Anti-CD79b antibodies and chimeric antigen receptors and methods of use thereof

Abstract

To provide anti-CD79B antibodies, chimeric antigen receptors, and methods of use thereof.SOLUTION: Provided herein are CD79b antibodies and CD79b-specific chimeric antigen receptors (CARs). Further provided herein are immune cells expressing the CD79b-specific CARs and methods of treating cancer by administering the CD79b-specific CAR immune cells.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] This application is the benefit of U.S. Provisional Patent Application No. 63 / 018,266, filed April 30, 2020. The entirety of this provisional application 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. In particular, the present disclosure relates to a CD79b antibody and related antibodies comprising at least a chimeric antigen receptor. The present invention relates to a series of compositions, as well as methods of using them. [Background technology]

[0003] 2. Description of Related Technology Chimeric antigen receptor (CAR) T cells targeting CD19 have been shown to be effective in B-cell malignancies. Recently, two anti-CD19 CAR T-cell therapy products have been developed for the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) and / or large B-cell leukemia approved by the U.S. Food and Drug Administration for the treatment of cell lymphoma. Long-term remissions lasting more than one year were observed in approximately 40-50% of patients. However, recurrence or progression occurs in approximately 50-60% of cases, and the main cause of resistance is loss of the CD19 antigen. Therefore, further improvements in outcomes in these patients are needed. To address this issue, there is an urgent need to develop CAR T cell therapies targeting novel targets. Summary of the Invention

[0004] The present disclosure relates to methods and compositions relating to certain antibodies. These antibodies are CD7 In any type of immunotherapy and for any medical application where targeting of 9b is therapeutically useful. In some embodiments, the present disclosure provides isolated monoclonal antibodies. antibodies, which specifically bind to CD79b; (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 Domain and at least about 80% Identical V 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 Domain and at least about 80 %Identical V H domain, and V of SEQ ID NO: 19 L At least approximately 80% identical to the domain V L In other embodiments, the antibody comprises the V domain of SEQ ID NO: 17. H Same as the domain Ichino V H domain, 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 Domain and at least about 80 %Identical V H domain, and V of SEQ ID NO: 29 L At least approximately 80% identical to the domain V L In other embodiments, the antibody comprises the V domain of SEQ ID NO: 27. H Same as the domain Ichino V 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. The antibody may be an IgG, IgM, IgA, or an antigen-binding fragment thereof. In this embodiment, the antibody is selected from the group consisting of Fab', F(ab'), F(ab'), monovalent scF In some embodiments, the antibody is a .sigma.v, bivalent scFv, or single domain antibody. , a human antibody, a humanized antibody, or a deimmunized antibody. is conjugated to an imaging agent, a chemotherapeutic agent, a toxin or a radionuclide.

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

[0012] In still other embodiments, the present disclosure provides a single antibody comprising a nucleic acid sequence encoding an antibody of the present disclosure. Isolated polynucleotide molecules are provided.

[0013] In yet another embodiment, the present disclosure provides a V H CDR1 of domain 3 (SEQ ID NOs: 1, 2 and 3) and V of clone T26 L CDR1-3 of the domain (sequence Antibody V containing columns 4, 5 and 6) H A recombinant polypeptide comprising the domain is provided.

[0014] In other embodiments, the present disclosure provides a V H CDR1-3 of the domain (sequence numbers 11, 12, and 13) and V of clone 5B L CDR1-3 of the domain (sequence no. Antibody V, including Nos. 14, 15, and 16 H and providing a recombinant polypeptide comprising the domain. .

[0015] In yet other embodiments, the present disclosure provides a V H CDR1 of domain 3 (SEQ ID NOs: 21, 22 and 23) and V of clone 28B L CDR1 of domain Antibody V comprising 3 (SEQ ID NOs: 24, 25 and 26) H Recombinant polypeptides containing domains to provide.

[0016] In yet other embodiments, the present disclosure provides nucleic acid sequences encoding the polypeptides of the present disclosure. The present invention provides an isolated polynucleotide molecule comprising:

[0017] In other embodiments, the present disclosure provides a method for the preparation of an antibody of the present disclosure or a recombinant polypeptide of the present disclosure. In some embodiments, a host cell is provided that contains one or more polynucleotide molecules encoding the In the present invention, the host cell is a mammalian cell, a yeast cell, a bacterial cell, a ciliated cell or an insect cell. It is a cell.

[0018] In still other embodiments, the present disclosure provides methods for treating a subject with cancer. The method includes administering to the subject an effective amount of an antibody of the present disclosure. In some embodiments, the cancer is a B-cell malignancy. is present in a pharmaceutically acceptable composition. In some embodiments, the antibody In some embodiments, the antibody is administered intravenously, intradermally, intratumorally, It is administered intramuscularly, intraperitoneally, subcutaneously, or topically. These methods further include administering to the subject at least a second anti-cancer treatment. In some embodiments, the second anti-cancer treatment is surgery, chemotherapy, radiation therapy, cryotherapy, or chemotherapy. In some embodiments, the second Anti-cancer treatments include 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 an engineered CD79b CAR or CD79b TC having an antigen-binding domain comprising Provide R.

[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 Domain and less Both are approximately 80% identical V H domain, and V of SEQ ID NO: 9 L Domain and at least about 80 %Identical V L In some embodiments, the antigen binding domain comprises the sequence Number 7 V H The same V as the domain H domain, and V of SEQ ID NO: 9 L Same as domain V 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: 12H 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 Domain and few At least about 80% identical V H domain, and V of SEQ ID NO: 19 L domain and at least about 80% identical V L In some embodiments, the antigen-binding domain comprises: V of SEQ ID NO: 17 H The same V as the domain H domain, and V of SEQ ID NO: 19 L domain The same V 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: 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.

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

[0026] In some embodiments, the antigen binding domains are connected via a linker to form a V L To the domain Concatenated V H In a further embodiment, the linker comprises a domain. 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). In embodiments, 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 Including some In embodiments, the CAR or TCR comprises a hinge. The hinges are 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: IgG4 CH2CH3 (SEQ ID NO: 64 or 65) or I gG4 CH1CH2CH3 (SEQ ID NO: 66 or 67). In a further embodiment, the CAR comprises a transmembrane domain. 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 the step of: In a further embodiment, the transduction marker is enhanced green fluorescent protein. In yet a further embodiment, the eGFP is SEQ ID NO: 83 In some embodiments, the transduction marker and / or stabilizer The whole switch is a truncated epidermal growth factor (EGFR). R has the amino acid sequence of SEQ ID NO: 41. In some embodiments, the transduction marker The CAR and / or safety switch are linked to the CAR by a truncation peptide. In a further embodiment, the truncated peptide is a 2A peptide. In a still further embodiment, the 2A peptide is a T2A peptide. The T2A peptide has the amino acid sequence of SEQ ID NO: 85. In a further embodiment, the CAR further comprises a second antigen-binding domain. The second antigen-binding domain is a CD19 antigen-binding domain, a CD20 antigen-binding domain, or CD22 antigen-binding domain.

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

[0029] In still other embodiments, the present disclosure provides a CD79b CAR or CD79b TC. In some embodiments, a host cell is provided that is engineered to express R. In some embodiments, the cell In some embodiments, the host cell is engineered to express a CAR of the present disclosure. The chief cell is an immune cell. In a further embodiment, the immune cell is a T cell. In a further embodiment, the T cells are primary human T cells or TILs. In some cases, the T cells are CD4+ T cells or CD8+ T cells. In some embodiments, the primary human T cells are obtained from a healthy donor. In some embodiments, the T cells are autologous T cells. In some embodiments, the cells are allogeneic T cells. It has been engineered using a transposase system.

[0030] In yet other embodiments, the present disclosure provides a method for the preparation of CD79b-targeted T cells and a pharmaceutical carrier. and a pharmaceutical composition comprising the CD79b-targeting T cells of the present disclosure. It has been engineered to express a TCR.

