Anti-CD79B antibodies and chimeric antigen receptors and methods of using them

Abstract

Anti-CD79B antibodies and chimeric antigen receptors and methods for their use are provided. Provided herein are CD79b antibodies and CD79b-specific chimeric antigen receptors (CARs). Also provided herein are immune cells expressing CD79b-specific CARs, and methods of treating cancer by administering CD79b-specific CAR immune cells.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 018,266, filed April 30, 2020, the entirety of which is incorporated herein by reference. background 1. Field

[0002] The present disclosure relates generally to the fields of immunology, cell biology, molecular biology, and medicine. More particularly, the present disclosure relates to CD79b antibodies and related compositions comprising at least a chimeric antigen receptor, and methods of their use. [Background technology]

[0003] 2. Description of Related Technology Chimeric antigen receptor (CAR) T cells targeting CD19 are highly effective in B-cell malignancies. Recently, two anti-CD19 CAR T-cell therapy products were approved by the U.S. Food and Drug Administration for relapsed or treatment-resistant B-cell acute lymphoblastic leukemia (ALL) and / or large B-cell lymphoma. Long-term remissions lasting more than one year were observed in approximately 40–50% of these patients in pivotal clinical trials. However, relapse or progression occurred in approximately 50–60% of patients, and the primary cause of resistance appears to be loss of the CD19 antigen. Therefore, there is an urgent need to develop novel CAR T-cell therapies to further improve outcomes in these patients. Summary of the Invention

[0004] The present disclosure relates to methods and compositions related to certain antibodies. These antibodies can be used in any type of immunotherapy and for any medical application where targeting CD79b is therapeutically useful. In some embodiments, the present disclosure provides isolated monoclonal antibodies that specifically bind to CD79b and (I): (a) a first V comprising SEQ ID NO: 1; H CDRs; (b) a second V comprising SEQ ID NO: 2 H CDRs; (c) a third V comprising SEQ ID NO: 3 H CDRs; (d) a first V comprising SEQ ID NO: 4 L CDRs; (e) a second V comprising SEQ ID NO: 5 L CDRs; and (f) a third V comprising SEQ ID NO: 6 L CDRs; (II): (a) a first V comprising SEQ ID NO: 11 H CDRs; (b) a second V comprising SEQ ID NO: 12 H CDRs; (c) a third V comprising SEQ ID NO: 13 H CDRs; (d) a first V comprising SEQ ID NO: 14 L CDRs; (e) a second V comprising SEQ ID NO: 15 L CDRs; and (f) a third V comprising SEQ ID NO: 16 L CDR; or (III): (a) a first V comprising SEQ ID NO: 21 H CDRs; (b) a second V comprising SEQ ID NO: 22 H CDRs; (c) a third V comprising SEQ ID NO: 23 H CDRs; (d) a first V comprising SEQ ID NO: 24 L CDRs; (e) a second V comprising SEQ ID NO: 25 L CDRs; and (f) a third V comprising SEQ ID NO: 26 L CDR Includes.

[0005] In some embodiments, the antibody comprises: (a) a first V comprising SEQ ID NO: 1; H CDRs; (b) a second V comprising SEQ ID NO: 2 H CDRs; (c) a third V comprising SEQ ID NO: 3 H CDRs; (d) a first V comprising SEQ ID NO: 4 L CDRs; (e) a second V comprising SEQ ID NO: 5 L CDRs; and (f) a third V comprising SEQ ID NO: 6 L CDR Includes.

[0006] In a further embodiment, the antibody comprises V of SEQ ID NO: 7 H V domains that are at least approximately 80% identical to H domain, and V of SEQ ID NO: 9 L V domains that are at least approximately 80% identical to L In other embodiments, the antibody comprises the V domain of SEQ ID NO:7. H The same V as the domain H domain, and V of SEQ ID NO: 9 L The same V as the domain L Includes the domain.

[0007] In some embodiments, the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 11; (b) a second VHCDR comprising SEQ ID NO: 12; (c) a third VH CDR comprising SEQ ID NO: 13; (d) a first VLCDR comprising SEQ ID NO: 14; (e) a second VLCDR comprising SEQ ID NO: 15; and (f) a third VLCDR comprising SEQ ID NO: 16 Includes.

[0008] In a further embodiment, the antibody comprises V of SEQ ID NO: 17 H V domains that are at least approximately 80% identical to H domain, and V of SEQ ID NO: 19 L V domains that are at least approximately 80% identical to L In other embodiments, the antibody comprises the V domain of SEQ ID NO: 17. H The same V as the domain Hdomain, and V of SEQ ID NO: 19 L The same V as the domain L Includes the domain. In some embodiments, the antibody comprises: (a) a first V comprising SEQ ID NO: 21 H CDRs; (b) a second V comprising SEQ ID NO: 22 H CDRs; (c) a third V comprising SEQ ID NO: 23 H CDRs; (d) a first V comprising SEQ ID NO: 24 L CDRs; (e) a second V comprising SEQ ID NO: 25 L CDRs; and (f) a third V comprising SEQ ID NO: 26 L CDR Includes.

[0009] In a further embodiment, the antibody comprises V of SEQ ID NO: 27 H V domains that are at least approximately 80% identical to H domain, and V of SEQ ID NO: 29 L V domains that are at least approximately 80% identical to L In other embodiments, the antibody comprises the V domain of SEQ ID NO: 27. H The same V as the domain H domain, and V of SEQ ID NO: 29 L The same V as the domain L Includes the domain.

[0010] In some embodiments, the antibody is recombinant. In some embodiments, the antibody is IgG, IgM, IgA, or an antigen-binding fragment thereof. In some embodiments, the antibody is a Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or single domain antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a deimmunized antibody. In some embodiments, the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

[0011] In other embodiments, the present disclosure provides compositions comprising an antibody of the present disclosure in a pharmaceutically acceptable carrier.

[0012] In still other embodiments, the present disclosure provides an isolated polynucleotide molecule comprising a nucleic acid sequence encoding an antibody of the present disclosure.

[0013] In yet another embodiment, the present disclosure provides a V H CDRs 1 to 3 (SEQ ID NOs: 1, 2, and 3) of the domain and V of clone T26 L Antibody V comprising CDRs 1 to 3 of the domain (SEQ ID NOs: 4, 5, and 6) H A recombinant polypeptide comprising the domain is provided.

[0014] In other embodiments, the present disclosure provides a V H CDRs 1 to 3 of the domain (SEQ ID NOs: 11, 12, and 13) and V of clone 5B L Antibody V comprising CDRs 1 to 3 of the domain (SEQ ID NOs: 14, 15, and 16) H A recombinant polypeptide comprising the domain is provided.

[0015] In yet other embodiments, the present disclosure provides a V H CDRs 1 to 3 of the domain (SEQ ID NOs: 21, 22, and 23) and V of clone 28B L Antibody V comprising CDRs 1 to 3 of the domain (SEQ ID NOs: 24, 25, and 26) H A recombinant polypeptide comprising the domain is provided.

[0016] In yet other embodiments, the present disclosure provides an isolated polynucleotide molecule comprising a nucleic acid sequence encoding a polypeptide of the present disclosure.

[0017] In other embodiments, the present disclosure provides host cells comprising one or more polynucleotide molecules encoding an antibody or recombinant polypeptide of the present disclosure. In some embodiments, the host cell is a mammalian cell, a yeast cell, a bacterial cell, a ciliated cell, or an insect cell.

[0018] In still other embodiments, the present disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject an effective amount of an antibody of the present disclosure. In some embodiments, the cancer is a B-cell malignancy. In some embodiments, the antibody is present in a pharmaceutically acceptable composition. In some embodiments, the antibody is administered systemically. In some embodiments, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. In some embodiments, the method further comprises administering at least a second anti-cancer therapy to the subject. In further embodiments, the second anti-cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In some embodiments, the second anti-cancer therapy comprises adoptive T-cell therapy.

[0019] In yet other embodiments, the present disclosure provides: (I): (a) a first V comprising SEQ ID NO: 1; H CDRs; (b) a second V comprising SEQ ID NO: 2 H CDRs; (c) a third V comprising SEQ ID NO: 3 H CDRs; (d) a first V comprising SEQ ID NO: 4 L CDRs; (e) a second V comprising SEQ ID NO: 5 L CDRs; and (f) a third V comprising SEQ ID NO: 6 L CDRs; (II): (a) a first V comprising SEQ ID NO: 11 H CDRs; (b) a second V comprising SEQ ID NO: 12 H CDRs; (c) a third V comprising SEQ ID NO: 13 H CDRs; (d) a first V comprising SEQ ID NO: 14 L CDRs; (e) a second V comprising SEQ ID NO: 15 L CDRs; and (f) a third V comprising SEQ ID NO: 16 L CDR; or (III): (a) a first V comprising SEQ ID NO: 21 H CDRs; (b) a second V comprising SEQ ID NO: 22 H CDRs; (c) a third V comprising SEQ ID NO: 23 H CDRs; (d) a first V comprising SEQ ID NO: 24 L CDRs; (e) a second V comprising SEQ ID NO: 25 L CDRs; and (f) a third V comprising SEQ ID NO: 26 L CDR The present invention provides an engineered CD79b CAR or CD79b TCR having an antigen binding domain comprising:

[0020] In some embodiments, the antigen binding domain comprises: (a) a first V comprising SEQ ID NO: 1; H CDRs; (b) a second V comprising SEQ ID NO: 2 H CDRs; (c) a third V comprising SEQ ID NO: 3 H CDRs; (d) a first V comprising SEQ ID NO: 4 L CDRs; (e) a second V comprising SEQ ID NO: 5 L CDRs; and (f) a third V comprising SEQ ID NO: 6 L CDR Includes.

[0021] In a further embodiment, the antigen binding domain comprises V of SEQ ID NO: 7 H V domains that are at least approximately 80% identical toH domain, and V of SEQ ID NO: 9 L V domains that are at least approximately 80% identical to L In some embodiments, the antigen-binding domain comprises the V domain of SEQ ID NO:7. H The same V as the domain H domain, and V of SEQ ID NO: 9 L The same V as the domain L Includes the domain.

[0022] In some embodiments, the antibody comprises: (a) a first V comprising SEQ ID NO: 11 H CDRs; (b) a second V comprising SEQ ID NO: 12 H CDRs; (c) a third V comprising SEQ ID NO: 13 H CDRs; (d) a first V comprising SEQ ID NO: 14 L CDRs; (e) a second V comprising SEQ ID NO: 15 L CDRs; and (f) a third V comprising SEQ ID NO: 16 L CDR Includes.

[0023] In a further embodiment, the antigen binding domain comprises V of SEQ ID NO: 17 H V domains that are at least approximately 80% identical to H domain, and V of SEQ ID NO: 19 L V domains that are at least approximately 80% identical to L In some embodiments, the antigen-binding domain comprises the V domain of SEQ ID NO: 17. H The same V as the domain H domain, and V of SEQ ID NO: 19 L The same V as the domain L Includes the domain.

[0024] In some embodiments, the antigen binding domain comprises: (a) a first V comprising SEQ ID NO: 21 H CDRs; (b) a second V comprising SEQ ID NO: 22H CDRs; (c) a third V comprising SEQ ID NO: 23 H CDRs; (d) a first V comprising SEQ ID NO: 24 L CDRs; (e) a second V comprising SEQ ID NO: 25 L CDRs; and (f) a third V comprising SEQ ID NO: 26 L CDR Includes.

[0025] In a further embodiment, the antigen binding domain comprises V of SEQ ID NO: 27 H V domains that are at least approximately 80% identical to H domain, and V of SEQ ID NO: 29 L V domains that are at least approximately 80% identical to L In some embodiments, the antigen-binding domain comprises the V domain of SEQ ID NO: 27. H The same V as the domain H domain, and V of SEQ ID NO: 29 L The same V as the domain L In some embodiments, the CAR comprises one or more signaling domains CD3ζ, CD28, OX40 / CD134, 4-1BB / CD137, or a combination thereof. In some embodiments, the CAR comprises a CD3ζ signaling domain and a CD28 signaling domain. In some embodiments, the CAR comprises a CD3ζ signaling domain and a 4-1BB signaling domain. In some embodiments, the CAR comprises a CD3ζ signaling domain and an OX-40 signaling domain. In some embodiments, the CAR or TCR is encoded by a viral vector. In further embodiments, the viral vector is a lentiviral vector.

[0026] In some embodiments, the antigen binding domains are connected via a linker to form a V L V linked to domain HIn further embodiments, the linker is Linker1 (SEQ ID NO: 44 or 45), Linker2 (SEQ ID NO: 46 or 47), Linker3 (SEQ ID NO: 48 or 49), or Linker4 (SEQ ID NO: 50 or 51). L -Linker 1-V H、 V L -Linker 2-V H , V L -Linker 3-V H , V L -Linker 4-V H , V H -Linker 1-V L , V H -Linker 2-V L , V H -Linker 3-V L or V H -Linker 4-V L In some embodiments, the CAR or TCR comprises a hinge. In further embodiments, 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), IgG4 CH2 (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 embodiments, the CAR comprises a transmembrane domain. In further embodiments, the transmembrane domain is CD8 TM1 (SEQ ID NO: 68 or 69), CD8 TM2 (SEQ ID NO: 70 or 71), or CD28 TM (SEQ ID NO: 72 or 73).

[0027] In some embodiments, the method further comprises a transduction marker and / or a safety switch. In further embodiments, the transduction marker is enhanced green fluorescent protein (eGFP). In still further embodiments, the eGFP has the amino acid sequence of SEQ ID NO: 83. In some embodiments, the transduction marker and / or safety switch is truncated epidermal growth factor (EGFR). In further embodiments, the EGFR has the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transduction marker and / or safety switch is linked to the CAR by a truncation peptide. In further embodiments, the truncation peptide is a 2A peptide. In still further embodiments, the 2A peptide is a T2A peptide. In still further embodiments, the T2A peptide has the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CAR further comprises a second antigen-binding domain. In further embodiments, the second antigen-binding domain is a CD19 antigen-binding domain, a CD20 antigen-binding domain, or a CD22 antigen-binding domain.

