connector 2-V L V H -Connector 3-V L or V H -Connector 4-V L .

31. The CAR or TCR of claim 19, wherein the CAR or TCR comprises a hinge.

32. The CAR or TCR of claim 31, wherein the hinge is a CD8 hinge 1 of SEQ ID NO: 53 or a hinge encoded by a polynucleotide of SEQ ID NO: 52, a CD8 hinge 2 of SEQ ID NO: 55 or a hinge encoded by a polynucleotide of SEQ ID NO: 54, a CD8 hinge 3 of SEQ ID NO: 57 or a hinge encoded by a polynucleotide of SEQ ID NO: 56, a CD28 hinge of SEQ ID NO: 59 or a hinge encoded by a polynucleotide of SEQ ID NO: 58, an IgG4 hinge of SEQ ID NO: 61 or a hinge encoded by a polynucleotide of SEQ ID NO: 60, an IgG4 CH2 of SEQ ID NO: 63 or a hinge encoded by a polynucleotide of SEQ ID NO: 62, an IgG4 CH2CH3 of SEQ ID NO: 65 or a hinge encoded by a polynucleotide of SEQ ID NO: 64, or an IgG4 CH1CH2CH3 of SEQ ID NO: 67 or a hinge encoded by a polynucleotide of SEQ ID NO:

52. The hinge is encoded by a polynucleotide of 66.

33. The CAR or TCR of claim 19, wherein the CAR comprises a transmembrane domain.

34. The CAR or TCR of claim 33, wherein the transmembrane domain is CD8™1 of SEQ ID NO: 69 or a transmembrane domain encoded by a polynucleotide of SEQ ID NO: 68, CD8™2 of SEQ ID NO: 71 or a transmembrane domain encoded by a polynucleotide of SEQ ID NO: 70, CD28™ of SEQ ID NO: 73 or a transmembrane domain encoded by a polynucleotide of SEQ ID NO: 72, or CD8α™ of SEQ ID NO: 87 or a transmembrane domain encoded by a polynucleotide of SEQ ID NO:

86.

35. The CAR or TCR of claim 19, further comprising a transduction marker and / or a safety switch.

36. The CAR or TCR of claim 35, wherein the transduction marker is enhanced green fluorescent protein (eGFP).

37. The CAR or TCR of claim 36, wherein the eGFP consists of the amino acid sequence of SEQ ID NO:

83.

38. The CAR or TCR of claim 35, wherein the transduction marker and / or safety switch is truncated epidermal growth factor (EGFR).

39. The CAR or TCR of claim 38, wherein the EGFR consists of the amino acid sequence of SEQ ID NO:

41.

40. The CAR or TCR of claim 35, wherein the transduction marker and / or safety switch is linked to the CAR via a cleavage peptide.

41. The CAR or TCR of claim 40, wherein the cleavage peptide is a 2A peptide.

42. The CAR or TCR of claim 41, wherein the 2A peptide is a T2A peptide.

43. The CAR or TCR of claim 42, wherein the T2A peptide comprises the amino acid sequence of SEQ ID NO:

85.

44. The CAR or TCR of claim 19, wherein the CAR further comprises a second antigen-binding domain.

45. The CAR or TCR of claim 44, wherein the second antigen-binding domain is a CD19, CD20, or CD22 antigen-binding domain.

46. ​​An expression vector encoding a CAR or TCR of any one of claims 19-45.

47. A host cell modified to express CD79b CAR, wherein the cell is modified to express the CAR of any one of claims 19-45.

48. The cell of claim 47, wherein the host cell is an immune cell.

49. The cell of claim 48, wherein the immune cell is a T cell.

50. The cell of claim 49, wherein the T cell is a primary human T cell or a TIL.

51. The cell of claim 49, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

52. The cell of claim 50, wherein the primary human T cell is obtained from a healthy donor.

53. The cell of claim 49, wherein the T cell is autologous.

54. The cell of claim 49, wherein the T cell is an allogeneic cell.

55. The cell of claim 47, wherein the cell is modified using a CRISPR or transposase system.

56. A pharmaceutical composition comprising CD79b-targeting T cells and a pharmaceutical carrier, wherein the CD79b-targeting T cells are modified to express the CAR or TCR of any one of claims 19-45.

57. A composition comprising an effective amount of CD79b-targeting T cells for treating cancer in a subject, wherein the CD79b-targeting T cells are modified to express the CAR or TCR of any one of claims 19-45.

58. Use of a composition comprising an effective amount of CD79b-targeting T cells in the preparation of a medicament for treating cancer in a subject, wherein the CD79b-targeting T cells are modified to express the CAR or TCR of any one of claims 19-45, and wherein the cancer is diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, or mantle cell lymphoma.

59. Use of CD79b-targeting T cells in the preparation of a medicament for treating cancer in a subject, wherein the CD79b-targeting T cells are modified to express the CAR or TCR of any one of claims 19-45, and wherein the cancer is diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, or mantle cell lymphoma.

60. The use of claim 59, wherein the subject has been previously administered CD19 CAR therapy.

61. The use of claim 60, wherein the subject is resistant to CD19 CAR therapy.

62. The use of claim 61, wherein the subject has CD19 antigen loss.

63. The use of claim 62, wherein the subject has a recurrent CD19-negative tumor.

64. The use of claim 59, wherein the CD79b-targeting T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

65. The use of claim 59, wherein the CD79b-targeting T cells are administered intravenously.

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