[0031] In other embodiments, the present disclosure provides an effective amount of a CD79b target to treat cancer in a subject. and a composition comprising a CD79b-targeted T cell, wherein the CD79b-targeted T cell is a CAR or has been engineered to express a TCR.

[0032] In yet other embodiments, the present disclosure provides an effective amount of CD7 The present invention provides a method for the treatment of a rhesus mast cell, comprising administering to a subject a subject in need thereof a therapeutically effective amount of CD79b-targeted T cells, the method ... These have been engineered to express a CAR or TCR.

[0033] In other embodiments, the present disclosure provides a method for treating cancer in a subject, the method comprising: The method includes administering to a subject an effective amount of CD79b-targeted T cells, The targeted T cells are engineered to express a CAR or TCR of the present disclosure. In yet a further embodiment, the cancer is a B-cell malignancy. B-cell malignancies include B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and Lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma In some embodiments, the subject has previously had C. In some embodiments, the subject has received CD19 CAR therapy. In a further embodiment, the subject is resistant to CAR therapy. In yet a further embodiment, the subject has recurrent CD19-negative tumors. In some embodiments, the CD79b-targeted T cells are administered intravenously, intradermally, intratumorally, In some embodiments, the C The D79b-targeting T cells are administered intravenously. further comprising administering at least a second anti-cancer treatment to the subject. Secondary anti-cancer treatments include surgery, chemotherapy, radiation therapy, cryotherapy, and hormone therapy. In some embodiments, the cancer is CD7 9b-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 It is an antigen-binding domain or a CD22 antigen-binding domain.

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

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

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

[0038] In a further embodiment, a method for treating cancer in a subject is provided, the method comprising: and administering an effective amount of CD79b CAR T cells (e.g., CAR T cells of the present embodiment) to a subject. 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 lymphoma or chronic In certain embodiments, the cancer expresses CD79b. It is a cancer that occurs.

[0039] In some embodiments, the subject has previously been administered a CD19 CAR therapy. In certain embodiments, the subject has a CD19 antigen loss or the like. In certain embodiments, the subject is resistant to CD19 CAR therapy. 19-negative tumors have recurred.

[0040] In certain embodiments, the CD79b CAR T cells are administered intravenously, intradermally, or intratumorally. In additional embodiments, the compound is administered intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. The method further comprises administering at least a second anti-cancer therapy to the subject. In some embodiments, the second anti-cancer therapy is selected from the group consisting of surgery, chemotherapy, radiation therapy, cryotherapy, Hormonal therapy, immunotherapy or cytokine therapy.

[0041] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, from this detailed description, those skilled in the art will appreciate that various modifications and variations within the spirit and scope of the invention are possible. Because various modifications and variations will become apparent, the detailed description and specific examples are intended to be illustrative of the preferred embodiments of the invention. It should be understood that the following statements are given by way of example only.

[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 is understood to be encompassed by the following claims, taken in conjunction with the detailed description of specific embodiments presented herein. This may be better understood by reference to one or more of these drawings. [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 essential component of the B cell receptor complex. CD79b is widely expressed in normal B cells and B cell malignancies, and Expression of CAR T-cell markers is typically observed in CD19-negative tumors that relapse after CD19-specific CAR T-cell therapy. Thus, in certain embodiments, the present disclosure provides a method for detecting CD79b-C CD79b monoclonal antibody and CD79b-specific CA, such as for AR T cells Provide R.

[0059] This study demonstrates the efficacy of CD79b-specific CAR T cells in in vitro and in vivo models. The efficacy of the cell product was demonstrated. Three mouse monoclonal antibodies against human CD79b These antibodies were developed using hybridoma technology and were specific for recombinant human CD79b. with high affinity (Kd range of 1.44-17.8 nM); and It was demonstrated that CD79b stains multiple lymphoma cell lines. The variable region of the light chain was cloned, and the CD3ζ and CD28 / 4-1BB costimulatory domains were cloned. We developed a lentiviral construct for an anti-CD79b CAR carrying the anti-CD79b CAR constructs were transduced using lentivirus into primary CD4+ and CD4+ cells from healthy donors. It was demonstrated that 8+ T cells could be transduced with a transduction efficiency of over 70%.

[0060] Anti-CD79b CAR T cells were used in combination with Daudi Burkitt lymphoma cell lines and Min o Compared to control anti-CD19 CAR T cells against mantle cell lymphoma cell lines The transduced T cells showed significant cytotoxic activity against the IgG4-dependent cytotoxic T cells, whereas non-transduced T cells showed no such activity. More importantly, anti-CD79b did not inhibit CD19-CD19. 79b+ lymphoma cells, whereas anti-CD19 CAR T cells inhibited CD19-CD7 It did not lyse 9b+ lymphoma cells. + 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 + Both anti-CD79b CAR T cells Significant degranulation was observed. The efficacy of anti-CD79b CAR T cells against M. lucifera was investigated in vivo. 2 × 10 6 Tumor cells / mouse NSG mice were injected IV with 10 × 10 6 T cells / mouse and non-transduced primary T cells, anti-CD19 CAR T cells, or anti-CD79b CAR T cells were administered via the tail vein. Bioluminescence imaging was used to assess tumor burden. The results showed that tumor growth progression was slower in mice treated with untransduced T cells. On the other hand, mice treated with anti-CD19 and anti-CD79b CAR T cells showed In the B Efficacy of novel anti-CD79b CAR T-cell therapy in patients with cellular malignancies This suggests that resistance due to CD19 loss after CD19-specific CAR T cell therapy may be due to: This could be a new strategy for overcoming gender.

[0061] In some embodiments, the anti-CD79b CAR construct is delivered via a lentiviral vector. The vector is capable of transducing immune cells such as T cells. The constructs contain CD28, CD3ζ and / or 4-1BB signaling domains. The construct may contain a transduction marker such as eGFP or a truncated EGFR domain. The transduction marker may comprise a cleavage peptide, such as the 2A peptide, that cleaves the CAR. can be linked to

[0062] Administering the CD79b-specific CAR immune cells (e.g., T cells) provided herein Further provided herein is a method of treating cancer by administering to a subject a cancer that expresses CD7 9b-expressing B-cell malignancies (e.g., B-cell acute lymphoblastic leukemia (ALL) , diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic The treatment may be anti-inflammatory, ... Patients with B-cell malignancies who have relapsed with CD19-negative tumors after CD19-CAR T-cell therapy It can be used to treat subjects with II. Definition

[0063] As used herein, "essentially free" with respect to a particular component means that Certain components are not intentionally formulated into the composition and / or are not intended to be contaminants. It is used herein to mean absent even at all or even in trace amounts. Therefore, the total amount of a particular component resulting from any unintentional inclusion of that component in a composition is , less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the component is undetectable.

[0064] As used herein, "a" or "an" may mean one or more. When used in a clause, the words "a" or "an" are used in conjunction with the word "including." It can mean one or more than one.

[0065] Use of the term "or" in the claims refers to alternatives only, unless expressly indicated to be so. or used to mean "and / or" unless the alternatives are mutually exclusive. However, the present disclosure supports the definition that refers to only alternatives and "and / or." As used herein, "another" means at least a second or more. The terms "about," "substantially," and "approximately" generally refer to This means a value plus or minus 5%.

[0066] "Treating" a disease or condition or treating a disease or condition means alleviating the symptoms of the disease. or a protocol that may involve administering one or more drugs to a patient for the purpose of alleviating symptoms. The desired effects of treatment include slowing the progression of the disease, improving the condition, Remission or alleviation of symptoms and remission or improvement of prognosis. It can occur before as well as after the signs or symptoms of a condition appear. "Placing" or "treating" a disease or unwanted condition is not "preventing" or "preventing" a disease or unwanted condition. Furthermore, "treat" or "treatment" can include the complete alleviation or amelioration of signs or symptoms. It does not require relief, it does not require a cure, and in particular it only has a marginal effect on the patient. Includes protocols.