[0028] In other embodiments, the present disclosure provides an expression vector encoding a CAR or TCR of the present disclosure.

[0029] In still other embodiments, the present disclosure provides host cells engineered to express a CD79b CAR or CD79b TCR. In some embodiments, the cell is engineered to express a CAR of the present disclosure. In some embodiments, the host cell is an immune cell. In further embodiments, the immune cell is a T cell. In still further embodiments, the T cell is a primary human T cell or TIL. In other embodiments, the T cell is a CD4+ T cell or a CD8+ T cell. In some embodiments, the primary human T cell is obtained from a healthy donor. In some embodiments, the T cell is an autologous T cell. In some embodiments, the T cell is an allogeneic T cell. In some embodiments, the cell is engineered using a CRISPR or transposase system.

[0030] In yet other embodiments, the present disclosure provides a pharmaceutical composition comprising CD79b-targeted T cells and a pharmaceutical carrier, wherein the CD79b-targeted T cells are engineered to express a CAR or TCR of the present disclosure.

[0031] In other embodiments, the present disclosure provides a composition comprising an effective amount of CD79b-targeted T cells for treating cancer in a subject, wherein the CD79b-targeted T cells are engineered to express a CAR or TCR of the present disclosure.

[0032] In yet other embodiments, the present disclosure provides for the use of a composition comprising an effective amount of CD79b-targeted T cells to treat cancer in a subject, wherein the CD79b-targeted T cells are engineered to express a CAR or TCR of the present disclosure.

[0033] In other embodiments, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of CD79b-targeted T cells, wherein the CD79b-targeted T cells are engineered to express a CAR or TCR of the present disclosure. In a further embodiment, the cancer is a B-cell malignancy. In yet a further embodiment, the B-cell malignancy is B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma, or chronic lymphocytic leukemia. In some embodiments, the subject has previously received CD19 CAR therapy. In some embodiments, the subject is resistant to CD19 CAR therapy. In a further embodiment, the subject has lost the CD19 antigen. In yet a further embodiment, the subject has relapsed with a CD19-negative tumor. In some embodiments, the CD79b-targeted T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. In some embodiments, the CD79b-targeted T cells are administered intravenously. In some embodiments, the methods further comprise administering at least a second anti-cancer treatment to the subject. In further embodiments, the second anti-cancer treatment is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, or cytokine therapy. In some embodiments, the cancer is a CD79b-expressing cancer.

[0034] In certain embodiments, the CAR further comprises a second antigen-binding domain. In some embodiments, the second antigen-binding domain is a CD19 antigen-binding domain, a CD20 antigen-binding domain, or a CD22 antigen-binding domain.

[0035] In another embodiment, an expression vector encoding the CD79b CAR of the present embodiment is provided.

[0036] Further provided herein is a host cell engineered to express a CD79b CAR, such as the CD79b of this embodiment. In some embodiments, the host cell is an immune cell, such as a T cell. In some embodiments, the T cell is a primary human T cell. In certain embodiments, the T cell is a CD4+ T cell or a CD8+ T cell. In some embodiments, the primary human T cell is obtained from a healthy donor. The T cell may be an autologous T cell or an allogeneic T cell.

[0037] Also provided herein is a pharmaceutical composition comprising CD79b CAR T cells (e.g., the CAR T cells of the present embodiment) and a pharmaceutical carrier. Further provided herein is a composition comprising an effective amount of CD79b CAR T cells (e.g., the CAR T cells of the present embodiment) for treating cancer in a subject. In another embodiment, provided is the use of a composition comprising an effective amount of CD79b CAR T cells (e.g., the CAR T cells of the present embodiment) for treating cancer in a subject.

[0038] In a further embodiment, a method for treating cancer in a subject is provided, the method comprising administering to the subject an effective amount of a CD79b CAR T cell (e.g., a CAR T cell of the present embodiment). In some embodiments, the cancer is a B-cell malignancy (e.g., B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma, or chronic lymphocytic leukemia). In certain embodiments, the cancer is a CD79b-expressing cancer.

[0039] In some embodiments, the subject has previously been administered CD19 CAR therapy. In certain embodiments, the subject is resistant to CD19 CAR therapy, such as due to loss of CD19 antigen. In certain embodiments, the subject has recurrent CD19-negative tumor.

[0040] In certain embodiments, the CD79b CAR T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. In additional embodiments, the method further comprises administering at least a second anticancer therapy to the subject. In some embodiments, the second anticancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0041] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0042] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0043] [Figure 1A] CD79b expression in cell lines. [Figure 1B] Expression of CD79b in human tissues. [Figure 1C] CD79b expression in leukemia. [Figure 1D] CD79b expression in lymphoma.

[0044] [Figure 2A] Flow cytometry analysis of CD79b-transduced cells. [Figure 2B] Binding affinity of CD79b monoclonal antibodies. [Figure 2C] Characterization of CD79b monoclonal antibodies. [Figure 2D] Staining of lymphoma cell line Clone 14.

[0045] [Figure 3A] Schematic depicting the construct for CD79b CAR. [Figure 3B] Flow cytometry analysis of CD79b CAR and CD19 CAR. [Figure 3C] Percent cytotoxicity of CD79b CAR and CD19 CAR with non-transduced T cells as a control. [Figure 3D] Flow cytometry of CD79b CAR and CD19 CAR with non-transduced T cells as a control.

[0046] [Figure 4A] T cells co-cultured with CD79b CAR and CD19 exon 2Δ splice variant. [Figure 4B] Flow cytometry analysis of the efficacy of CARs incubated for 4 days at an effector:target ratio of 5:1. [Figure 4C] Absolute cell counts of Daudi cells bearing CD79b CAR. [Figure 4D] Absolute cell counts of CD19 knockdown cells with CD79b CAR.

[0047] [Figure 5A] Schematic of preclinical studies. [Figure 5B] Bioluminescence images of mice under study. [Figure 5C] Percent survival of mice during the study.

[0048] [Figure 6A] A CD79b CAR construct of certain embodiments. [Figure 6B] The graphs and histograms show that anti-CD79b CAR T cells exhibit cytotoxicity against Daudi lymphoma cells in vitro. [Figure 6C]The graphs and histograms show that anti-CD79b CAR T cells exhibit cytotoxicity against Daudi lymphoma cells in vitro. [Figure 6D] Graphs and imaging show that anti-CD79b CAR T cells demonstrate efficacy against Daudi lymphoma xenografts in vivo. [Figure 6E] Graphs and imaging show that anti-CD79b CAR T cells demonstrate efficacy against Daudi lymphoma xenografts in vivo.

[0049] [Figure 7] Binding of anti-CD79b antibodies (clones 5B and 28B) to human CD79b.

[0050] [Figure 8A] Domain map of the anti-CD79b CAR used in the embodiments herein. [Figure 8B] Map of the CAR construct in a lentiviral vector (pLVEG) containing the EF1α promoter.

[0051] [Figure 9A] Transduction of CAR in NFAT reporter cells. [Figure 9B] Luciferase activity in transduced NFAT reporter cells cocultured with antibodies or Dadui Burkitt cells. [Figure 9C] Luciferase activity in transduced NFAT reporter cells cocultured with SUDHL6. [Figure 9D] Luciferase activity in transduced NFAT reporter cells cocultured with SUDHL6.

[0052] [Figure 10A] Representative transduction efficiencies of CARs in T cells. [Figure 10B]Phosphorylation of CD3ζ and ERK1 / 2 in T cells expressing or not expressing CAR.

[0053] [Figure 11A] Expansion of T cells expressing or not expressing CAR. [Figure 11B] Expansion of T cells expressing or not expressing CAR.

[0054] [Figure 12] Cytokine expression of T cells expressing or not expressing CAR.

[0055] [Figure 13A] Degranulation of T cells expressing or not expressing CAR in response to lymphoma cells. [Figure 13B] Degranulation of T cells expressing or not expressing CAR in response to lymphoma cells.

[0056] [Figure 14A] Cytotoxic activity of T cells expressing or not expressing CAR against lymphoma cells. [Figure 14B] Lysis of SUDHL6 cells by CAR T cells.

[0057] [Figure 15A] Bioluminescence imaging of tumor burden in mice treated with T cells expressing or not expressing CAR. [Figure 15B] Probability of survival in mouse cancer models treated with cells expressing or not expressing CAR. DETAILED DESCRIPTION OF THE INVENTION

[0058] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 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 usually retained in CD19-negative tumors that recur after CD19-specific CAR T cell therapy. Thus, in certain embodiments, the present disclosure provides CD79b monoclonal antibodies and CD79b-specific CARs, such as for CD79b-CAR T cells.

[0059] This study demonstrated the efficacy of this CD79b-specific CAR T cell product in in vitro and in vivo models. Three mouse monoclonal antibodies against human CD79b were developed using hybridoma technology. These antibodies were demonstrated to specifically bind to recombinant human CD79b, have high affinity (Kd range of 1.44–17.8 nM), and stain multiple lymphoma cell lines. Next, we cloned the heavy and light chain variable regions of the CD79b antibody and developed a lentiviral construct for an anti-CD79b CAR containing CD3ζ and CD28 / 4-1BB costimulatory domains. We demonstrated that the anti-CD79b CAR construct could be transduced into primary CD4+ and CD8+ T cells from healthy donors using lentivirus with a transduction efficiency of over 70%.

[0060] We observed that anti-CD79b CAR T cells, but not untransduced T cells, exhibited significant cytotoxic activity against Daudi Burkitt's lymphoma and Mino mantle cell lymphoma cell lines, comparable to that of control anti-CD19 CAR T cells. More importantly, anti-CD79b lysed CD19-CD79b+ lymphoma cells, whereas anti-CD19 CAR T cells did not. Furthermore, CD4 + Anti-CD79b CAR T cells and CD8 + Co-culture of anti-CD79b CAR T cells with lymphoma cells resulted in CD4 + Anti-CD79b CAR T cells and CD8 +Significant degranulation was observed in both anti-CD79b CAR T cells and NSG mice. The efficacy of anti-CD79b CAR T cells was also investigated in vivo against a Mino lymphoma xenograft model in NSG mice. Luciferase-labeled Mino mantle cell lymphoma cells were injected at 2 × 10 6 NSG mice were IV injected with 10 x 10 tumor cells / mouse. After 18 days, mice were 6 T cells / mice were treated via the tail vein 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 progression of tumor growth in mice treated with untransduced T cells. Meanwhile, tumor growth was inhibited and survival time improved in mice treated with anti-CD19 and anti-CD79b CAR T cells. Therefore, these results demonstrated the efficacy of this novel anti-CD79b CAR T cell therapy in patients with B cell malignancies. This may represent a novel strategy for overcoming resistance due to CD19 loss after CD19-specific CAR T cell therapy.

[0061] In some embodiments, the anti-CD79b CAR construct is encoded by a lentiviral vector. The vector can be transduced into immune cells such as T cells. The construct can include CD28, CD3ζ, and / or 4-1BB signaling domains. The construct can include a transduction marker such as eGFP or a truncated EGFR domain. The transduction marker can be linked to the CAR by a truncated peptide such as the 2A peptide.

[0062] Further provided herein is a method for treating cancer by administering the CD79b-specific CAR immune cells (e.g., T cells) provided herein. The cancer may be a B-cell malignancy expressing CD79b (e.g., B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma, or chronic lymphocytic leukemia). This treatment may be used to treat a subject with a B-cell malignancy who has relapsed with a CD19-negative tumor after anti-CD19-CAR T-cell therapy. II. Definition

[0063] As used herein, "essentially free" of a specific component means that the specific component is not intentionally formulated into the composition and / or is not present even as a contaminant or in trace amounts.Therefore, the total amount of the specific component due to any unintentional contamination of a composition is less than 0.05%, preferably less than 0.01%.Most preferred is a composition in which the amount of the specific component cannot be detected by standard analytical methods.

[0064] As used herein, "a" or "an" can mean one or more. When used in the claims, the words "a" or "an," when used in conjunction with the word "comprising," can mean one or more than one.

[0065] The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, although the present disclosure supports the definition to refer to alternatives only and "and / or." As used herein, "another" can mean at least a second or more. The terms "about," "substantially," and "approximately" generally mean the stated value plus or minus 5%.

[0066] "Treating" a disease or condition or treatment of a disease or condition refers to carrying out a protocol that may include administering one or more drugs to a patient with the goal of alleviating the signs or symptoms of the disease. Desirable effects of treatment include slowing the rate of disease progression, reversing or alleviating the disease state, and alleviating or improving prognosis. Alleviation can occur before, as well as after, the signs or symptoms of the disease or condition manifest. Thus, "treating" or "treatment" can include "preventing" or "prevention" of a disease or undesirable condition. Furthermore, "treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0067] As used throughout this application, the term "therapeutic effect" or "therapeutically effective" refers to anything that promotes or improves the well-being of a subject with respect to medical treatment for that condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer can include, for example, reducing tumor size, reducing the invasiveness of a tumor, reducing the rate of cancer growth, or preventing metastasis. Treating cancer can also refer to extending the survival time of a subject with cancer.

[0068] "Subject" and "patient" refer to humans or non-humans, e.g., primates, mammals, and vertebrates. In certain embodiments, the subject is a human.

[0069] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered appropriately to animals, such as humans. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients will be known to those of skill in the art in light of the present disclosure. Furthermore, it is understood that for administration to animals (e.g., humans), preparations should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0070] As used herein, "pharmaceutically acceptable carriers" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, 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, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, flow and nutritional supplements, such similar materials, and combinations thereof, as known to those skilled in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters. III.CD79b antibody

[0071] In certain embodiments, antibodies or fragments thereof that bind to at least a portion of CD79b and inhibit any of CD79b activities, including at least signal transduction, are contemplated. As used herein, the term "antibody" is intended to refer broadly to any immunological binding agent (e.g., IgG, IgM, IgA, IgD, IgE, and genetically modified IgG) and polypeptides comprising antibody CDR domains that retain antigen-binding activity. The antibody may be selected from the group consisting of a chimeric antibody, an affinity-matured antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or an antigen-binding antibody fragment, or a natural or synthetic ligand. In certain cases, the anti-CD79b antibody is a monoclonal antibody or a humanized antibody.