[0067] The term "therapeutic effect" or "therapeutically effective" as used throughout this application means Refers to something that promotes or improves the well-being of a subject in relation to medical treatment for a condition This includes reducing the frequency or severity of signs or symptoms of a disease, but For example, cancer treatment includes, for example, reducing tumor size, reducing tumor invasiveness, and the like. Treatment of cancer may include administering to a subject with cancer, reducing the rate of cancer growth, or preventing metastasis. It can also refer to an extension of survival time.

[0068] "Subject" and "patient" refer to a human or non-human, e.g., a primate, mammal, and It refers to a vertebrate. In certain embodiments, the subject is a human.

[0069] The phrase "pharmaceutically or pharmacologically acceptable" means a compound that is suitable for administration to an animal, such as a human. Molecular entities and compositions that do not produce adverse, allergic or other untoward reactions. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients is within the scope of the present disclosure. In addition, for administration to animals (e.g., humans), the preparations , as required by the FDA Office of Biological Standards It is understood that sterility, pyrogenicity, general safety and purity standards should be met. .

[0070] As used herein, "pharmaceutically acceptable carrier" refers to a compound known to those skilled in the art. As such, any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, food saline, parenteral vehicles (e.g., sodium chloride, Ringer's dextrose, etc.), non-aqueous Solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable Suitable organic esters (e.g., ethyl oleate), dispersion media, coatings, surfactants, acids anti-oxidants, preservatives (e.g., antibacterial or antifungal agents), antioxidants, chelating agents and inert gas), isotonicity agent, absorption retardant, salt, drug, drug stabilizer, gel, binder, excipient, disintegrant, Lubricants, sweeteners, flavorings, colorants, flow and nutritional supplements, and similar materials; The pH and exact concentration of the various components in the pharmaceutical composition is adjusted according to known parameters. III.CD79b antibody

[0071] In certain embodiments, the method binds to at least a portion of CD79b and inhibits CD79b activity. an antibody or a fragment thereof that inhibits activity including at least signal transduction As used herein, the term "antibody" refers to any immunological Binding agents (e.g., IgG, IgM, IgA, IgD, IgE and genetically modified Ig IgG) and a polypeptide containing an antibody CDR domain that retains antigen-binding activity. Antibodies include chimeric antibodies, affinity matured antibodies, polyclonal antibodies, and clonal, monoclonal, humanized, human antibody or antigen-binding antibody fragment or may be selected from the group consisting of natural or synthetic ligands. The CD79b antibody is a monoclonal antibody or a humanized antibody.

[0072] Thus, by known means and as described herein, CD79b antibodies The compound or epitope thereof has been isolated from a natural source or is a synthetic derivative of a natural compound. CD79b, whether monoclonal or variant, one or more of its respective epitopes Polyclonal or monoclonal antibodies specific for the topope or any of the aforementioned conjugates. Monoclonal antibodies, antibody fragments, and binding domains and CDRs (manipulated from any of the foregoing) (including modified forms) can be produced.

[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 Consists of a domain (iii) the V of a single antibody; L and V H "Fv" consisting of domains fragment; (iv) V H (v) an isolated "dAb" fragment consisting of the (vi) a bivalent fragment comprising two linked Fab fragments; (vii) a F(ab')2 fragment, H Domains and V L Domain and that and a peptide linker that allows the two domains to associate to form the binding domain. (viii) bispecific single chain Fv molecules ("scFv"); (ix) a gene encoding a nucleotide sequence of ... Diabodies (USA), which are multivalent or multispecific fragments constructed by fusion Patent Application Publication No. 20050214860), but are not limited to Fv, The scFv or diabody molecule is V H Domains and V L Disulfides linking the domains The antibody can be stabilized by the incorporation of a peptide bridge. Minibodies containing the nucleus can also be created.

[0074] Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al. 2003) reported that it acts as a truncated antibody, has a longer serum half-life, and "Antibody-like binding peptides" are peptides that have the advantage that their synthesis methods are less cumbersome. The term "ABiP" (Antimicrobial Immunosorbent Assay) is described.

[0075] To generate antibodies specific to CD79b, we cloned the CD79b extracellular domain (ECD) tag. Animals can be inoculated with antigens such as proteins. Antigens can be administered in a variety of ways to boost the immune response. As used herein, a conjugate is a molecule that is often attached or conjugated to a molecule of the formula: , any peptide bound to an antigen used to elicit an immune response in an animal; It is a polypeptide, protein or non-protein substance that is produced in animals in response to antigen challenge. The antibodies produced in the antibody-producing cells are derived from various non-identical B lymphocytes. Polyclonal antibodies contain multiple antibodies (polyclonal antibodies) that each bind to different antigens. Polyclonal antibodies in a given animal are a mixed population of antibody species that can recognize different epitopes. When strict conditions for antibody production are given, most of the antibodies in the serum of the animal The specificity of the antigenic compound against which the animal is immunized is known as the collective epitope. Further, affinity purification can be used to select only those antibodies that recognize the target antigen or epitope. It can be further improved.

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

[0077] Hybridoma technology involves the production of single B lymphoma cells derived from mice pre-immunized with the CD79b antigen. This technique requires the fusion of spheres with immortal myeloma cells (usually mouse myelomas). provides a method for propagating a single antibody-producing cell over an indefinite number of generations, allowing the same antigen to be expressed Unlimited amounts of structurally identical antibodies (monoclonal antibodies) with specificity or epitope specificity Antibodies (antibodies) can be generated.

[0078] Plasma B cells (CD45 + CD5 - CD19 + ) was prepared from immunized rabbits. The CD79b-binding cells can be isolated from rabbit peripheral blood mononuclear cells and further selected for CD79b-binding cells. After enrichment of antibody-producing B cells, total RNA can be isolated and cDNA synthesized. The DNA sequences of antibody variable regions of both the nucleotide and light chains can be amplified and used to generate phage-displayed Fab fragments. It can be constructed into an expression vector and transformed into E. coli. Binding Fabs can be selected through multiple rounds of enrichment panning and sequenced. The identified hits that bind to CD79b were then analyzed in human embryonic kidney (HEK293) cells (Invitrogen). Using a mammalian expression vector system, rabbit and rabbit / human chimeric genes were expressed in It can be expressed as a full-length IgG of the IgG type and analyzed by fast protein liquid chromatography (FPLC). ) separation unit and can be purified using Protein G resin.

[0079] In one embodiment, the antibody is a chimeric antibody, e.g., a chimeric antibody in which heterologous non-human sequences, human sequences, or humanized sequences (e.g., framework sequences and / or constant domain sequences) Antibodies containing antigen-binding sequences derived from the transplanted non-human donor. The constant domains of the heavy and the low chains were replaced with similar domains of human origin. Methods have been developed to preserve the variable regions of the foreign antibody. "Fully human" monoclonal antibodies are produced in transgenic mice. Antibodies having both rodent, e.g., murine, and human amino acid sequences are possible. The variable domains of monoclonal antibodies can be further enhanced by recombinantly constructing the variable domains. Methods have also been developed to convert the antibody into a human form. Only the variable CDRs are derived from a mouse monoclonal antibody, while the framework and constant regions The regions are derived from human amino acid sequences (U.S. Patent Nos. 5,091,513 and 6, (See US Pat. No. 881,557.) Amino acid sequences within antibodies characteristic of rodents are compared with those of humans. By substituting the amino acid sequence found at the corresponding position in the antibody, It is believed that this reduces the likelihood of a harmful immune response during vaccination. The hybridoma or other cell is capable of expressing the binding specificity of the antibody produced by the hybridoma. It may also be subject to genetic mutations or other alterations that may or may not alter the

[0080] Methods for producing polyclonal antibodies in various animal species, as well as humanized antibodies We produce various types of monoclonal antibodies, including chimeric and fully human antibodies. Methods for preparing hydroxybenzoates are well known and highly predictable in the art. The patents and US patent applications provide useful descriptions of such methods: US Pat. No. 5,629,399; Application Nos. 2004 / 0126828 and 2002 / 0172677; and U.S. Pat. No. 3,817,837; No. 3,850,752; No. 3,939,350; No. 3,939,350; No. 3,996,345; No. 4,196,265; No. 4,275,149; No. 4 , 277,437; 4,366,241; 4,469,797; 4, No. 472,509; No. 4,606,855; No. 4,703,003; No. 4,7 No. 42,159; No. 4,767,720; No. 4,816,567; No. 4,86 No. 7,973; No. 4,938,948; No. 4,946,778; No. 5,021 , No. 5,164,296; No. 5,196,066; No. 5,223, No. 409; No. 5,403,484; No. 5,420,253; No. 5,565,3 No. 32; No. 5,571,698; No. 5,627,052; No. 5,656,43 No. 4; No. 5,770,376; No. 5,789,208; No. 5,821,337 No. 5,844,091; No. 5,858,657; No. 5,861,155 ; Same No. 5,871,907; Same No. 5,969,108; Same No. 6,054,297; Same No. 6,165,464; Same No. 6,365,157; ​​Same No. 6,406,867; Same No. 6,406,867; Same No. 6,406,867; No. 6,709,659; No. 6,709,873; No. 6,753,407; No. No. 6,814,965; No. 6,849,259; No. 6,861,572; No. 6 ,875,434; and 6,891,024. All references cited herein are hereby incorporated by reference. Patents, patent applications and other publications, and all patents, patents, patent applications and other publications cited therein. Published patent applications and other publications are incorporated herein by reference.

[0081] Antibodies may be produced from any animal origin, including birds and mammals. The body may be from sheep, murines (e.g., mice and rats), rabbits, goats, guinea pigs, camel, horse or chicken antibodies. Furthermore, newer technologies have led to the development of human antibodies. This will enable the development and screening of human combinatorial antibody libraries. For example, see U.S. Pat. No. 6,946,546, incorporated herein by reference. As reported, bacteriophage antibody expression technology allows specific antibodies to be produced without animal immunization. It is possible to generate specific antibodies.

[0082] Antibodies against CD79b may be obtained from a variety of animal species, monoclonal cell lines, or other sources of the antibodies. Regardless of the type of CD79b, it is highly likely that it will have the ability to neutralize or counteract the effects of CD79b. Certain animal species are allergic to antibodies due to the activation of the complement system via the "Fc" portion of the antibody. are not preferred for generating therapeutic antibodies because they may be more likely to cause a vasoconstriction reaction. However, enzymatic digestion of the whole antibody may not be sufficient to obtain the "Fc" (complement fixation) fragment. ) fragments, and antibody fragments having the binding domain or CDRs. By removing the Fc portion, the antigen-antibody fragment can be prevented from reacting with undesired immunological Fc-less antibodies are less likely to elicit a response and are therefore less suitable for prophylactic or therapeutic treatment. As noted above, preference may be given to antibodies raised in other species or Reduces adverse immunological consequences resulting from administering antibodies with sequences from other species to animals To reduce or eliminate antibodies to be chimeric or partially human or fully human may be constructed.

[0083] Substitution variants typically involve a change of one amino acid to another at one or more sites within a protein. amino acid replacements, with or without loss of other functions or properties of the polypeptide. Substitutions may be conservative and may be designed to modulate one or more properties of the That is, one amino acid is replaced with one that has a similar shape and charge. Conversions are well known in the art and include, for example, alanine to serine; lysine to lysine; asparagine to glutamine or histidine; aspartic acid to to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamine acid to aspartic acid; glycine to proline; histidine to asparagine or to glutamine; isoleucine to leucine or valine; leucine to valine or to 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 and valine to isoleucine or leucine. Alternatively, the substitution may be carried out such that the function or activity of the polypeptide is affected. Non-conservative changes usually involve replacing a residue with a similar one, but with a different structure. Substitution of a non-polar amino acid, e.g., a polar or charged amino acid with a non-polar amino acid or uncharged amino acids, and vice versa.

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

[0085] Approximately 0.001 mg to approximately 10 mg of total polypeptides, peptides and / or It is contemplated that the protein is present in the composition. The concentration of is 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 It may be greater than or equal to (or any range derivable therein). At most 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, 2 6, 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, 6 6, 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% of the antibodies that bind to CD79b It can be a body.

[0086] The antibody, or preferably an immunological portion of the antibody, may be fused to another protein. It can be chemically conjugated or expressed as a fusion protein with another protein. In this specification and the appended claims, all such fusion proteins are referred to as antibodies. or included in the definition of the immunological portion of an antibody.

[0087] Embodiments include linking at least one agent to form an antibody conjugate or payload. The present invention provides antibodies and antibody-like molecules, polypeptides and peptides against CD79b, which To enhance the effectiveness of antibody molecules as diagnostic or therapeutic agents, It is possible to link or covalently bond or conjugate a desired molecule or moiety. Such molecules or moieties comprise at least one effector molecule or receptor. Effector molecules can be, but are not limited to, receptor molecules. For example, it includes molecules with cytotoxic activity. Typical examples include toxins, therapeutic enzymes, antibiotics, and radiolabeled nucleotides. In contrast, a reporter molecule is any moiety that can be detected using an assay. Non-limiting examples of reporter molecules that may be conjugated to antibodies include enzymes, Radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules , photoaffinity molecules, colored particles, or ligands such as biotin.

[0088] Several methods are known in the art for attaching or conjugating antibodies to binding moieties. Some attachment methods include, for example, the use of organic chelators, e.g., dienes, attached to the antibody. Ethylenetriaminepentaacetic anhydride (DTPA); Ethylenetriaminetetraacetic acid; N-chloro- p-Toluenesulfonamide; and / or tetrachloro-3,6-diphenylglycol Also, glutaraldehyde or The enzyme is reacted with the monoclonal antibody in the presence of a coupling agent such as periodate. In the presence of these coupling agents, or with isothiocyanates, The reaction prepares a conjugate bearing a fluorescein marker. IV. Cell therapy

[0089] Certain embodiments of the present disclosure involve obtaining T cells and administering them to target cancer cells. The present invention relates to administering T cells to a subject as immunotherapy. The cells may be administered to a subject in a manner consistent with the invention encompassed herein. The body may deliver a composition that may or may not itself be an immune cell. In the example, 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 Examples include:

[0090] Some fundamentals for the induction, activation, and expansion of functional antitumor effector T cells Several approaches have been reported in the past 20 years. These include tumor-infiltrating lymphocytes ( autologous cells such as TILs; autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs), or T Beads coated with cellular ligands and activating antibodies, or for capturing target cell membranes The isolated cells were used to generate ex vivo activated T cells; anti-host tumor T cell receptors. allogeneic cells that naturally express the TCR; and known as "T-bodies" The tumor-specific TCR molecules are designed to express tumor-reactive or chimeric TCR molecules that exhibit antibody-like tumor recognition ability. Genetically reprogrammed or "redirected" non-tumor-specific autologous cells or These approaches can be used in the methods of the present disclosure. , which has become the basis for numerous protocols for preparing and immunizing T cells. Preparation of AT cells

[0091] In some embodiments, the T cells are present in the blood, bone marrow, lymph, or lymphoid organs. In some embodiments, the cells are human cells. The cells are typically primary cells. and, for example, cells isolated directly from a subject and / or cells isolated from a subject, In some embodiments, the cells include T cells or other cells. One or more subsets of a type (e.g., the entire T cell population, CD4 + cells, CD8 + Cells, and their subpopulations, e.g., function, activation state, maturity, differentiation potential, expansion, recycling The range of antigenicity, localization and / or persistence, antigen specificity, type of antigen receptor, and specific organs may also be important. or compartments, markers or cytokine secretion profiles The subjects to be treated include those defined by the degree of differentiation and / or the type of tumor. With respect to the body, the cells can be allogeneic and / or autologous cells. In the case of existing technologies, cells are pluripotent and / or or multipotent (e.g., induced pluripotent stem cells (iPSCs) In some embodiments, the method further comprises isolating cells from a subject. and processes, 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 subsets The group includes 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 Molly T(T CM ), Effector Memory T(T EM ) or terminally differentiated effectors Memory T(T TEMRA ) cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells cells, helper T cells, cytotoxic T cells, mucosa-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 be.