[0072] Thus, by known means and as described herein, polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs (including engineered forms of any of the foregoing) can be made specific for CD79b, one or more of its respective epitopes, or binding domains and CDRs (including engineered forms of any of the foregoing) whether the CD79b antigen or epitopes thereof are isolated from natural sources or are synthetic derivatives or variants of natural compounds.

[0073] Examples of antibody fragments suitable for this embodiment include: (i) V L , V H , C L and C H1 (ii) a Fab fragment consisting of the V domain; H and C H1 (iii) an “Fd” fragment consisting of a single antibody domain; L and V H (iv) an "Fv" fragment consisting of the V domain; H (v) an isolated CDR region; (vi) an F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; (vii) a V H Domains and V L (viii) bispecific single-chain Fv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies, which are multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Application Publication No. 20050214860). Fv, scFv, or diabody molecules include, but are not limited to, single-chain Fv molecules ("scFv"), in which two Fv domains are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bispecific single-chain Fv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies, which are multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Application Publication No. 20050214860). H Domains and V L The scFv can be stabilized by the incorporation of disulfide bridges linking the domains. Minibodies can also be made that contain an scFv connected to a CH3 domain.

[0074] Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al. (2003) describe "antibody-like binding peptidomimetics" (ABiPs), which are peptides that act as truncated antibodies and have certain advantages, such as a longer serum half-life and less cumbersome synthesis methods.

[0075] To generate antibodies specific to CD79b, animals can be inoculated with an antigen, such as the CD79b extracellular domain (ECD) protein. Antigens are often bound or conjugated to another molecule to enhance the immune response. As used herein, a conjugate is any peptide, polypeptide, protein, or nonproteinaceous substance bound to an antigen used to elicit an immune response in an animal. Antibodies produced in an animal in response to antigen inoculation include various non-identical molecules (polyclonal antibodies) produced by various individual antibody-producing B lymphocytes. Polyclonal antibodies are a mixed population of antibody species, each capable of recognizing a different epitope on the same antigen. Given stringent conditions for polyclonal antibody production in an animal, most of the antibodies in the animal's serum will recognize a collective epitope on the antigenic compound to which the animal was immunized. This specificity can be further enhanced by affinity purification, which selects only those antibodies that recognize the antigen or epitope of interest.

[0076] Monoclonal antibodies are antibodies of a single species, and all antibody-producing cells are derived from a single B lymphocyte cell line, so each antibody molecule recognizes the same epitope. The method for producing monoclonal antibodies (MAbs) generally begins with the same principles as those for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used in the production of monoclonal antibodies. In some embodiments, rabbit cells, sheep cells, or frog cells are used in the production of monoclonal antibodies. The use of rats is well known and may offer certain advantages. Mice (e.g., BALB / c mice) are routinely used and generally produce a high rate of stable fusions.

[0077] Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with the CD79b antigen with an immortal myeloma cell (usually a mouse myeloma). This technology provides a method for propagating a single antibody-producing cell for an indefinite number of generations, allowing the production of unlimited quantities of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

[0078] Plasma B cells (CD45 + CD5 - CD19 + ) can be isolated from freshly prepared rabbit peripheral blood mononuclear cells from immunized rabbits and further selected for CD79b-binding cells. After enrichment of antibody-producing B cells, total RNA can be isolated and cDNA synthesized. DNA sequences of both heavy and light chain antibody variable regions can be amplified, constructed into phage display Fab expression vectors, and transformed into E. coli. Fabs that specifically bind to CD79b can be selected through multiple rounds of enrichment panning and sequenced. Selected CD79b-binding hits can be expressed as rabbit and rabbit / human chimeric full-length IgG in human embryonic kidney (HEK293) cells (Invitrogen) using a mammalian expression vector system and purified using Protein G resin with a fast protein liquid chromatography (FPLC) separation unit.

[0079] In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising antigen-binding sequences derived from a non-human donor grafted onto heterologous non-human, human, or humanized sequences (e.g., framework and / or constant domain sequences). Methods have been developed to replace the light and heavy chain constant domains of a monoclonal antibody with analogous domains of human origin, leaving the variable regions of the foreign antibody intact. Alternatively, "fully human" monoclonal antibodies have been produced in mice transfected with 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 containing both rodent, e.g., murine, and human amino acid sequences. In "humanized" monoclonal antibodies, only the hypervariable CDRs are derived from a murine monoclonal antibody, while the framework and constant regions are derived from human amino acid sequences (see U.S. Pat. Nos. 5,091,513 and 6,881,557). It is believed that replacing amino acid sequences within an antibody that are characteristic of rodents with amino acid sequences found at the corresponding positions in human antibodies reduces the likelihood of adverse immune reactions during therapeutic use. Hybridomas or other cells that produce antibodies may also be subject to genetic mutations or other alterations that may or may not change the binding specificity of the antibodies produced by the hybridoma.

[0080] Methods for producing polyclonal antibodies in various animal species, as well as methods for making various types of monoclonal antibodies, including humanized antibodies, chimeric antibodies, and fully human antibodies, are well known and highly predictable in the art. For example, the following U.S. patents and patent applications provide useful 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. ; Same No. 4,366,241; Same No. 4,469,797; Same No. 4,472,509; Same No. 4,606,855; Same No. 4,703,003; Same No. 4,742,159; Same No. 4,767,72 No. 0; No. 4,816,567; No. 4,867,973; No. 4,938,948; No. 4,946,778; No. 5,021,236; No. 5,164,296; No. 5,196,0 No. 66; No. 5,223,409; No. 5,403,484; No. 5,420,253; No. 5,565,332; No. 5,571,698; No. 5,627,052; No. 5,656 ,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,87 Nos. 1,907; 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, published patent applications and other publications cited herein and all patents, published patent applications and other publications cited therein are hereby incorporated by reference.

[0081] Antibodies can be produced from any animal origin, including birds and mammals. Preferably, the antibodies are sheep, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken antibodies. Furthermore, newer technologies allow for the development of human antibodies and screening from human combinatorial antibody libraries. For example, as described in U.S. Patent No. 6,946,546, incorporated herein by reference, bacteriophage antibody expression technology allows for the production of specific antibodies without the need for animal immunization.

[0082] It is fully expected 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 origin of the antibody. Certain animal species may be undesirable for the production of therapeutic antibodies because they may be more likely to cause allergic reactions due to activation of the complement system via the "Fc" portion of the antibody. However, whole antibodies may be enzymatically digested to produce "Fc" (complement-binding) fragments and antibody fragments containing the binding domain or CDRs. Removal of the Fc portion reduces the likelihood that the antigen-antibody fragment will elicit an undesirable immunological response, so antibodies without Fc may be preferred for prophylactic or therapeutic treatment. As described above, antibodies may be engineered to be chimeric or partially or fully human to reduce or eliminate adverse immunological consequences resulting from administering to animals antibodies produced in other species or antibodies with sequences derived from other species.

[0083] Substitutional variants usually involve exchanging one amino acid for another at one or more sites within the protein, and can be designed to modulate one or more properties of the polypeptide, with or without loss of other functions or properties. Substitutions can be conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art, and include, for example, 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, substitution may be non-conservative substitution, such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with a chemically dissimilar residue, for example, substituting a polar or charged amino acid for a non-polar or uncharged amino acid, and vice versa.

[0084] The protein may be a recombinant protein, i.e., a protein synthesized in vitro. Alternatively, non-recombinant or recombinant proteins may be isolated from bacteria. It is also contemplated that bacteria containing such variants may be implemented in compositions and methods. Consequently, the protein need not be isolated.

[0085] It is contemplated that the composition will have from about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. Thus, the concentration of protein in the composition can be about, at least about, or at most about 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 derivable therein). Of these, about, at least about, or at most about 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.

[0086] Antibodies, or preferably immunological portions of antibodies, may be chemically conjugated to or expressed as fusion proteins with other proteins, and all such fusion proteins are included in the definition of an antibody or immunological portion of an antibody for purposes of this specification and the appended claims.

[0087] Embodiments provide antibodies and antibody-like molecules, polypeptides, and peptides against CD79b that are linked to at least one agent to form an antibody conjugate or payload. To enhance the effectiveness of antibody molecules as diagnostic or therapeutic agents, it is conventional to link, covalently bind, or conjugate at least one desired molecule or moiety to the antibody molecule. Such molecules or moieties can be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules attached to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, and the like. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include ligands such as enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or biotin.

[0088] Several methods for attaching or conjugating antibodies to conjugate moieties are known in the art. Some attachment methods include, for example, the use of metal chelate complexes using organic chelating agents attached to antibodies, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6-diphenylglycouril-3. Alternatively, monoclonal antibodies may be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. IV. Cell therapy

[0089] Certain embodiments of the present disclosure relate to obtaining T cells and administering them to a subject as immunotherapy against targeted cancer cells. The cells may deliver antibody compositions encompassed herein, but may or may not themselves be immune cells. In certain embodiments, the cells are immune cells. Examples of cells include T cells (including T cells or T 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.

[0090] Several basic approaches for the induction, activation, and expansion of functional anti-tumor effector T cells have been reported in the past 20 years. These include autologous cells such as tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by target cell membrane capture; 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 "redirected" to express tumor-reactive or chimeric TCR molecules that exhibit antibody-like tumor recognition capabilities, known as "T-bodies." These approaches have led to numerous protocols for preparing and immunizing T cells that can be used in the methods of the present disclosure. Preparation of AT cells

[0091] In some embodiments, the T cells are derived from blood, bone marrow, lymph, or lymphoid organs. In some embodiments, the cells are human cells. The cells are typically primary cells, e.g., cells isolated directly from a subject and / or cells isolated and frozen from a subject. In some embodiments, the cells include one or more subsets of T cells or other cell types (e.g., the entire T cell population, CD4 + cells, CD8 +The present invention includes cells and subpopulations thereof, e.g., those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject being treated, the cells can be allogeneic and / or autologous. In some embodiments, such as with existing technologies, the cells are pluripotent and / or multipotent (e.g., stem cells such as induced pluripotent stem cells (iPSCs)). In some embodiments, the method includes isolating cells from a subject, preparing, treating, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0092] T cells (e.g., CD4 + and / or CD8 + T cell) subtypes and subpopulations include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes (e.g., stem cell memory T (TSC) M ), Central Memory T(T CM ), Effector Memory T(T EM ) or terminally differentiated effector memory T (T TEMRA ) cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, intrinsic and adaptive regulatory T (Treg) cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), alpha / beta T cells, and delta / gamma T cells.

[0093] In some embodiments, one or more of the T cell populations are enriched or depleted for cells that are positive for a specific marker, such as a surface marker, or negative for a specific marker. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain T cell populations (e.g., non-memory cells), but present or expressed at relatively high levels on certain other T cell populations (e.g., memory cells). In one embodiment, the cells (e.g., CD8 + cells or CD3 + The cells) are enriched (i.e., positively selected) for cells that are CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L positive or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or are depleted (e.g., negatively selected) for cells that are CD45RA positive or express high surface levels of CD45RA. In some embodiments, the cells are enriched or depleted for cells that are CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127) positive or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some examples, CD8 + T cells are enriched for CD45RO positive (or CD45RA negative) and CD62L positive cells.

[0094] In some embodiments, T cells are isolated from PBMC samples by negative selection of markers (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes or other leukocytes). + or CD8 + By using a selection process, CD4 + Helper T cells and CD8 + Cytotoxic T cells are isolated. + and CD8+ The population can be further sorted into subpopulations by positive or negative selection of markers that are expressed or expressed to a relatively high degree on one or more subpopulations of naive, memory and / or effector T cells.

[0095] In some embodiments, CD8 + The cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM Enrichment of T cells is performed to enhance efficacy, such as improving long-term survival, expansion, and / or engraftment after administration, which in some embodiments is particularly robust in such subpopulations. CM Enriched CD8 + T cells and CD4 + Combining it with T cells further increases efficacy.

[0096] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension is purified in any suitable manner, for example, mechanically (e.g., by gentleMACS TM Tumors can be disaggregated using a dissociator (Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). A single cell suspension of the enzymatic digest of the tumor is cultured in interleukin-2 (IL-2). The cells are grown to confluence (e.g., approximately 2 x 10 6The cells are cultured for, for example, about 5 to about 21 days, preferably about 10 to about 14 days, until they become lymphocytes. For example, the cells can be cultured for 5 days, 5.5 days, or 5.8 days to 21 days, 21.5 days, or 21.8 days, for example, 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days, or 14.8 days.

[0097] The cultured T cells can be pooled and rapidly expanded. Rapid expansion over about 10 to about 14 days, preferably about 14 days, results in an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or more). More preferably, rapid expansion over about 10 to about 14 days, preferably about 14 days, results in an increase of at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900, or more).

[0098] Expansion can be achieved by any of several methods known in the art. For example, T cells can be readily expanded using nonspecific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. The nonspecific T cell receptor stimulation can include approximately 30 ng / ml of OKT3 (available from Ortho-McNeil®, Raritan, NJ), a mouse monoclonal anti-CD3 antibody. Alternatively, T cells can be readily expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more cancer antigens (including antigenic portions thereof, such as epitopes, or cells) in the presence of a T cell growth factor (e.g., 300 IU / ml IL-2 or IL-15 (IL-2 is preferred)), which can optionally be expressed from a vector, e.g., a human leukocyte antigen A2 (HLA-A2)-binding peptide. The in vitro-induced T cells are rapidly expanded by restimulation with the same cancer antigen pulsed on antigen-presenting cells expressing HLA-A2. Alternatively, the T cells can be restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0099] The autologous T cells can be modified to express T cell growth factors that promote the proliferation 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. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rded., 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 certain embodiments, the modified autologous T cells express high levels of T cell growth factors. T cell growth factor coding sequences (e.g., the coding sequence for IL-12) are readily available in the art, as are promoters whose operably linked sequences promote high level expression of the T cell growth factor coding sequence. B. Genetically Engineered Antigen Receptors

[0100] The cells can be genetically engineered to express an engineered antigen receptor, such as an engineered TCR or chimeric antigen receptor (CAR). For example, autologous T cells can be engineered to express a T cell receptor (TCR) with antigen specificity for a cancer antigen, such as CD79b. Suitable TCRs include those with antigen specificity for melanoma antigens, such as gp100 or MART-1. Suitable engineering methods are known in the art. See, for example, Sambrook and Ausubel, supra. For example, the T cells can be transduced to express a TCR with antigen specificity for a cancer antigen using the transduction methods described in Heemskerk et al. Hum Gene Ther. 19:496-510 (2008) and Johnson et al. Blood 114:535-46 (2009).