[0093] In some embodiments, one or more of the T cell populations are identified by a specific marker, such as a surface marker. The cells are enriched for marker-positive or specific marker-negative cells, and In some cases, such markers are associated with certain T cell populations. are absent or expressed at relatively low levels on cells (e.g., non-memory cells), It is present or present at relatively high levels on certain other T cell populations (e.g., memory cells). In one embodiment, the marker is expressed in those cells (e.g., CD4+ cells). 8 + cells or CD3 + cells) express CD45RO, CCR7, CD28, CD27, CD 44, CD127 and / or CD62L positivity or high surface levels of C D45RO, CCR7, CD28, CD27, CD44, CD127 and / or C Cells expressing D62L are enriched (i.e., positively selected) and / or CD45RA positive or expressing high surface levels of CD45RA The expressing cells are depleted (e.g., negatively selected). In morphology, the cells express CD122, CD95, CD25, CD27 and / or I L7-Ra (CD127) positivity or high surface levels of CD122, CD95 , cells expressing CD25, CD27 and / or IL7-Ra (CD127) In some instances, CD8 + T cells are CD4 5RO-positive (or CD45RA-negative) and CD62L-positive cells The cells are concentrated.

[0094] In some embodiments, T cells are non-T cells (e.g., B cells, monocytes or other by negative selection of markers (e.g., CD14) expressed on leukocytes (white blood cells). In some embodiments, CD4 + or CD8 + Selection process By using CD4 + Helper T cells and CD8 + Cytotoxic T cells are isolated Such CD4 + and CD8 + A population can consist of one or more naive, memory and and / or expressed or expressed to a relatively high degree on a subpopulation of effector T cells Further subpopulation sorting can be achieved by positive or negative selection of the markers identified. It can be tagged.

[0095] In some embodiments, CD8 + The cells express surface antibodies associated with each subpopulation. Positive or negative selection based on the origin of the naive, central Further enrichment of memory, effector memory and / or central memory stem cells In some embodiments, the central memory T( T CM ) Cell enrichment may improve long-term survival, expansion, and / or engraftment after administration. This is done to increase efficacy, which in some embodiments is done to target such subpopulations. In some embodiments, T CM Enriched CD8 + T thin Cells and CD4 + Combining it with T cells further increases efficacy.

[0096] In some embodiments, the T cells are autologous T cells. A sample is obtained from the patient to obtain a single cell suspension. The single cell suspension can be cultured in any suitable medium. in a suitable manner, e.g. mechanically (e.g. gentleMACS TM Dissociate Tumors were decongested using a r, Miltenyi Biotec, Auburn, Calif. can be obtained either enzymatically (e.g., by condensation) or enzymatically (e.g., collagenase or DNase) Single cell suspensions of enzymatic digests of tumors were cultured in interleukin-2 (IL-2). The cells were cultured until they reached confluence (e.g., approximately 2 × 10 6 lymphocytes) until For example, the cells are cultured for about 5 to about 21 days, preferably about 10 to about 14 days. These cells were cultured for 5 days, 5.5 days, or 5.8 days to 21 days, 21.5 days, or 21 days. 8 days, e.g., 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days The cells may be cultured for 14.8 days or 15.8 days.

[0097] The cultured T cells can be pooled and rapidly expanded, preferably for about 10 to about 14 days. or rapid expansion over a period of approximately 14 days to produce a population of at least approximately 50 antigen-specific T cells. 0-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or more) increase More preferably, rapid expansion occurs over a period of about 10 to about 14 days, preferably about 14 days. by at least about 200 times (e.g., 200, 300, 400, 500, 600, 700, 800, 900 or more) increase.

[0098] Expansion can be achieved by any of several methods known in the art. For example, T cells can be produced by the administration of feeder lymphocytes and interleukin-2 (IL-2). or interleukin-15 (IL-15), with IL-2 being preferred. They can be easily expanded using nonspecific T cell receptor stimulation in the presence of T cell receptor stimulation was performed with approximately 30 ng / ml of mouse monoclonal anti-CD3 antibody. OKT3 (Ortho-McNeil®, Raritan, NJ) Alternatively, the T cells may be treated with a T cell growth factor (e.g., 300 IU / ml of IL-2 or IL-15 (IL-2 is preferred) Cancer antigens (including their antigenic parts, such as epitopes, or cells) are transfected into peripheral blood mononuclear cells (PBMs). C) can be easily expanded by stimulating it in vitro, and the cancer antigens The vector can be expressed in accordance with the method of the present invention, for example, human leukocyte antigen A2 (HLA-A2) binding peptide. The in vitro induced T cells express HLA-A2 antigens. They are rapidly expanded by restimulation with the same cancer antigen pulsed onto the presenting cells. Alternatively, the T cells may be, for example, irradiated autologous lymphocytes or irradiated HLA-A They can be restimulated with 2+ allogeneic lymphocytes and IL-2.

[0099] The autologous T cells express T cell growth factors that promote the proliferation and activation of these autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL-1), IL-2, and IL-3. Suitable modification methods include IL-2, IL-7, IL-15, and IL-12. Methods are known in the art. See, e.g., Sambrook et al., Molecular Biology, vol. lar Cloning:A Laboratory Manual,3 rd ed., Cold Spring Harbor Press,Cold Spring Har bor, NY2001; and Ausubel et al., Current P rotocols in Molecular Biology,Greene Pub lishing Associates and John Wiley & Sons , NY, 1994. In certain embodiments, the modified autologous T cells are T cells High levels of growth factors are expressed. T cell growth factor coding sequences (e.g., the coding sequences for IL-12) The promoter sequence (promoter sequence) is a promoter whose operably linked to a T cell growth factor coding sequence promotes high level expression. Like promoters, these are readily available in the art. B. Genetically Engineered Antigen Receptors

[0100] The cells may contain engineered antigen receptors, such as engineered TCRs or chimeric antigen receptors (CARs). For example, autologous T cells can be genetically engineered to express a specific antigen receptor. Expresses T cell receptors (TCRs) with antigen specificity for cancer antigens such as 79b Suitable TCRs include those that target, for example, melanoma antigens, such as gp100 or or MART-1. These methods are well known in the art. See, e.g., Sambrook and Ausubel, supra. For example, the above T cells are Ther.19:496-510(2008) and Johnson et al.Bl The transduction method described in

[114] ,

[114] , [535-46] (2009) was used to generate anti-cancer The antigen-specific TCR can be transduced to express a TCR with antigen specificity for the antigen.

[0101] In some embodiments, the T cells comprise one or more T cells introduced via genetic engineering. one or more nucleic acids encoding the above antigen receptors, and genetic manipulation of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., that is, they are not normally present in the cell or in a sample obtained from the cell, but are present in, for example, another organism. or nucleic acids obtained from cells, e.g., cells that have been engineered and / or or is not normally found in the organism from which such cells originate. These nucleic acids are non-naturally occurring, such as nucleic acids not found in nature (eg, chimeras).

[0102] In some embodiments, the CAR comprises an extracellular polypeptide that specifically binds to CD79b. In some embodiments, the antigen is expressed on the cell surface. In some embodiments, the CAR is a TCR-like C AR, and antigens are expressed in the context of major histocompatibility complex (MHC) molecules, similar to TCRs. processed peptide antigens (e.g., intracellular antigens) that are recognized on the cell surface in It is a peptide antigen of the protein.