[0101] In some embodiments, the T cells comprise one or more nucleic acids encoding one or more antigen receptors, introduced via genetic engineering, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in the cell or a sample obtained from the cell, e.g., nucleic acids obtained from another organism or cell that are not normally found in, e.g., the cell being engineered and / or the organism from which such cell originates. In some embodiments, the nucleic acids are non-naturally occurring, such as nucleic acids not found in nature (e.g., chimeras).

[0102] In some embodiments, the CAR comprises 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 (e.g., a peptide antigen of an intracellular protein) that is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule, similar to a TCR.

[0103] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing those receptors into cells are described, for example, in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US2013014 9337, U.S. Patent Nos. 6,451,995, 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 European Patent Application No. EP 2537416, and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some embodiments, genetically engineered antigen receptors include CARs such as those described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668A1. 1. Chimeric antigen receptor

[0104] In some embodiments, the CAR comprises a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen-binding region.

[0105] In some embodiments, engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). These CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some embodiments via a linker and / or transmembrane domain. Such molecules typically mimic or mimic signaling through a natural antigen receptor, through such a receptor in conjunction with a costimulatory receptor, and / or through a costimulatory receptor alone.

[0106] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides (including humanized CARs (hCARs) to reduce immunogenicity) that include an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope that includes a space shared between one or more antigens. In certain embodiments, the binding region may include 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, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0107] It is contemplated that the human CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present disclosure includes the full-length cDNA or coding region of the CAR. The antigen-binding region or domain is the V of a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody. H Chain and V LThe fragments may include fragments of the chains (e.g., those described in U.S. Patent No. 7,109,304, incorporated herein by reference). The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0108] The configuration can be a multimer (e.g., a diabody or multimer). The multimer is most likely formed by cross-pairing of the variable portions of the light and heavy chains into a diabody. The hinge portion of the construct can have several options, ranging from a complete deletion, to maintaining the first cysteine, to substituting proline instead of serine, to truncating up to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerizing protein can serve this purpose. Only one of the Fc domains can be used, for example, the CH2 or CH3 domain from a human immunoglobulin. The hinge, CH2, and CH3 regions of a human immunoglobulin modified to improve dimerization can also be used. Only the hinge portion of an immunoglobulin can also be used. A portion of CD8 alpha can also be used.

[0109] In some embodiments, the CAR nucleic acid comprises a sequence encoding another costimulatory receptor, such as a transmembrane domain and a modified CD28 intracellular signaling domain, including, but not limited to, one or more of CD28, CD27, OX-40 (CD134), and 4-1BB (CD137).

[0110] In some embodiments, CARs are constructed to have specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker and / or an antigen intended to induce an attenuated response (e.g., an antigen expressed in a normal or non-diseased cell type). Thus, the CAR typically comprises, in its extracellular portion, one or more antigen-binding molecules (e.g., one or more antigen-binding fragments, antigen-binding domains, or antigen-binding portions) or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR comprises an antigen-binding portion of an antibody molecule (e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb)).

[0111] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA, cDNA, or synthesized (e.g., via PCR), or a combination thereof. Since introns have been found to stabilize mRNA, depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof. It may also be beneficial to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0112] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector.Methods for stably transfecting cells by electroporation using naked DNA are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding the chimeric receptor that is contained in a plasmid expression vector in the appropriate orientation for expression.

[0113] Alternatively, viral vectors (such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors or lentiviral vectors) can be used to introduce chimeric constructs into immune cells.The vector suitable for use according to the method of the present disclosure is a non-replicating vector in immune cells.There are many known virus-based vectors (such as HIV, SV40, EBV, HSV or BPV-based vectors), and the copy number of the virus that is maintained in cells is low enough to maintain the viability of the cells.

[0114] In some embodiments, the antigen-specific binding component, i.e., the recognition component, is linked to one or more transmembrane domains and intracellular signaling domains.In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR.In one embodiment, the transmembrane domain that naturally associates 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 avoid such domains from binding to the transmembrane domains of the same or different surface membrane proteins, in order to minimize interaction with other members of the receptor complex.

[0115] In some embodiments, the transmembrane domain is derived from natural or synthetic sources. If the origin is natural, the domain is derived from any membrane-bound or transmembrane protein in some embodiments. The transmembrane region includes (i.e., includes at least) a transmembrane region derived from the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, and CD3 delta. Alternatively, the transmembrane domain is a synthetic transmembrane domain in some embodiments. In some embodiments, synthetic transmembrane domains primarily contain hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine may be found at each end of a synthetic transmembrane domain.

[0116] In a specific embodiment, the CAR construct comprises light chain-linker-heavy chain-hinge-transmembrane domain-signaling domain, where the linker comprises, consists of, or consists essentially of linker 1 (SEQ ID NO: 44 or 45), linker 2 (SEQ ID NO: 46 or 47), linker 3 (SEQ ID NO: 48 or 49), or linker 4 (SEQ ID NO: 50 or 51). The hinge comprises, consists of, or consists essentially of 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 IgG4 CH1CH2CH3 (SEQ ID NO: 66 or 67). The transmembrane domain comprises, consists of, or consists essentially of 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 certain embodiments, a CD8TM lacking (contiguous) the amino acid sequence of LYC and / or NHRN, such as at its C-terminus, is utilized. The signaling domain comprises, consists of, or consists essentially of 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 CD3 intracellular (SEQ ID NO:80 or 81). The CAR construct may further comprise, consist of, or consist essentially of GFP (SEQ ID NO:82 or 83), T2A (SEQ ID NO:84 or 85), and / or EGFR (SEQ ID NO:40 or 41). Exemplary heavy chain (HC), linker and light chain (LC) combinations may include, but are not limited to, LC-linker1-HC; LC-linker2-HC; LC-linker3-HC; LC-linker4-HC; HC-linker1-LC; HC-linker2-LC; HC-linker3-LC; or HC-linker4-LC. 2. T cell receptor (TCR)

[0117] In some embodiments, genetically engineered antigen receptors include recombinant TCRs and / or TCRs cloned from naturally occurring T cells. "T cell receptor" or "TCR" refers to a molecule that contains a variable α chain and a variable β chain (also known as TCRα and TCRβ, respectively) or a variable γ chain and a variable δ chain (also known as TCRγ and TCRδ, respectively), and that can specifically bind to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type.

[0118] TCRs, which typically exist as αβ and γδ types, are generally similar in structure, although the T cells expressing them may differ in anatomical location or function. TCRs may be found on the cell surface or in soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes) and are typically involved in recognizing antigens bound to major histocompatibility complex (MHC) molecules on the surface. In some embodiments, TCRs may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., 1997). For example, in some embodiments, 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 associate with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs of the αβ or γδ types.

[0119] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment (e.g., an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e., an MHC-peptide complex). An "antigen-binding portion" or antigen-binding fragment of a TCR, which may be used interchangeably, refers to a molecule that includes only a portion of the structural domain of the TCR but binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion includes sufficient variable domains of the TCR (e.g., the variable a chain and variable β chain of the TCR) to form a binding site for binding to a specific MHC-peptide complex, e.g., each chain typically includes three complementarity-determining regions.

[0120] In some embodiments, the variable domains of TCR chains associate to form loops, or complementarity-determining regions (CDRs) similar to immunoglobulins, which mediate antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Typically, like immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the primary CDR involved in recognizing processed antigens, although CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, whereas CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the beta chain may contain an additional hypervariable (HV4) region.

[0121] In some embodiments, a TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., a chain, β chain) comprises two immunoglobulin domains: a variable domain (e.g., V) at the N-terminus; aor Vp; usually Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest," US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed., amino acids 1-116), and one constant domain adjacent to the cell membrane (e.g., the a-chain constant domain or C a The TCR may comprise a β-chain constant domain or Cp, typically amino acids 117-259 according to Kabat, and a β-chain constant domain or Cp, typically amino acids 117-295 according to Kabat. For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two membrane-proximal constant domains and two membrane-distal variable domains containing the CDRs. The constant domain of the TCR domain contains a short connective sequence in which cysteine ​​residues form disulfide bonds, thereby forming a link between the two chains. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α-chain and β-chain, such that the TCR comprises two disulfide bonds in the constant domain.

[0122] 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. In some cases, due to its structure, the TCR can associate with other molecules such as CD3. For example, a TCR that includes a constant domain along with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0123] Generally, CD3 is a multiprotein complex that can have three distinct chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, this complex can contain a homodimer of CD3γ, CD3δ, two CD3ε, and a CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a property 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 known as an immunoreceptor tyrosine-based activation motif (ITAM), while each CD3ζ chain contains three. Generally, ITAMs are involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in transmitting signals from the TCR to the cell. The CD3 chain and the ζ chain associate with the TCR to form a complex known as the T cell receptor complex.

[0124] In some embodiments, the TCR can be a heterodimer of two chains, α and β (or optionally γ and δ), or can be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α and β or γ and δ) linked, such as by a disulfide bond. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, nucleic acids encoding the TCR can be obtained from various 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, e.g., a cell, e.g., a T cell (e.g., a cytotoxic T cell), T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from an in vivo isolated cell. In some embodiments, a high-affinity T cell clone can be isolated from a patient and the TCR isolated. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, TCR clones against a target antigen are produced in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). In some embodiments, phage display is used to isolate TCRs against the target antigen. In some embodiments, TCRs or antigen-binding portions thereof can be synthetically produced with knowledge of the sequence of the TCR. C. Delivery method

[0125] Those skilled in the art would be fully capable of constructing vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) to express the antigen receptors of the present disclosure. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors (including replication-competent, replication-deficient, and gutless forms thereof), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors, and the like. These include, but are not limited to, adenovirus (A virus) vectors and group B adenovirus enadenotucirev vectors. 1. Viral Vectors

[0126] A viral vector encoding an antigen receptor can be provided in certain embodiments of the present disclosure. When creating a recombinant viral vector, non-essential genes are usually replaced with genes or coding sequences for heterologous (or non-native) proteins. A viral vector is a type of expression construct that uses viral sequences to introduce nucleic acids and, in some cases, proteins into cells. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into the genome of host cells and stably and efficiently express viral genes, makes these viruses attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain embodiments of the present disclosure are described below.

[0127] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136).

[0128] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo.For example, recombinant lentiviruses that can infect non-dividing cells (wherein suitable host cells are transfected with two or more vectors that have packaging functions, i.e., gag, pol and env, and rev and tat) are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. 2. Regulatory elements

[0129] The expression cassette contained in the vector useful in the present disclosure includes, inter alia, a eukaryotic transcription promoter operably linked to a protein-coding sequence, a splice signal including an intervening sequence, and a transcription termination / polyadenylation sequence (5' to 3' direction). The promoters and enhancers that control the transcription of protein-coding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information carried by each element, allowing different genes to exert different, often complex, patterns of transcriptional control. Promoters used in the context of the present disclosure include constitutive promoters, inducible promoters, and tissue-specific promoters. a. Promoter / enhancer

[0130] The expression constructs provided herein include a promoter that drives expression of an antigen receptor. Promoters generally contain sequences that function to locate the start site for RNA synthesis. The most well-known example of this is the TATA box, but some promoters, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, lack a TATA box and instead use discrete elements overlapping the start site itself to anchor the start location. Additional promoter elements control the frequency of transcription initiation. These are usually located in the region 30-110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription initiation site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

[0131] The spacing between promoter elements is often variable, and promoter function is preserved even when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decrease. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0132] A promoter may be a promoter naturally associated with a nucleic acid sequence, such as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as an "endogenous" promoter. Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not naturally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not naturally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not "naturally occurring," i.e., contain various elements of various transcriptional control regions and / or expression-altering mutations. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to synthetically producing promoter and enhancer nucleic acid sequences, recombinant cloning and / or PCR TMNucleic acid amplification techniques, including but not limited to, can be used to generate sequences in connection with the compositions disclosed herein. Additionally, it is contemplated that regulatory sequences that direct transcription and / or expression of sequences in organelles other than the nucleus (e.g., mitochondria, chloroplasts, etc.) can be used as well.

[0133] Naturally, it will be important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are aware that a combination of promoter, enhancer, and cell type is generally used for protein expression (see, e.g., Sambrook et al. 1989, incorporated herein by reference). The promoter used can be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter that directs high-level expression of the introduced DNA segment and is useful under appropriate conditions (e.g., a promoter useful in the large-scale production of recombinant proteins and / or recombinant peptides). The promoter can be heterologous or endogenous.

[0134] Additionally, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Data Base EPDB via the World Wide Web at epd.isb-sib.ch / ) can be used to drive expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another viable embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if an appropriate bacterial polymerase is provided as part of the delivery complex or as an additional genetic expression construct.

[0135] Non-limiting examples of promoters include early or late viral promoters (e.g., SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter); eukaryotic promoters (e.g., beta-actin promoter, GADPH promoter, metallothionein promoter); and chain-like response element promoters (e.g., cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and minimal TATA box proximal response element promoter (tre)). Human growth hormone promoter sequences (e.g., human growth hormone minimal promoter listed in GenBank, Accession No. X05244, nucleotides 283-341) or mouse mammary tumor promoter (available from ATCC, Cat. No. ATCC 45007) can also be used. In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter; however, any other promoter useful for driving expression of therapeutic genes is also applicable to the practice of the present disclosure.

[0136] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have the ability to act in cis and in any orientation over relatively long distances (up to several kilobases away from the target promoter).However, enhancer function is not necessarily limited to such long distances, as enhancers can also function proximal to a given promoter. b. Initiation signal and linked expression

[0137] Specific initiation signals may also be used in the expression constructs provided herein for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals (including the ATG initiation codon) may need to be provided. One of ordinary skill in the art would be readily able to determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in-frame" with the reading frame of the desired coding sequence. The exogenous translational control signals and initiation codons may be natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcriptional enhancer elements.

[0138] In certain embodiments, the use of internal ribosome entry site (IRES) elements is used to generate multigene messages, i.e., polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornaviridae family (polio and encephalomyocarditis) and IRESs from mammalian messages have been reported. IRES elements can link heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together, generating polycistronic messages. IRES elements allow each open reading frame to be accessible to ribosomes for efficient translation. Multiple genes can also be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0139] Furthermore, certain 2A sequence elements can be used to achieve linked or simultaneous expression of genes within the constructs provided herein. For example, a cleavage sequence can be used to link open reading frames to form a single cistron, thereby co-expressing genes. An exemplary cleavage sequence is F2A (foot-and-mouth disease virus 2A) or a "2A-like" sequence (e.g., Thosea asigna virus 2A; T2A). 3. Replication starting point To propagate a vector in a host cell, the vector may contain one or more origins of replication (often referred to as "ori"), such as a nucleic acid sequence corresponding to the EBV oriP described above, which is a specific nucleic acid sequence at which replication is initiated, or a genetically engineered oriP with a similar or enhanced function in programming. Alternatively, origins of replication of other viruses that replicate extrachromosomally, as described above, or autonomously replicating sequences (ARS) can be used. 4. Selectable and Screenable Markers

[0140] In some embodiments, cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker causes an identifiable change in the cell that allows cells containing the expression vector to be easily identified. Generally, a selectable marker is a marker that confers a selectable characteristic. A positive selectable marker is a marker whose presence allows its selection, and a negative selectable marker is a marker whose presence prevents selection. An example of a positive selectable marker is a drug resistance marker.

[0141] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of conditions, other types of markers are contemplated, including colorimetrically based screenable markers such as GFP. Alternatively, screenable enzymes can be used as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, perhaps in conjunction with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are well known to those skilled in the art. 5. Other nucleic acid delivery methods

[0142] In addition to viral delivery of nucleic acids encoding antigen receptors, the following methods are contemplated in this disclosure as additional methods of recombinant gene delivery into a given host cell.

[0143] The introduction of nucleic acid such as DNA or RNA into immune cells of the present disclosure can be carried out by any method suitable for nucleic acid delivery to transform cells, as described herein or known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA (for example, ex vivo transfection, injection (including microinjection)); electroporation; calcium phosphate precipitation; DEAE-dextran followed by polyethylene glycol; direct sonication; liposome-mediated transfection and receptor-mediated transfection; microprojectile bombardment; agitation with silicon carbide fiber; Agrobacterium-mediated transformation; desiccation / inhibition-mediated DNA uptake, and any combination of such methods. By applying these methods and the like, organelles, cells, tissues or organisms can be stably or transiently transformed. V. Treatment Method

[0144] Certain aspects of this embodiment can be used to prevent or treat diseases or disorders associated with CD79b signaling, including those in which killing CD79b-positive cells can ameliorate at least one symptom of the disease or disorder. CD79b signaling can be reduced by any suitable composition that prevents the proliferation of cancer cells. In certain cases, such a substance can be an anti-CD79b antibody or a cell expressing an anti-CD79b CAR.

[0145] In some embodiments, the present disclosure provides a method for immunotherapy, comprising administering an effective amount of a composition comprising an antibody (comprising at least CAR T cells) of the present disclosure. In one embodiment, a medical disease or disorder is treated by administering a CAR-expressing cell population that induces an immune response. In certain embodiments of the present disclosure, cancer is treated by administering a CAR immune cell population that induces an immune response. Provided herein is a method for treating or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of an antigen-specific cell therapy. The method may be applied to the treatment of immune disorders, solid cancers, and hematological cancers, for example. In particular, the cancer may 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's lymphoma, and chronic lymphocytic leukemia.

[0146] The tumors for which this treatment method is useful include any malignant cell type, such as those found in solid tumors or blood tumors.Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary blood tumors include bone marrow tumors, T- or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including digestive cancer and gastrointestinal stromal cancer), 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.

[0147] The cancer may specifically be cancer of the following histological types, but is not limited to: neoplasia, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular adenocarcinoma Follicular carcinoma; Follicular adenocarcinoma; Papillary-follicular adenocarcinoma; Non-encapsulated sclerosing carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma; Adnexal carcinoma; Apocrine adenocarcinoma; Sebaceous gland carcinoma; Ceruminous adenocarcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant; Granulosa cell tumor, malignant; Androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma Tumor, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Glomus angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Lentigo maligna melanoma; Acral lentiginous melanoma; Nodular melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Mixed Müllerian tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Fetal Cancer; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Paracortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillar astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal;Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Lateral granuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cleaved cell NHL; Bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; 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.

[0148] Certain embodiments relate to methods for treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by the abnormal proliferation (production by division) of blood cells, usually white blood cells (leukocytes). Leukemia is part of a broad group of diseases called hematological malignancies. Leukemia is a broad term that encompasses a wide variety of diseases. Leukemia is clinically and pathologically divided into acute and chronic forms.

[0149] In some embodiments of the methods of the present disclosure, the activated CD4 T cells and / or CD8 T cells in the individual are characterized by increased cytolytic activity compared to before administration of the γ-IFN-producing CD4 T cells and / or CD8 T cells, and / or a combination thereof. γ-IFN can be measured by any means known in the art, including, for example, intracellular cytokine staining (ICS), which involves fixing, permeabilizing, and staining cells with an antibody against γ-IFN. Cytolytic activity can be measured by any means known in the art, for example, using a cell killing assay with a mixture of effector and target cells.

[0150] In some embodiments, the subject may be administered non-myeloablative lymphodepleting chemotherapy prior to T cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable such treatment, administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma, which may be metastatic. An exemplary administration route for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain embodiments, approximately 60 mg / kg of cyclophosphamide is administered for two days, followed by approximately 25 mg / m 2 of fludarabine was administered for 5 days.

[0151] In certain embodiments, a T cell growth factor that promotes the proliferation and activation of autologous T cells is administered to a subject simultaneously with or subsequent to the administration of autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the proliferation and activation of autologous T cells. Examples of suitable T cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations (e.g., IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2). IL-12 is a preferred T cell growth factor.

[0152] Therapeutically effective amounts of immune cells can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal or intraarticular injection or infusion.

[0153] For accessible, discrete solid tumors, intratumoral injection, i.e., injection into the tumor vasculature, is particularly contemplated. Local, regional, or systemic administration may also be appropriate. For tumors >4 cm, the administered volume is about 4-10 ml (especially 10 ml), and for tumors <4 cm, a volume of about 1-3 ml is used (especially 3 ml). Multiple injections delivered as a single dose comprise a volume of about 0.1 to about 0.5 ml.

[0154] The T cell population may be administered in a treatment regimen consistent with the disease, e.g., in a single dose or multiple doses over one to several days to ameliorate the disease state, or in periodic doses over a longer period to inhibit disease progression and prevent disease recurrence. The precise dose employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be decided according to the judgment of the physician and each patient's circumstances. The therapeutically effective amount of T cells will depend on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, the dose that may be used in treating a human subject is at least 3.8 x 10 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 In certain embodiments, the dose used in treating a human subject is in the range of about 3.8 x 10 9 ~Approx. 3.8×10 10 T cells / m 2 In additional embodiments, the therapeutically effective amount of T cells is in the range of about 5×10 6 cells / kg body weight~approx. 7.5×10 8 cells / kg body weight (e.g., approximately 2 x 10 7 Cells ~ approx. 5 x 10 8 cells / kg body weight or approximately 5 x 10 7 cells ~ approx. 2 x 10 8 The amount of T cells can vary (cells / kg body weight). The exact amount of T cells can be easily determined by those skilled in the art based on the age, weight, sex and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0155] In certain embodiments of the present disclosure, an effective amount of CD79b CAR-expressing immune cells is delivered to an individual in need thereof, such as an individual with cancer.The cells then strengthen the individual's immune system to attack cancer cells.In some embodiments, an individual is provided with one or more doses of immune cells.When an individual is provided with two or more doses of immune cells, the period between doses should be sufficient to allow time for the immune cells to grow within the individual, and in certain embodiments, the period between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0156] In a specific embodiment, the cells engineered to express a CD79b CAR are administered in a therapeutically effective amount (10 3 ~10 10A therapeutically effective amount is provided to an individual in the range of 10 3 ~10 10 , 10 3 ~10 9 , 10 3 ~10 8 , 10 3 ~10 7 , 10 3 ~10 6 , 10 3 ~10 5 , 10 3 ~10 4 , 10 4 ~10 10 , 10 4 ~10 9 , 10 4 ~10 8 , 10 4 ~10 7 , 10 4 ~10 6 , 10 4 ~10 5 , 10 5 ~10 10 , 10 5 ~10 9 , 10 5 ~10 8 , 10 5 ~10 6 , 10 6 ~10 10 , 10 6 ~10 9 , 10 6 ~10 8 , 10 7 ~10 10 , 10 7 ~10 8 , 10 8 ~10 10 , 10 9 ~10 10 cells, or 10 9 ~10 10 Thus, in certain embodiments, an individual with a cancer is provided one or more times with a therapeutically effective amount of cells expressing a CD79b CAR. A. Pharmaceutical Compositions

[0157] Also provided herein are pharmaceutical compositions and formulations comprising the CAR T cells and a pharmaceutically acceptable carrier.

[0158] Pharmaceutical compositions and formulations as described herein comprise the active ingredient (e.g., antibody or polypeptide) having a desired degree of purity in one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 ndThe pharmaceutical composition may be prepared in the form of a lyophilized formulation or an aqueous solution by mixing with (Illegible, 2012 edition). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers (e.g., phosphate, citric acid, and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride); hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol alcohol, butyl alcohol, or benzyl alcohol; alkyl parabens (e.g., methylparaben or propylparaben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues). polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., polyethylene glycol (PEG)). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as neutral active soluble hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). In one embodiment, the sHASEGP is used in combination with one or more additional glycosaminoglycanases, for example, chondroitinases. B. Combination Therapy

[0159] In certain embodiments, the compositions and methods of this embodiment comprise a T cell population that is combined with at least one additional treatment.The additional treatment can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof.The additional treatment can be in the form of adjuvant therapy or neoadjuvant therapy.

[0160] In some embodiments, the additional treatment is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is administration of a side effect limiting agent (e.g., an agent aimed at reducing the incidence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment 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 treatment may be one or more chemotherapeutic agents known in the art.

[0161] Immune cell therapy can be administered before, during, after, or in various combinations with an additional cancer treatment, such as immune checkpoint therapy. These administrations can occur at intervals ranging from simultaneous administration to minutes, days, or weeks. In embodiments in which immune cell therapy is provided to a patient separately from an additional therapeutic agent, it is common to ensure that no significant time elapses between delivery times so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is contemplated that the antibody therapy and anticancer therapy may be provided to the patient within about 12 to 24 or 72 hours of each other, more particularly, within about 6 to 12 hours of each other. In such situations, if several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between administrations, it may be desirable to significantly extend the treatment period.

[0162] Various combinations can be used. In the example below, immune cell therapy is "A" and anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0163] Administration of any compound or treatment of the present embodiments to a patient follows typical protocols for administering such compounds, taking into account any toxicities of those agents. Thus, in some embodiments, there is a step of monitoring for toxicities that may result from the combination therapy. 1.Chemotherapy

[0164] A wide variety of chemotherapeutic agents may be used in accordance with this embodiment. Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclosphosphamide); alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa); ethyleneimines and methylamelamines (altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylamelamine); acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatins; kallistatins; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (especially the synthetic analogs KW-2189 and C including B1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquinone) chin, phenesterine, prednimustine, trofosfamide, and uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine); antibiotics (e.g., enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11)); dynemicins (including dynemicin A);Bisphosphonates (e.g., clodronate); esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarubicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomicinis, dactinomycin, daunorubicin, detorubicin rubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalarrhinicin, olivomycin, peplomycin, potfilomycin ycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, pteropterin, and trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine, iamiprine and thioguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine); androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone); anti-adrenals (e.g., mitotane and trilostane); folic acid supplements (e.g., frolinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid;Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diazicon; Elformithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids (e.g., maytansin and ansamitocins); Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerin; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid acid); 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecines (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids (e.g., retinoic acid); capecitabine;Carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein tranferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above; 2. Radiation therapy

[0165] Other widely used agents that cause DNA damage include gamma rays, X-rays, and / or what are commonly known as directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwaves, proton beam irradiation, and UV irradiation, are also contemplated. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Radioisotope dose ranges vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells. 3. Immunotherapy

[0166] Those skilled in the art will understand that immunotherapy can be used in conjunction with or in conjunction with the methods of the above embodiments. In the context of cancer treatment, immunotherapeutics typically rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody can function alone as a therapeutic effector or can recruit other cells to actually affect cell killing. The antibody can also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0167] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) covalently linked to cytotoxic drugs, which can be used in combination therapy. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver payloads (drugs) to tumor cells with abundant levels of antigen. Targeted delivery of the drug also minimizes exposure to normal tissues, resulting in reduced toxicity and an improved therapeutic index. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).