[0103] Exemplary antigen receptors, including CARs and recombinant TCRs, and their Methods for manipulating receptors and for introducing them into cells include those described in, for example, International Patent Publication No. Patent application publication numbers WO200014257, WO2013126726, WO2012 / 1 29514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication No. US2002131 960, US2013287748, US20130149337, U.S. Patent No. 6,45 No. 1,995, No. 7,446,190, No. 8,252,592, No. 8,339 , No. 645, No. 8,398,282, No. 7,446,179, No. 6,410, No. 319, No. 7,070,995, No. 7,265,209, No. 7,354,7 Nos. 62, 7,446,191, 8,324,353 and 8,479, 118, and those described in European Patent Application No. EP2537416; and / or Sadelain et al.,2013;Davila et al.,2 013; Turtle et al., 2012; Wu et al., 2012 In some embodiments, genetically engineered antigen receptors include those described in The target includes CARs, such as those described in U.S. Pat. No. 7,446,190, and international Including those described in patent application publication number WO / 2014055668Al. 1. Chimeric antigen receptor

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

[0105] In some embodiments, the engineered antigen receptor includes an activating or stimulatory receptor. CARs, costimulatory CARs (see WO2014 / 055668) and / or is an inhibitory CAR (iCAR, see Fedorov et al., 2013) These CARs generally react with one or more intracellular signals. In some embodiments, the nucleic acid is linked to a nucleic acid signaling component via a linker and / or a transmembrane domain. Such molecules contain extracellular antigen (or ligand) binding domains linked by a nucleotide sequence. , usually signaling through natural antigen receptors, together with costimulatory receptors. signaling via receptors and / or via costimulatory receptors only To imitate or imitate.

[0106] Certain embodiments of the present disclosure include intracellular signaling domains, transmembrane domains, and and an extracellular domain containing one or more signaling motifs. Polypeptides (including humanized CARs (hCARs) to reduce immunogenicity) In certain embodiments, the CAR comprises a nucleic acid encoding It may recognize epitopes that contain spaces shared between one or more antigens. In this embodiment, the binding region is a complementarity determining region of a monoclonal antibody, In another embodiment, the antibody may comprise a variable region, and / or an antigen-binding fragment thereof. The specificity of the peptides (e.g., cytokines) that bind to the receptors is derived from the do.

[0107] Human CAR nucleic acids are human genes used to enhance cellular immunotherapy in human patients. In a specific embodiment, the present disclosure provides a full-length cCAR. The DNA or coding region contains the antigen-binding region or domain, which is specific to a specific human monoclonal antibody. V of single-chain variable fragment (scFv) derived from a monoclonal antibody H Chain and V L Chain fragments (e.g., U.S. Pat. No. 7,109,304, incorporated herein by reference). The fragment may comprise any of the human antigen-specific antibodies. In a more specific embodiment, the fragments may be different in number of antigen-binding domains. The fragment is encoded by a sequence optimized for human codon usage for expression in human cells. It is an antigen-specific scFv encoded by

[0108] The configuration can be a multimer (e.g., a diabody or multimer). The possibility that the variable portions of the heavy and low chains are formed by cross-pairing into diabodies The hinge region of the construct retained the first cysteine ​​from the full deletion. The substitution is proline rather than serine, and the residue is cut up to the first cysteine. There are several options available, ranging from the Fc portion being deleted. Any protein that binds and / or dimerizes may serve this purpose. Only one of the domains, e.g., the CH2 domain or CH3 domain from a human immunoglobulin Domains can be used. Human immunoglobulins engineered to improve dimerization The hinge, CH2 and CH3 regions of immunoglobulins can also be used. You can use just the CD8 portion, or you can use the CD8 alpha portion.

[0109] In some embodiments, the CAR nucleic acid comprises a transmembrane domain and a modified C It contains sequences encoding other costimulatory receptors, such as the D28 intracellular signaling domain. Other costimulatory receptors include CD28, CD27, OX-40 (CD134) and and 4-1BB (CD137). .

[0110] In some embodiments, the CAR is directed to a specific cell type to be targeted by adoptive therapy. Specific antigens (or markers or ligands), such as antigens expressed in, e.g., Cancer markers and / or antigens intended to induce attenuated responses (e.g., normal cells These antibodies are constructed to have specificity for antigens expressed on affected or non-affected cell types. Thus, the CAR typically contains one or more antigen-binding molecules (e.g., one or more antigen-binding fragments, antigen-binding domains or antigen-binding portions) or one In some embodiments, the antibody variable domains and / or antibody molecules include: The CAR may be an antigen-binding portion of an antibody molecule (e.g., a monoclonal antibody (mAb)). Single-chain antibody fragments (scFv) derived from variable heavy (VH) and variable light (VL) chains ) is included.

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

[0112] The chimeric construct is then injected into immune cells either as naked DNA or in a suitable vector. It is contemplated that the vector can be introduced into the host by electroporation using naked DNA. Methods for stably transfecting cells are known in the art. See, e.g., U.S. Pat. See US Pat. No. 6,410,319. Naked DNA generally refers to DNA that has been prepared in a manner suitable for expression. The DNA encoding the chimeric receptor contained in the plasmid expression vector is called Point.

[0113] Alternatively, viral vectors (e.g., recombinant human leukocytes) can be used to introduce the chimeric construct into immune cells. retroviral vector, adenoviral vector, adeno-associated viral vector or Suitable vectors for use in accordance with the methods of the present disclosure include: The vectors are non-replicating vectors in immune cells. Many are known (e.g., based on HIV, SV40, EBV, HSV, or BPV). vector), the number of copies of the virus maintained within the cell is sufficient to maintain the viability of the cell. It is low enough that

[0114] In some embodiments, the component that specifically binds to the antigen, i.e., the recognition component, , linked to one or more transmembrane domains and an intracellular signaling domain. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the CAR comprises a domain that is naturally associated with one of the domains in the CAR. In some cases, the transmembrane domain is a such domains to minimize interactions with other members of the Sceptor complex to avoid binding to the transmembrane domains of the same or different surface membrane proteins. It is selected or modified by amino acid substitution.

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

[0116] In a specific embodiment, the CAR construct comprises a light chain-linker-heavy chain-hinge-transmembrane construct. The linker comprises a signaling domain and a signaling domain. or 45), linker 2 (SEQ ID NO: 46 or 47), linker 3 (SEQ ID NO: 48 or or linker 4 (SEQ ID NO: 50 or 51), The hinge is a CD8 hinge 1 (SEQ ID NO: 52 or 5 3), CD8 hinge 2 (SEQ ID NO: 54 or 55), CD8 hinge 3 (SEQ ID NO: 56 or 57), CD28 hinge (SEQ ID NO: 58 or 59), IgG4 hinge (SEQ ID NO: 60 or 61), IgG4CH2 (SEQ ID NO: 62 or 63), IgG4CH2CH3 (SEQ ID NO: 64), Sequence No. 64 or 65) or IgG4CH1CH2CH3 (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) , CD28™ (SEQ ID NO: 72 or 73), or CD8α™ (SEQ ID NO: :87). In this study, CD8 TM is a lyc- and / or NHRN receptor agonist, e.g., at its C-terminus. The signaling domain is C. D28 (SEQ ID NO: 74 or 75), 4-1BB (SEQ ID NO: 76 or 77), OX-4 0 (SEQ ID NO: 78 or 79) and / or CD3 intracellular (SEQ ID NO: 80 or 81) The CAR construct comprises, consists of, or consists essentially of: GFP (SEQ ID NO: 82 or 83), T2A (SEQ ID NO: 84 or 85) and / or further comprising, consisting of, or essentially consisting of EGFR (SEQ ID NO: 40 or 41). Exemplary heavy chain (HC), linker, and light chain (LC) combinations include: The combination is LC-Linker 1-HC; LC-Linker 2-HC; LC-Linker 3- HC;LC-Linker4-HC;HC-Linker1-LC;HC-Linker2-LC;H C-Linker 3-LC; or HC-Linker 4-LC. It will not be done. 2. T cell receptor (TCR)

[0117] In some embodiments, the genetically engineered antigen receptor includes a recombinant TCR. and / or TCRs cloned from naturally occurring T cells. "TCR" refers to a transmembrane receptor (TCR) that is composed of a variable alpha chain and a variable beta chain (TCRα and TCRβ, respectively). and TCRβ) or variable γ and variable δ chains (TCRγ and TCRβ, respectively) and TCRδ), which bind specifically to antigenic peptides bound to MHC receptors. In some embodiments, the TCR refers to a molecule that can specifically bind to It is an αβ type.