[0168] In one aspect of immunotherapy, tumor cells must have some marker that is amenable to targeting, i.e., some marker that is not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. 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 to combine anti-cancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, and gamma-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0169] Examples of immunotherapies include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapy, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapy, such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0170] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either strengthen or weaken signals (e.g., costimulatory molecules). 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 V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0171] The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant ligand or receptor, or may be an antibody, particularly a human antibody. Known inhibitors of immune checkpoint proteins or their analogs may be used, particularly chimeric, humanized, or human antibodies. As those skilled in the art will recognize, alternative and / or equivalent names may be used for certain antibodies described in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0172] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand-binding partner. In a specific embodiment, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits PDL1 from binding to its binding partner. In a specific embodiment, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits PDL2 from binding to its binding partner. In a specific embodiment, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0173] 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 the 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 OPDIVO®, is an anti-PD-1 antibody that may be used. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

[0174] Another immune checkpoint that can be targeted in the methods provided herein 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 bound 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 resembles the T cell costimulatory protein CD28; 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, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for the function of these cells. Activation of T cells via the T cell receptor and CD28 leads to high expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0175] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human, humanized, or chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0176] Anti-human CTLA-4 antibodies (or VH and / or VL domains therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or heavy and light chain 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 for binding to the same epitope on CTLA-4 as the above-mentioned antibodies and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to the above-described antibody (eg, at least about 90%, 95%, or 99% variable region identity to ipilimumab). 4.Surgery

[0177] Approximately 60% of people with cancer undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, which physically removes, excises, and / or destroys all or part of the cancerous tissue, and may be used in conjunction with other treatments (e.g., the treatment of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies). Lumpectomy refers to the physical removal of at least part of the tumor. In addition to lumpectomy, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0178] When removing part or all of cancerous cells, tissue or tumor, cavity can be formed in the body.Treatment can be achieved by perfusion, direct injection or local application of additional anti-cancer therapy to the area.Such treatment 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 can also be at various dosages. 5. Other agents

[0179] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions may enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents may be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. VI. Products or Kits

[0180] Also provided herein are articles of manufacture or kits containing immune cells, antibodies, reagents, buffers, or combinations thereof. The articles of manufacture or kits may further include a package insert containing instructions for using the immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein may be included in the articles of manufacture or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds a formulation and a label, which may be attached to or associated with the container and indicate usage. The articles of manufacture or kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. In some embodiments, the article of manufacture further comprises one or more additional agents (e.g., chemotherapeutic agents and anti-neoplastic agents). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes. VII. SEQUENCES USED IN CERTAIN EMBODIMENTS Clone T26 VH region: nucleic acid GAGGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACCAGGCCTTGAGTGGATCGGAGCAATTGATCCTTCAGATAGTTATACTGGCTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTACAAGAAGCTACTATGGTAACTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (357 nt) (SEQ ID NO: 8) Amino acid sequence EVQLQESGAELVKPGASVKMSCKASGYTFTSYWMHWVKQRPGPGLEWIGAIDPSDSYTGYNQKFKGKATLTVDTSSSTAYMHLSSLTSEDSAVYFCTRSYYGNSWFAYWGQGTLVTVSA (SEQ ID NO: 7) (119 aa) T26 VH CDR1: GYTFTSYW (SEQ ID NO: 1) T26 VH CDR2: IDPSDSYT (SEQ ID NO: 2) T26 VH CDR3: NSWFAYWGQGTLV (SEQ ID NO: 3) Clone T26 VL region: nucleic acid ACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATAAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGAATCCCAGCCAGGTTTAGTGCCAGTGGGTCTGGGACAGACTTCCACCCTCAACATCCTCTGGGAGGAGGATGTTGCAGCCTATTACTGTCAAGTAATGAGGACCATTCACGTTCGGCTCGGGGACAAGGTTGGAAATAAAAC ( 330 nt) (SEQ ID NO: 10) amino acid sequence IVLTQSPASLAVSLGQRATICSKASQSVDYDGDSYINWYQQKPGQPPKLLIYAASNLESGIPARFSASGSGTDFTLNIHPVEEDVAAYYCQQSNEDPFTFGSGTRLEIK (110 aa) (SEQ ID NO: 9) T26 VL CDR1: QSVDYDGDSY (SEQ ID NO: 4) T26 VL CDR2: AAS (SEQ ID NO: 5) T26 VL CDR3: QQSNEDPFT (SEQ ID NO: 6) Clone 5B VH region: nucleic acid GAGGTGCAGCTGCAGGATCTGGGGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGCAATTGATCCTTCAGATAGTTAACTGGCT ACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTACAAGAAGCTACTATGGTAACTCCTGGTTTGATTACTGGGGCCAAGGACTCTGGTCACTGTCTCTGCA (357 nt) (SEQ ID NO: 18) amino acid sequence EVQLQESGAELVKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIDPSDSYTGYNQKFKGKATLTVDTSSSTAYMHLSSLTSEDSAVYFCTRSYYGNSWFDYWGQGTLVTVSA (119aa) (SEQ ID NO: 17) 5B VH CDR1: GYTFTSYW (SEQ ID NO: 11) 5B VH CDR2: IDPDSYT (SEQ ID NO: 12) 5B VH CDR3: NSWFDYWGQGTLV (SEQ ID NO: 13) Clone 5B VL region: nucleic acid GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGAAGGTGATAGTTATATGAACTGGTACCAACAGAAAACCAGGACAGCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGAATCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCTGTGGAGGAGGATGCTGCAACCTATCACTGTCAGCAAAGTAATGAGGACCCGTTCACGTTCGAGGGGGGACCAAGTTGGAAATAAA (333 nt) (SEQ ID NO: 20) amino acid sequence DIVLTQSPASLAVSLGQRATICSKASQSVDYEGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEDAATYHCQQSNEDPFTFGGGTKLEIK (111 aa) (SEQ ID NO: 19) 5B VL CDR1: QSVDYEGDSY (SEQ ID NO: 14) 5B VL CDR2: AAS (SEQ ID NO: 15) 5B VL CDR3: QQSNEDPFT (SEQ ID NO: 16) Clone 28B VH region: nucleic acid GAGGTGCAGCTGCAGGATCTGGGGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGCAATTGATCCTTCAGATAGTTAACTGGCT ACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCACATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTACAAGAAGCTACTATGGTAACTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (357 nt) (SEQ ID NO: 28) amino acid sequence EVQLQESGAELVKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIDPSDSYTGYNQKFKGKATLTVDTSSSTAYMHLSSLTSEDSAVYFCTRSYYGNSWFAYWGQGTLVTVSA (119 aa) (SEQ ID NO: 27) 28B VH CDR1: GYTFTSYW (SEQ ID NO: 21) 28B VH CDR2: DPSDSYT (SEQ ID NO: 22) 28B VH CDR3: SWFAYWGQGTLV (SEQ ID NO: 23) Clone 28B VL region: nucleic acid GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATTTATGTTGCATCCAATCTAGAATCTGGAATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAC (333 nt) (SEQ ID NO: 30) Amino acid sequence DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYVASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLEIN (111 aa) (SEQ ID NO: 29) 28B VL CDR1: QSVDYDGDSY (SEQ ID NO: 24) 28B VL CDR2: VAS (SEQ ID NO: 25) 28B VL CDR3: QQSNEDPFT (SEQ ID NO: 26) SEQ ID NO: 40 Truncated human EGFR SEQ ID NO: 41 EGFRIII-IV RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKA TGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR SEQ ID NO: 42 CD8Leader ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCC SEQ ID NO: 43 CD8Leader MALPVTALLLPLALLLHAARP SEQ ID NO: 44 リンカー1 GGTGGCGGAGGTTCT SEQ ID NO: 45 リンカー1 GGGGS SEQ ID NO: 46 リンカー2 GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC SEQ ID NO: 47 リンカー2 GGGGSGGGGS SEQ ID NO: 48 リンカー3 GGAGGGTGGTAGTGGTGGGGGGGGGGGGGG SEQ ID NO: 49 Linker 3 GGGGSGGGGSGGGGS SEQ ID NO: 50 Linker 4 GGAGGAGGTGGTAGTGGTGGAGGAGGAAGTGGTGGCGGAGGTTTCTGGAGGTGGAGGTTCC SEQ ID NO: 51 Linker 4 GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 52 CD8 Hinge 1 ACAACTACTCCAGCACCACGACCACCAACACCTGCTCCAACTATCGCATCTCAACCACTTTCTCTACGTCCAGAAGCATGCCGACCAGCTGCAGGAGGTGCAGTTCATACGAGAGGTCTAGATTTCGCATGTGAT SEQ ID NO: 53 CD8 Hinge 1 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 54 CD8 Hinge 2 AAGCCCACAACTACTCCAGCACCACGACCACCAACACCTGCTCCAACTATCGCATCTCAACCACTTTCTCTACGTCCAGAAGCATGCCGACCAGCTGCAGGAGGTGCAGTTCATACGAGAGGTCTAGATTTCGCATGTGAT SEQ ID NO: 55 CD8 Hinge 2 KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 56 CD8 Hinge 3 TTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACAACTACTCCAGCACCACGACCACACACCTGCTCCAACTATCGCATCTCAACCACTTTCTCTACGTCCAGAAGCATGCCGACCAGCTGCAGGAGGTGCAGTTCATACGAGAGGTCTAGATTTCGCATGTGAT SEQ ID NO: 57 CD8 factor FSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 58 CD28 ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGG SEQ ID NO: 59 CD28 IEVMYPPPYLDNEXNGTIIHVKG SEQ ID NO: 60 IgG4 ヒ GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCA SEQ ID NO: 61 IgG4 ヒ ESKYGPPCPSCP SEQ ID NO: 62 IgG4 CH2 GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGG SEQ ID NO: 63 IgG4 CH2 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG SEQ ID NO: 64 IgG4 CH2CH3 GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 65 IgG4 CH2CH3 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 66 IgG4 CH1CH2CH3 GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 67 IgG4 CH1CH2CH3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 68 CD8 TM 1 ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCCTTCTCCTATCACTGGTTATCACCCTTTACTGC SEQ ID NO: 69 CD8 TM 1 IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 70 CD8 TM 2 ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCCTTCTCCTATCACTGGTTATCACCCTTTACTGCAACCACAGGAAC SEQ ID NO: 71 CD8 TM 2 IYIWAPLAGTCGVLLLSLVITLYCNHRN SEQ ID NO: 72 CD28 TM AAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC SEQ ID NO: 73 CD28 TM KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF SEQ ID NO: 74 CD28 AGAAGTAAAAGAAGTAGGCTACTTCATAGTGATTACATGAATATGACTCCTCGACGACCTGGTCCCACCCGTAAGCATTATCAGCCCTATGCACCACCACGAGATTTCGCAGCCTATCGCTCC SEQ ID NO: 75 CD28 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 76 4-1BB AAACGAGGTAGAAAAAAACTTCTTTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGATGTAGTTGTCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG SEQ ID NO: 77 4-1BB RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 78 OX-40 AGGCGCGACCAGCGGCTGCCACCTGATGCACACAAGCCACCAGGAGGAGGCTCTTTCCGGACCCCAATCCAGGAGGAGCAGGCAGACGCACACAGCACACTGGCCAAGATC SEQ ID NO: 79 OX-40 RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO: 80 CD3ζ intracellular AGAGTTAAATTTAGCAGAAGTGCAGATGCTCCTGCGTATAAACAGGGTCAAAACCAACTATATAATGAACTAAATCTAGGACGAAGAGAAGAATATGATGTTTTAGATAAAAGACGTGGTCGAGATCCTGAAATGGGAGGAAAACCTAGAAGAAAAAATCCTCAAGAAGGCCTATATAATGAACTACAAAAAGATAAGATGGCAGAAGCTTATAGTGAAATTGGAATGAAAGGAGAACGTCGTAGAGGTAAAGGTCATGATGGTCTTTATCAAGGTCTTAGTACAGCAACAAAAGATACATATGATGCACTTCATATGCAAGCACTTCCACCTCGT SEQ ID NO: 81 CDζ intracellular RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 82 Enhanced GFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG SEQ ID NO: 83 Enhanced GFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 84 T2A GAGGGCAGAGGCAGTCTGCTGACATGCGGTGACGTGGAAGAGAATCCCGGCCCT SEQ ID NO: 85 T2A EGRGSLLTCGDVEENPGP SEQ ID NO: 86 CD8α TM (nucleic acid) ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCCTTCTCCTATCACTGGTTATCACC SEQ ID NO: 87 CD8α TM (amino acid sequence) IYIWAPLAGTCGVLLLSLVIT [Example]

[0181] VIII. Working Examples The following examples are included to demonstrate specific embodiments of the present disclosure. It should be recognized by those skilled in the art that the procedures disclosed in the following examples are procedures discovered by the inventors to work well in the practice of the invention and can therefore be considered preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments disclosed which will still yield the same or similar results without departing from the spirit and scope of the invention. Example 1 - CD79b Antibody and CAR Development

[0182] CD79b expression is restricted to the B cell lineage Using real-time PCR, we found that CD79b is expressed in a wide range of B-cell lymphoma cell lines, including Mino, Daudi, HBC-1, Jeko, SUDHL6, SUDHL4, and U2932, but not in Jurkat and J76 T-cell lymphoma / leukemia cell lines (Figure 1A). To determine whether CD79b is expressed in normal tissues, we obtained the FirstChoice Human Total RNA Survey Panel from Applied Biosystems, which contains total RNA from 20 normal human tissues. 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 exclusively in lymphoid tissues, such as spleen and lymph nodes, and absent from all non-lymphoid normal tissues (Figure 1B).

[0183] Using publicly available gene expression datasets (Oncomine), CD79b was found to be highly expressed in ALL and chronic lymphocytic leukemia (Figure 1C) and multiple B-cell lymphoma subtypes (e.g., Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma) (Figure 1D). The number of samples for each tumor type is indicated in parentheses.

[0184] Generation of multiple monoclonal antibodies against human CD79b and characterization of their heavy and light chain sequences Anti-human CD79b monoclonal antibodies were generated using hybridoma technology by immunizing mice with mouse fibroblast L cells expressing human CD79b (Figure 2A). Three clones with high binding ability to recombinant human CD79b protein were identified by ELISA (Figure 2B). The affinities of these three monoclonal antibodies were further measured by Octet Assay, and three clones, 14 (IgG1), 16A (IgG2), and 45 (IgG2), with Kd values ​​of 1.44, 17.8, and 2.0 nM, respectively, were selected for further development (Figure 2C). Monoclonal antibody clone #14 was conjugated to a fluorescent dye and showed staining of B cell lymphoma cell lines comparable to that of a commercially available anti-CD79b antibody from BD Biosciences (Figure 2D). Total RNA from the hybridomas of the above monoclonal antibodies was extracted, and cDNA was synthesized. The heavy and light chain V genes were cloned using 5'-RACE PCR (rapid amplification of cDNA ends). Protein sequences were predicted from DNA sequences. Hybridoma culture supernatants were purified, and the heavy and light chain protein sequences were confirmed by mass spectrometry at the MD Anderson Proteomics Core Facility.

[0185] Generation of anti-CD79b CAR T cells We generated several anti-CD79b CAR constructs using specific sequences from the single-chain fragment (scFv) variable region. To detect CAR expression in transduced T cells, we used a CAR-enhanced green fluorescent protein (eGFP) fusion construct or a truncated form of the human epidermal growth factor receptor (huEGFRt) (Figure 3A). The latter could also serve as a safety switch to remove CAR T cells in the event of severe toxicity. We incorporated the CD3-zeta (CD3z) chain to provide signal 1 for T cell activation, and the costimulatory domains CD28 or 4-1BB to provide signal 2 (Figure 3A).

[0186] These constructs were cloned into the lentiviral vector pHR_SFFV, which was then used to transduce primary healthy donor T cells. Clone 45-CD79b-CD28-CAR was used to transduce CD4 + and CD8 + Representative transduction efficiencies (>70%) as measured by eGFP expression in T cells are shown in Figure 3B. Anti-CD19-CAR T cells were used as a control.

[0187] The cytotoxic activity of CAR T cells against CellTrace Far Red-labeled Daudi cells was measured by Aqua staining in a 16-hour flow cytometry assay at the indicated effector:target (E:T) ratios (Figure 3C). Representative dot plots (upper right quadrant) with cell death rates for various culture conditions at an E:T ratio of 20:1 are shown in Figure 3D. The data show that both anti-CD19- and anti-CD79b-CAR T cells were highly cytotoxic to Daudi Burkitt lymphoma cells compared to untransduced control T cells.

[0188] Anti-CD79b CAR T cells target CD19 + Lymphoma cells and CD19 - are cytotoxic to both lymphoma cells :CD19 negative (CD19 - To measure the efficacy of anti-CD79b CAR T cells against lymphoma cells, degranulation and cytotoxicity assays were performed using SUDHL6, a diffuse large B-cell lymphoma cell line lacking CD19. CD19 was first knocked out using CRISPR-Cas9 (CD19KOSUDHL6), and then these lymphoma cells were transduced with a CD19 splice variant (CD19Dex2) lacking exon 2, the binding site for the anti-CD19 antibody clone FMC63 used in the anti-CD19 CAR construct.

[0189] Non-transduced primary T cells (control T), clone-14-CD79b-CD28 CAR, 14-CD79b-4-1BB CAR, and FMC63-CD19-CD28 CAR were cocultured with Daudi cells or the above-mentioned 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 4 days after coculture. 14-CD79b-CD28 and 14-CD79b-4-1BB CAR T cells exhibited significantly higher degranulation and cytotoxic activity against both cell lines, whereas control T cells did not (Figure 4A and Figure 4B). In contrast, the FMC63-CD19-CD28 CAR expresses CD19 + It was cytotoxic to Daudi but not to CD19KOSUDHL6-CD19Dex2 tumor cells.

[0190] After 4 days of coculture, the absolute number of viable tumor cells was also measured using CountBright Absolute Counting Beads for flow cytometry (Figure 4C). The results were consistent with the observed percentage of viable tumor cells (Figure 4B). Representative dot plots of target and effector T cells are shown (Figure 4A and Figure 4B). These experiments were repeated at least three times with similar results.

[0191] Anti-CD79b CAR T cells demonstrate in vivo efficacy against lymphoma xenografts To test the efficacy of anti-CD79b CAR T cells in vivo, NSG mice were injected with 2 × 10 Mino mantle cell lymphoma cell lines expressing the firefly luciferase gene. 6 10 × 10 tumor cells / mouse. 18 days later, mice were transduced with untransduced primary T cells, anti-CD19-CD28 CAR T cells, or clone 45 anti-CD79b-CD28 CAR T cells. 6 Cars+ Mice were treated with either transduced or untransduced T cells via tail vein injection. Tumor burden was assessed using bioluminescence imaging (Figure 5A). The results showed progressive tumor growth in mice treated with untransduced T cells. In contrast, favorable tumor control and a significant improvement in survival time (p<0.05) were observed in mice treated with both anti-CD19 and anti-CD79b CAR T cells (Figure 5B and Figure 5C). In vitro results were validated at least three times for each individual experiment, and in vivo results were validated twice. Therefore, anti-CD79b CAR therapy can be used for the treatment of B-cell malignancies, both with and without CD19 expression. Example 2 - Development of further CD79b antibodies and CARs

[0192] Generation of anti-CD79b CAR T cells using new anti-CD79b antibody clones: Several anti-CD79b CAR constructs were generated using specific sequences of single-chain fragments (scFv) of the variable region from additional antibody clones (T26, 5B, and 28B). To detect CAR expression in transduced T cells, we used a CAR-enhanced green fluorescent protein (eGFP) fusion construct or a truncated human epidermal growth factor receptor (huEGFRt) (Figure 6A). The latter can also serve as a safety switch to remove CAR T cells in the event of severe toxicity. We incorporated the CD3-zeta (CD3z) chain to provide signal 1 for T cell activation, and the costimulatory domains CD28, 4-1BB, or OX-40 to provide signal 2 (Figure 6A).

[0193] Anti-CD79b CAR T cells are cytotoxic to Daudi lymphoma cells in vitro. Anti-CD79b-CAR T cells were generated by cloning the above CAR constructs (T26 and 28B) into the lentiviral vector pLVEG and then transducing primary healthy donor T cells with it. Anti-CD19-CAR T cells were used as a control. The cytotoxic activity of CAR T cells (labeled with CellTrace Far Red) against Daudi cells (labeled with CellTrace Violet) was measured by flow cytometry assay after 1 and 4 days of coculture at the indicated effector:target (E:T) ratios (Figure 6B). Representative dot plots are shown in Figure 6C. The data show that both anti-CD19-CAR T cells and anti-CD79b-CAR T cells were highly cytotoxic to Daudi Burkitt lymphoma cells.

[0194] Anti-CD79b CAR T cells demonstrate in vivo efficacy against Daudi lymphoma xenografts: To test the efficacy of anti-CD79b CAR T cells (clone T26) in vivo, 2 × 10 Daudi Burkitt lymphoma cell lines expressing the firefly luciferase gene were transfected into NSG mice. 4 3x10 tumor cells / mouse. Ten days later, mice were either untreated or injected IV with anti-CD19-CAR T cells, or clone T26 anti-CD79b-CAR T cells. 6 Mice were treated with 10 CAR T cells / mouse via tail vein injection. Tumor burden was assessed using bioluminescence imaging (Figure 6D). The results showed progressive tumor growth in untreated mice. In contrast, good tumor control and a significant improvement in survival time (p=0.01) were observed in both mice treated with anti-CD19 CAR T cells and mice treated with anti-CD79b CAR T cells (Figure 6E). These results demonstrate that anti-CD79b CAR T cells have strong anti-tumor activity in vivo. Example 3 - Further development and investigation of CD79b antibodies and CARs

[0195] Binding of anti-CD79b antibodies to human CD79b: Full-length anti-CD79b monoclonal antibodies (clones 5B and 28B) isolated from hybridoma supernatants were tested by ELISA for binding to human CD79b expressed on mouse fibroblast L cells (Figure 7).

[0196] CAR Construct Generation: Using the specific sequences of single-chain fragments (scFv) of the variable regions derived from clone 28B (shown in Figure 7), several anti-CD79b CAR constructs were generated using the CD8α or CD28 hinge / transmembrane domains. To detect CAR expression in transduced T cells, CAR-enhanced green fluorescent protein (eGFP) fusion constructs or a truncated human epidermal growth factor receptor (huEGFRt) were used (Figure 8A). An anti-CD19 CAR construct was used as a control. The latter can also serve as a safety switch to remove CAR T cells in the event of severe toxicity. The CD3-zeta (CD3ζ) chain was incorporated to provide signal 1 for T cell activation, and the costimulatory domains CD28, 4-1BB, or OX40 were incorporated to provide signal 2 (Figure 8A). These constructs were cloned into the lentiviral vector pLVEG containing the EF1α promoter (Figure 8B), which was then used to transduce T cells and tested in various in vitro and in vivo assays described below.

[0197] Anti-CD79b CARs specifically recognize human CD79b: To measure the signaling capacity of anti-CD79b CARs, we used lentivirus to transfect these CARs (clone 28B was used for CD79b CARs) into Jurkat-Lucia mice. TMWe transduced these CARs into NFAT reporter cells (Figure 9A). In this cell line, the Lucia gene, encoding a secreted coelenterazine-utilizing luciferase, is driven by an ISG54 minimal promoter fused to six copies of the NFAT consensus transcriptional response element. Culturing these transduced Jurkat cells with anti-CD3 / anti-CD28 antibodies or Daudi Burkitt lymphoma cells for 24 hours induced stronger luciferase activity than untreated cells (Figure 9B). To establish the specificity of CD79b recognition by these CAR molecules, we used CRISPR / Cas9 to generate isogenic versions 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). CAR-transduced Jurkat-Lucia TM Coculture of NFAT reporter cells with the SUDHL6 isogenic cell line showed that the CD79b CAR recognized both parental and CD19KO cells, but showed significantly reduced reactivity with CD79bKO or CD19KOCD79bKO cells. In contrast, the CD19 CAR recognized both parental and CD79bKO cells, but not CD19KO or CD19KOCD79bKO cells (Figure 9D).

[0198] Transduction of CARs into primary human T cells: Representative transduction efficiencies measured by eGFP expression and / or recombinant human CD79b-Fc protein staining using the CAR constructs described above (Figure 8A) are shown in Figure 10A. Anti-CD19-CAR T cells were used as a control. Anti-CD79b CAR T cells were either untreated or stimulated with anti-CD3 / CD28 antibodies or recombinant human CD79b-Fc protein. After 15 minutes, CD3ζ and ERK1 / 2 phosphorylation were assessed by flow cytometry. The results show no significant difference in baseline phosphorylation between CAR+ and CAR-T cells, suggesting the absence of sustained signaling. Stimulation with anti-CD3 / CD28 antibodies induced CD3ζ phosphorylation in both CAR+ T cells but not in CAR-T cells. In contrast, stimulation with recombinant human CD79b-Fc protein induced phosphorylation of CD3ζ and ERK1 / 2 in CAR+ T cells but not in CAR- T cells, suggesting that these CARs can induce signaling in primary T cells in response to recognition of CD79b protein (Figure 10B).

[0199] Proliferative activity of anti-CD79b CAR T cells: Anti-CD79b CAR T cells generated from healthy donor T cells using the CAR constructs described above (Figure 8A) were labeled with CellTrace Far Red and cocultured with CD79b-expressing Daudi Burkitt lymphoma tumor cells at an effector-to-target (E:T) ratio of 1:1. After 4 days, CAR T cell proliferation was assessed by flow cytometry using the dye dilution method. The results showed that both CD4+ and CD8+ anti-CD79b CAR T cells proliferated significantly more in response to Daudi tumor cells than non-transduced T cells. Proliferation of anti-CD79b CAR T cells was comparable to that of anti-CD19 CAR T cells (Figures 11A and 11B).

[0200] Cytokine production by anti-CD79b CAR T cells: Anti-CD79b CAR T cells generated from healthy donor T cells using the CAR constructs described above (Figure 8A) were cocultured with Daudi Burkitt lymphoma tumor cells at an E:T ratio of 1:1. After 24 hours, cytokine levels in the supernatant were assessed by multiplex cytokine assay. The results showed that anti-CD79b CAR T cells produced significant amounts of IL-2, GM-CSF, IFN-γ, and IL-17A in response to Daudi tumor cells compared with non-transduced T cells. Cytokine production by anti-CD79b CAR T cells was comparable to that of anti-CD19 CAR T cells (Figure 12).

[0201] Anti-CD79b CAR T cells degranulate in response to lymphoma cells: Anti-CD79b CAR T cells generated from healthy donor T cells using the CAR constructs described above (Figure 8A) were cocultured with Daudi Burkitt lymphoma tumor cells at an E:T ratio of 1:1. After 6 hours, degranulation was assessed by flow cytometry after staining for CD107a / b. The results showed that both CD4+ and CD8+ anti-CD79b CAR T cells significantly degranulated in response to Daudi tumor cells compared with untransduced T cells. Degranulation by anti-CD79b CAR T cells was comparable to that of anti-CD19 CAR T cells. Degranulation was numerically greater in anti-CD79b CAR T cells containing the CD8α hinge / transmembrane domain compared to anti-CD79b CAR T cells containing the CD28 hinge / transmembrane domain (Figures 13A and 13B).

[0202] Anti-CD79b CAR T cells are cytotoxic to lymphoma cells: Anti-CD79b CAR T cells, generated from healthy donor T cells using the CAR construct described above (Figure 8A) and labeled with CellTrace Far Red, were cocultured with Daudi Burkitt lymphoma tumor cells or SUDHL6 isogenic cell lines (parental, CD19KO, CD79bKO, and CD19 / CD79b double KO) labeled with CellTrace Violet at a 1:1 E:T ratio. After 4 days, cytotoxicity was assessed by flow cytometry by measuring the absolute number of remaining viable tumor cells and by calculating percent lysis. The results showed that CD79b CAR T cells induced significant cytotoxicity of Daudi tumor cells compared with untransduced T cells. The cytotoxic activity of anti-CD79b CAR T cells was comparable to that of anti-CD19 CAR T cells. Cytotoxic activity was superior for 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 14A). Furthermore, anti-CD79b CAR T cells induced significant lysis of both parental and CD19KO SUDHL6 cells, but lysis was significantly reduced in CD79bKO or CD19KOCD79bKO cells. In contrast, anti-CD19 CAR T cells induced significant lysis of both parental and CD79bKO cells, but lysis was significantly reduced in CD19KO or CD19KOCD79bKO cells (Figure 14B).