[0118] TCRs, which usually exist as αβ and γδ types, generally have similar structures, but T cells expressing TCRs may differ in anatomical location or function. Generally, TCRs are expressed on the surface of T cells (or T lymphocytes). found on the surface of the body, usually by binding to major histocompatibility complex (MHC) molecules In some embodiments, a TCR comprises a constant domain, a transmembrane domain, and a It may also contain a cytoplasmic domain, a short cytoplasmic tail, and / or a short cytoplasmic tail (e.g., Janeway e (See, e.g., et al., 1997). For example, in some embodiments, each chain of the TCR is , one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, transmembrane In some embodiments, the T CR associates with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise stated, the term "TCR" includes functional TCR fragments thereof. This term should be understood to include TCRs of the αβ or γδ types. It also encompasses intact or full-length TCRs.

[0119] Therefore, for purposes herein, reference to a TCR includes any TCR or functional TCR. Fragments (e.g., specific antigenic peptides bound in MHC molecules, i.e., The antigen-binding portion of the TCR that binds to the MHC-peptide complex is included. The "antigen-binding portion" or antigen-binding fragment of a TCR may be expressed as a structural domain of the TCR. antigens that contain only a portion of the main domain but that the complete TCR binds (e.g., MHC-peptide In some cases, the antigen-binding moiety refers to a molecule that binds to a specific The potential for sufficient TCR binding to form a binding site for binding to the appropriate MHC-peptide complex. variable domains (e.g., the variable a and variable β chains of a TCR), e.g., typically each chain It contains three complementarity determining regions.

[0120] In some embodiments, the variable domains of the TCR chains associate to form loops, or immunoglobulins. It forms a complementarity determining region (CDR) similar to that of immunoglobulin, which allows antigen recognition. The peptide specificity is determined by forming the binding site of the TCR molecule. Typically, as with immunoglobulins, CDRs are separated by framework regions. The retinal septum is separated by a retinal septum (FR) (e.g., Jores et al., 1990; Cho See thia et al., 1988; Lefranc et al., 2003 In some embodiments, CDR3 is involved in recognition of processed antigens. The CDR1 of the alpha chain interacts with the N-terminal part of the antigen peptide. It has also been shown that CDR1 of the beta chain binds to the C-terminal portion of the peptide. CDR2 is thought to recognize the MHC molecule. In some embodiments, the variable region of the β chain may include an additional hypervariable (HV4) region.

[0121] In some embodiments, the TCR chain comprises a constant domain. Like the phospholipase A, the extracellular portions of the TCR chains (e.g., a chain, β chain) bind to two immunoglobulins. a variable domain at the N-terminus (e.g., V a or Vp; usually Ka bat numbering, Kabat et al., “Sequences of P roteins of Immunological Interest, US Dep. t.Health and Human Services,Public Healt h Service National Institutes of Health, May 1991 th ed.-based amino acids 1–116), and one adjacent to the cell membrane the constant domain of (e.g., the a chain constant domain or C a , usually based on Kabat amino acids 117-259, β-chain constant domain or Cp, usually amino acids according to Kabat 117-295). For example, in some cases, the two chains The extracellular portion of the TCR, which is expressed as a TCR, consists of two membrane-proximal constant domains and two CDR-containing domains. The membrane-distal variable domain is a TCR domain with cysteine ​​residues that form disulfide bonds. It contains a short connecting sequence that forms a nucleotide bond, thereby creating a link between the two strands. In some embodiments, the TCR contains an additional sequence in each of the α and β chains. TCRs contain two disulfide bonds in the constant domain, which may contain stearate residues. .

[0122] In some embodiments, the TCR chain may include a transmembrane domain. In embodiments, the transmembrane domain is positively charged. The R chain contains the cytoplasmic tail. In some cases, due to its structure, the TCR It can associate with other molecules such as CD3. For example, it contains a constant domain along with a transmembrane region. The TCR, which contains the cytochrome P454, can anchor its proteins to the cell membrane and interact with the CD3 signaling machinery or can associate with the invariant subunit of the complex.

[0123] Generally, CD3 is composed of three different chains (γ, δ, and ε) and the ζ chain in mammals. For example, in mammals, this complex may comprise CD3γ It may comprise a homodimer of a CD3 chain, a CD3δ chain, two CD3ε chains, and a CD3ζ chain. The γ, CD3δ, and CD3ε chains are immunoglobulins containing a single immunoglobulin domain. The CD3γ chain, CD4γ, and CD5γ are highly related cell surface proteins of the phospholipase C superfamily. The transmembrane regions of the CD3δ and CD3ε chains are negatively charged, which is why these chains are This is a property that allows it to associate with the highly charged T cell receptor chains. The intracellular tails of the δ chain and CD3ε chain each contain an immunoreceptor activation tyrosine motif or Each CD3 ζ chain contains a single conserved motif known as an ITAM, whereas In general, ITAMs are involved in the signaling activity of the TCR complex. The accessory molecule has a negatively charged transmembrane region and is involved in the propagation of signals from the TCR to the cell. The CD3 chain and ζ chain work together with the TCR to form the T cell receptor They form complexes known as complexes.

[0124] In some embodiments, the TCR comprises two chains, α and β (or optionally γ and δ) or may be a single chain TCR construct. In some embodiments, the TCR comprises two separate TCRs linked by, for example, a disulfide bond. In some embodiments, the α-chain and β-chain or γ-chain and δ-chain heterodimer are used. In this method, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, the nucleic acid encoding the TCR is a publicly available TCR DNA. A sequences can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification. In some embodiments, the TCR is derived from a biological source, e.g., a cell, e.g., , T cells (e.g., cytotoxic T cells), T cell hybridomas or other publicly available In some embodiments, the T cells are isolated in vivo. In some embodiments, the high affinity T cell clones can be obtained from: In some embodiments, T cells can be isolated from a patient and TCRs can be isolated. The T cell hybridoma or clone may be cultured. Therefore, TCR clones against target antigens are synthesized by integrating human immune system genes (e.g., human leukocyte antigen receptors). or HLA) engineered transgenic mice. In embodiments, phage display is used to isolate TCRs against target antigens. In some embodiments, the TCR or antigen-binding portion thereof is identified using a method that uses a TCR sequence. Thus, it can be made synthetically. C. Delivery method

[0125] Those skilled in the art will appreciate that standard recombinant techniques (e.g., recombinant vectors) can be used to express the antigen receptors of the present disclosure. For example, Sambrook et al., 2001 and Ausubel et al. , 1996 (both of which are incorporated herein by reference) As vectors, plasmids, cosmids, etc. , viruses (bacteriophages, animal viruses and plant viruses) and artificial chromosomes (e.g., YAC), e.g., retroviral vectors (e.g., Moloney murine leukemia Derived from viral vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc. lentiviral vectors (e.g., HIV-1, HIV-2, SIV, BIV) Adenovirus (Ad) vectors (including those derived from replication-competent, replication-competent, and replication-competent vectors) Adeno-associated viruses (including production-deficient and gutless forms) AAV vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors virus vector, Epstein-Barr virus vector, herpes virus vector, vaccine Xenopus virus vector, Harvey murine sarcoma virus vector, mouse mammary tumor virus vector, Rous sarcoma virus vector, parvovirus vector, poliovirus vector vector, vesicular stomatitis virus vector, Maraba virus vector vectors and group B adenovirus enadenotucirev vectors. Not limited to these. 1. Viral Vectors

[0126] Viral vectors encoding antigen receptors are provided in certain embodiments of the present disclosure. When constructing a recombinant viral vector, non-essential genes are usually not heterologous. The gene or coding sequence for a (or non-native) protein is replaced by a viral vector. The marker utilizes viral sequences to introduce nucleic acids and sometimes proteins into cells. It is a type of expression construct that expresses the ability of a particular virus to infect cells or its receptor. their ability to enter cells via endocytosis and to infect the host cell genome Their ability to integrate and stably and efficiently express viral genes has made them These viruses are attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Viral vectors that can be used to deliver nucleic acids of certain embodiments of the present disclosure Non-limiting examples are listed below:

[0127] Lentiviruses are complex retroviruses and are a common retroviral gene. In addition to gag, pol, and env, other genes with regulatory or structural functions are also Lentiviral vectors are well known in the art (see, e.g., U.S. Patent No. 6,011,229). (See US Pat. Nos. 3,516 and 5,994,136).