[0203] Anti-CD79b CAR T cells exert antitumor effects in vivo: To examine the efficacy of anti-CD79b-CAR T cells in vivo, 2 × 10 Daudi Burkitt lymphoma cells expressing the firefly luciferase gene were transfected with CAR T cells. 4 NSG mice were IV injected with 1000 sera from 1000 CAR T cells / mouse. Eleven days later, the mice were transduced with 5x10 untransduced T cells, or anti-CD19-CAR T cells or anti-CD79b-CAR T cells generated using the CAR constructs described above (Figure 8A). 6Mice were treated with CAR+ T cells per mouse via tail vein injection. Tumor burden was assessed using bioluminescence imaging (Figure 15A). The results showed rapid tumor growth in mice treated with untransduced T cells. In contrast, good tumor control and significantly improved survival time were observed in both mice treated with anti-CD19 CAR T cells and mice treated with anti-CD79b CAR T cells. This tumor control and survival time was superior for anti-CD79b CAR T cells containing the CD8α hinge / transmembrane domain compared to anti-CD79b CAR T cells containing the CD28 hinge / transmembrane domain, with the best survival time observed for anti-CD79b CAR T cells containing the CD8α hinge / transmembrane domain and OX40 costimulatory domain (Figure 15B).

[0204] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More particularly, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. literature The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Chothia et al., 1988 . Davila et al., 2013. European patent application number EP2537416 Heemskerk et al. Hum Gene Ther. 19:496-510, 2008. International Patent Publication No. WO / 2014055668 Al. International Patent Publication No. WO200014257 International Patent Publication No. WO2012 / 129514 International Patent Publication No. WO2013 / 071154 International Patent Publication No. WO2013 / 123061 International Patent Publication No. WO2013 / 166321 International Patent Publication No. WO2013126726 International Patent Publication No. WO2014031687 Johnson et al. Blood 114:535-46, 2009. Jores et al., 1990. Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed. Lefranc et al., 2003. Liu et al., 2003. Remington's Pharmaceutical Sciences 22 nd edition, 2012. Sadelain et al., 2013. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001. Turtle et al., 2012. U.S. Patent No. 5,091,513 U.S. Patent No. 5,091,513 U.S. Patent No. 5,994,136 U.S. Patent No. 6,013,516 U.S. Patent No. 6,410,319 U.S. Patent No. 6,410,319 U.S. Patent No. 6,451,995 U.S. Patent No. 6,881,557 U.S. Patent No. 6,946,546 U.S. Patent No. 7,070,995 U.S. Patent No. 7,265,209 U.S. Patent No. 7,354,762 U.S. Patent No. 7,446,179 U.S. Patent No. 7,446,190 U.S. Patent No. 7,446,191 U.S. Patent No. 8,252,592 U.S. Patent No. 8,324,353 U.S. Patent No. 8,339,645 U.S. Patent No. 8,398,282 U.S. Patent No. 8,479,118 U.S. Patent No.: 7,446,190 U.S. Patent Publication No. US2002131960 U.S. Patent Publication No. US20050214860 U.S. Patent Publication No. US20130149337 U.S. Patent Publication No. US2013287748 Wu et al., 2012

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to CD79b; (I): (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 1; Ｈ CDRs; (b) a second V comprising the amino acid sequence set forth in SEQ ID NO:2; Ｈ CDRs; (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 3 Ｈ CDRs; (d) a first V comprising the amino acid sequence set forth in SEQ ID NO:4; Ｌ CDRs; (e) a second V comprising the amino acid sequence set forth in SEQ ID NO:5; Ｌ CDRs; and (f) a third V comprising the amino acid sequence set forth in SEQ ID NO:6 Ｌ CDRs; an isolated monoclonal antibody or an antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof is selected from the group consisting of V of SEQ ID NO: 7 Ｈ V domains that are at least 80% identical to Ｈ domain, and V of SEQ ID NO:9 Ｌ V domains that are at least 80% identical to Ｌ 2. The antibody or antigen-binding fragment thereof of claim 1, comprising the domain.

3. The antibody or antigen-binding fragment thereof is selected from the group consisting of V of SEQ ID NO: 7 Ｈ The same V as the domain Ｈ domain, and V of SEQ ID NO:9 Ｌ The same V as the domain Ｌ 2. The antibody or antigen-binding fragment thereof of claim 1, comprising the domain.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is recombinant.

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

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

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is a humanized antibody or a deimmunized antibody.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

9. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in a pharmaceutically acceptable carrier.

10. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 7.

11. V of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3 Ｈ Antibody V comprising CDRs 1-3 of domain Ｈ Domains and V of the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6 Ｌ Antibody V comprising CDRs 1-3 of domain Ｌ A recombinant polypeptide comprising a domain.

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

13. A host cell comprising one or more polynucleotide molecules encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 7 or the recombinant polypeptide of claim 11.

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

15. A composition for use in a method for treating a subject with cancer, comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

16. 16. The composition of claim 15, wherein the cancer is a B-cell malignancy.

17. 16. The composition of claim 15, wherein the antibody or antigen-binding fragment thereof is present in a pharmaceutically acceptable composition.

18. The composition of claim 15 , which is administered systemically to a subject.

19. 16. The composition of claim 15, wherein the composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically to a subject.

20. The method of claim 15, further comprising administering at least a second anti-cancer treatment to the subject.

21. 21. The composition of claim 20, wherein the second anti-cancer treatment is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

22. 21. The composition of claim 20, wherein the second anti-cancer treatment comprises adoptive T cell therapy.

23. (I): (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 1; Ｈ CDRs; (b) a second V comprising the amino acid sequence set forth in SEQ ID NO:2; Ｈ CDRs; (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 3 Ｈ CDRs; (d) a first V comprising the amino acid sequence set forth in SEQ ID NO:4; Ｌ CDRs; (e) a second V comprising the amino acid sequence set forth in SEQ ID NO:5; Ｌ CDRs; and (f) a third V comprising the amino acid sequence set forth in SEQ ID NO:6 Ｌ an engineered CD79b CAR or TCR having an antigen binding domain comprising:

24. 24. The CAR or TCR of claim 23, wherein the antigen-binding domain comprises a VH domain that is at least 80% identical to the VH domain of SEQ ID NO: 7 and a VL domain that is at least 80% identical to the VL domain of SEQ ID NO:

9.

25. 24. The CAR or TCR of claim 23, wherein the antigen binding domain comprises a VH domain identical to the VH domain of SEQ ID NO:7 and a VL domain identical to the VL domain of SEQ ID NO:

9.

26. The CAR or TCR of claim 23, wherein the CAR comprises one or more signaling domains selected from the group consisting of CD3ζ, CD28, OX40 / CD134, 4-1BB / CD137, and combinations thereof.

27. The CAR or TCR of claim 23, wherein the CAR comprises a CD3ζ signaling domain and a CD28 signaling domain.

28. The CAR or TCR of claim 23, wherein the CAR comprises a CD3ζ signaling domain and a 4-1BB signaling domain.

29. The CAR or TCR of claim 23, wherein the CAR comprises a CD3ζ signaling domain and an OX-40 signaling domain.

30. 24. The CAR or TCR of claim 23, wherein the CAR or TCR is encoded by a viral vector.

31. The CAR or TCR of claim 30, wherein the viral vector is a lentiviral vector.

32. The antigen-binding domain is connected to a V Ｌ V linked to domain Ｈ The CAR or TCR according to any one of claims 23 to 31, comprising a domain.

33. The CAR or TCR of claim 32, wherein the linker comprises linker 1 (SEQ ID NO:45) or a linker encoded by the polynucleotide of SEQ ID NO:44, linker 2 (SEQ ID NO:47) or a linker encoded by the polynucleotide of SEQ ID NO:46, linker 3 (SEQ ID NO:49) or a linker encoded by the polynucleotide of SEQ ID NO:48, or linker 4 (SEQ ID NO:51) or a linker encoded by the polynucleotide of SEQ ID NO:

50.

34. The CAR is V Ｌ -Linker 1-V Ｈ、 V Ｌ -Linker 2-V Ｈ , V Ｌ -Linker 3-V Ｈ , V Ｌ -Linker 4-V Ｈ , V Ｈ -Linker 1-V Ｌ , V Ｈ -Linker 2-V Ｌ , V Ｈ -Linker 3-V Ｌ or V Ｈ -Linker 4-V Ｌ 34. The CAR or TCR of claim 33, comprising:

35. The CAR or TCR of any one of claims 23 to 34, wherein the CAR or TCR comprises a hinge.

36. The CAR or TCR of claim 35, wherein the hinge is CD8 hinge 1 (SEQ ID NO:53) or a hinge encoded by the polynucleotide of SEQ ID NO:52, CD8 hinge 2 (SEQ ID NO:55) or a hinge encoded by the polynucleotide of SEQ ID NO:54, CD8 hinge 3 (SEQ ID NO:57) or a hinge encoded by the polynucleotide of SEQ ID NO:56, CD28 hinge (SEQ ID NO:59) or a hinge encoded by the polynucleotide of SEQ ID NO:58, IgG4 hinge (SEQ ID NO:60 or 61), IgG4 CH2 (SEQ ID NO:63) or a hinge encoded by the polynucleotide of SEQ ID NO:62, IgG4 CH2CH3 (SEQ ID NO:65) or a hinge encoded by the polynucleotide of SEQ ID NO:64, or IgG4 CH1CH2CH3 (SEQ ID NO:67) or a hinge encoded by the polynucleotide of SEQ ID NO:

66.

37. The CAR or TCR of any one of claims 23 to 36, wherein the CAR comprises a transmembrane domain.

38. The transmembrane domain is a transmembrane domain encoded by the polynucleotide of CD8 TM1 (SEQ ID NO: 69) or SEQ ID NO: 68, a transmembrane domain encoded by the polynucleotide of CD8 TM2 (SEQ ID NO: 71) or SEQ ID NO: 70, a transmembrane domain encoded by the polynucleotide of CD28 38. The CAR or TCR of claim 37, wherein the transmembrane domain is a transmembrane domain encoded by the polynucleotide of CD8α TM (SEQ ID NO: 73) or SEQ ID NO: 72, or a transmembrane domain encoded by the polynucleotide of CD8α TM (SEQ ID NO: 87) or SEQ ID NO:

86.

39. 24. The CAR or TCR of claim 23, further comprising a transduction marker and / or a safety switch.

40. 40. The CAR or TCR of claim 39, wherein the transduction marker is enhanced green fluorescent protein (eGFP).

41. The CAR or TCR of claim 40, wherein the eGFP has the amino acid sequence of SEQ ID NO:

83.

42. 40. The CAR or TCR of claim 39, wherein the transduction marker and / or safety switch is a truncated epidermal growth factor receptor (EGFR).

43. 43. The CAR or TCR of claim 42, wherein the EGFR has the amino acid sequence of SEQ ID NO:

41.

44. 40. The CAR or TCR of claim 39, wherein the transduction marker and / or safety switch is linked to the CAR by a truncation peptide.

45. 45. The CAR or TCR of claim 44, wherein the truncated peptide is a 2A peptide.

46. 46. ​​The CAR or TCR of claim 45, wherein the 2A peptide is a T2A peptide.

47. 47. The CAR or TCR of claim 46, wherein the T2A peptide has the amino acid sequence of SEQ ID NO:

85.

48. 24. The CAR or TCR of claim 23, wherein the CAR further comprises a second antigen-binding domain.

49. 49. The CAR or TCR of claim 48, wherein the second antigen-binding domain is a CD19 antigen-binding domain, a CD20 antigen-binding domain, or a CD22 antigen-binding domain.

50. The CAR or TCR of any one of claims 23 to 49, wherein the CAR or TCR comprises a CD8α hinge comprising the amino acid sequence set forth in SEQ ID NO: 87, and a transmembrane domain or a transmembrane domain encoded by the polynucleotide of SEQ ID NO: 86, a human OX-40 signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 79 or a signaling domain encoded by the polynucleotide of SEQ ID NO: 78, and a CD3ζ domain comprising the amino acid sequence set forth in SEQ ID NO: 81 or a domain encoded by the polynucleotide of SEQ ID NO:

82.

51. An expression vector encoding the CAR or TCR according to any one of claims 23 to 50.

52. A host cell engineered to express the CD79b CAR or CD79b TCR of any one of claims 23 to 50.

53. The cell of claim 52, wherein the cell is engineered to express the CAR of any one of claims 23 to 50.

54. 53. The cell of claim 52, wherein the host cell is an immune cell.

55. 55. The cell of claim 54, wherein the immune cell is a T cell.

56. 56. The cell of claim 55, wherein the T cell is a primary human T cell or a TIL.

57. 56. The cell of claim 55, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

58. 57. The cell of claim 56, wherein the primary human T cells are obtained from a healthy donor.

59. 56. The cell of claim 55, wherein the T cell is an autologous T cell.

60. 56. The cell of claim 55, wherein the T cell is an allogeneic T cell.

61. 53. The cell of claim 52, wherein the cell is engineered with a CRISPR or transposase system.

62. 51. A pharmaceutical composition comprising CD79b-targeted T cells and a pharmaceutical carrier, wherein the CD79b-targeted T cells are engineered to express the CAR or TCR of any one of claims 23 to 50.

63. 51. A composition comprising an effective amount of CD79b-targeted T cells for treating cancer in a subject, wherein the CD79b-targeted T cells are engineered to express the CAR or TCR of any one of claims 23 to 50.

64. 51. A composition for use in a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of CD79b-targeted T cells, wherein the CD79b-targeted T cells have been engineered to express the CAR or TCR of any one of claims 23 to 50.

65. 65. The composition of claim 64, wherein the cancer is a B-cell malignancy.

66. 66. The composition of claim 65, wherein the B-cell malignancy is B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma, or chronic lymphocytic leukemia.

67. 65. The composition of claim 64, wherein the subject has previously received CD19 CAR therapy.

68. 68. The composition of claim 67, wherein the subject is refractory to CD19 CAR therapy.

69. 69. The composition of claim 68, wherein the subject has lost the CD19 antigen.

70. 70. The composition of claim 69, wherein the subject has a recurrent CD19-negative tumor.

71. 65. The composition of claim 64, wherein the CD79b-targeted T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

72. 65. The composition of claim 64, wherein the CD79b-targeted T cells are administered intravenously.

73. 65. The composition of claim 64, wherein the method further comprises administering at least a second anti-cancer treatment to the subject.

74. 74. The composition of claim 73, wherein the second anti-cancer treatment is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

75. 65. The composition of claim 64, wherein the cancer is a CD79b-expressing cancer.

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