[0128] Recombinant lentiviral vectors are capable of infecting non-dividing cells and are useful in in vivo and ex vivo expression. They can be used for gene transfer and nucleic acid sequence expression both in vivo and in vivo. Recombinant lentivirus capable of infecting dividing cells (wherein suitable host cells are Packaging functions, namely gag, pol and env, and rev and tat (transfected with two or more vectors having the formula (I)) is incorporated herein by reference. No. 5,994,136, which is hereby incorporated by reference. 2. Regulatory elements

[0129] Expression cassettes included in vectors useful in the present disclosure include, inter alia, protein-encoding a eukaryotic transcription promoter operably linked to a sequence, a splice signal including an intervening sequence, Null, and transcription termination / polyadenylation sequences (5' to 3'). Promoters and enhancers that control transcription of genes encoding proteins in eukaryotic cells. A gene is made up of multiple genetic elements. The cellular machinery carried by each element It can collect and integrate regulatory information from different genes, often This allows for the development of complex patterns of different transcriptional regulation. The promoters used in this context include constitutive promoters and 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. A promoter generally defines a sequence that locates the start site for RNA synthesis. The best known example of this is the TATA box, but mammalian Mammalian terminal deoxynucleotidyl transferase gene promoter and Some promoters, such as those of the SV40 late genes, contain a TATA box. The discontinuous elements, lacking a sequence and overlapping the initiation site itself, fix the location of initiation. Additional promoter elements control the frequency of transcription initiation. These are located in the region 30110 bp upstream of the start site, but several promoters It has also been shown that the coding sequence contains functional elements downstream of the initiation site. To be "under the control" of a transcriptional open reading frame, the 5' end of the transcription start site must be aligned with the selected promoter. The promoter is placed "downstream" (i.e., 3') of the motor. The "upstream" promoter controls the transcription of DNA. It stimulates transcription and promotes expression of the encoded RNA.

[0131] The spacing between promoter elements is often variable, and elements can be inverted or The function of the promoters is preserved even when they are separated or moved relative to each other. In the case of promoters, the spacing between promoter elements can be up to 50° before activity begins to decline. Depending on the promoter, individual elements may function cooperatively or independently. A promoter may function to activate transcription of a nucleic acid sequence. It may be used in conjunction with the term "enhancer," which refers to a cis-acting regulatory sequence involved in the regulation of and may not be used.

[0132] A promoter is a 5' non-coding sequence located upstream of a coding segment and / or exon. A nucleic acid sequence that is naturally associated with a nucleic acid sequence, such as may be obtained by isolating a code sequence. Such a promoter is called an "endogenous" promoter. Similarly, an enhancer can be located downstream or upstream of a nucleic acid sequence. It may be an enhancer naturally associated with the sequence. A recombinant promoter is a promoter that is not naturally associated with a particular environment. By placing the coding nucleic acid segment under the control of a heterologous promoter, certain advantages are possible. Also, a recombinant or heterologous enhancer may be a nuclear enhancer in its natural environment. It also refers to enhancers that are not naturally associated with a promoter or sequence. The promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers of other genes. A promoter or enhancer isolated from any virus or prokaryotic or eukaryotic cell. sensors, as well as non-"naturally occurring" i.e., various elements of various transcription control regions. promoters or enhancers containing mutations that alter expression and / or For example, the promoters most commonly used in recombinant DNA construction are β-lactamase (penicillinase), lactose and tryptophan (trp-) Promoter systems include: Synthesizing promoter and enhancer nucleic acid sequences In addition to being produced by recombinant cloning and / or PCR TM Starting with Using nucleic acid amplification techniques such as those described herein, a sequence may be generated in connection with the compositions disclosed herein. Furthermore, in organelles other than the nucleus (e.g., mitochondria, chloroplasts, etc.), It is contemplated that regulatory sequences that direct the transcription and / or expression of the sequences may be used as well. .

[0133] Naturally, the D in the organelle, cell type, tissue, organ or organism selected for expression Use a promoter and / or enhancer that effectively directs expression of the NA segment. Those skilled in the art of molecular biology generally use promoters for protein expression. We are aware of the use of promoter, enhancer, and cell type combinations (e.g., See, for example, Sambrook et al. 1989, incorporated herein by reference. The promoters used are constitutive promoters, tissue-specific promoters, and , an inducible promoter, and / or a promoter that directs high-level expression of the introduced DNA segment. A useful promoter (e.g., a promoter for the expression of recombinant proteins and / or or a promoter useful in the large-scale production of recombinant peptides). The motor may be heterologous or endogenous.

[0134] Additionally, any promoter / enhancer combination (e.g., epd.isb- Eukaryotic Promote via the World Wide Web at sib.ch / Genes encoding nucleotide sequences (according to the EPDB Data Base) can also be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another viable embodiment. Bacterial polymerases may be provided as part of a delivery complex or as additional genetic expression constructs. When provided with a nucleotide sequence, eukaryotic cells support cytoplasmic transcription from certain bacterial promoters. obtain.

[0135] Non-limiting examples of promoters include early viral promoters or late viral promoters. Promoters (e.g., SV40 early or late promoters, cytomegalovirus ( CMV immediate early promoter, Rous sarcoma virus (RSV) early promoter); eukaryotic Cellular promoters (e.g., beta-actin promoter, GADPH promoter, the tarothionine promoter); and cascade response element promoters (e.g., Cyclic AMP response element promoter (cre), serum response element promoter sre, phorbol ester promoter (TPA) and minimal TATA promoter Human growth hormone (HGH) is a transcription factor that regulates the transcription of thyroid hormone receptors (THRs). Promoter sequences (e.g., human growth hormone minimal promoter sequences listed in Genbank) promoter, accession number X05244, nucleotides 283 to 341) or Mouse mammary tumor promoter (available from ATCC, Cat. No. ATCC 4500 7) can also be used. In certain embodiments, the promoter is CM V IE, dectin-1, dectin-2, human CD11c, F4 / 80, SM22, RS V, SV40, Ad MLP, beta-actin, MHC class I or MHC class I I promoter, however, useful for driving expression of therapeutic genes. Any other promoters may also be applied to the practice of the present disclosure.

[0136] In certain embodiments, the methods of the present disclosure involve the use of enhancer sequences, i.e., promoter sequences. A nucleic acid sequence that increases the activity of a gene, and that is located in cis and in any orientation, over a relatively long distance. It has the ability to act over long distances (up to several kilobases away from the target promoter). However, enhancers may also be located close to a given promoter. Enhancer function is not necessarily limited to such long distances, as it can function at long distances. stomach. b. Initiation signal and linked expression

[0137] For efficient translation of the coding sequence, in the expression constructs provided in this disclosure, specific Other initiation signals may also be used. These signals may be derived from the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, must be provided. Those skilled in the art will be able to determine this and provide the necessary signals. To ensure translation of the entire insert, the initiation codon must be It is well known that the reading frame of the coding sequence must be "in frame" with the reading frame of the coding sequence. The native translational control signals and initiation codons can be natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[0138] In certain embodiments, the use of an internal ribosome entry site (IRES) element The use of ribosomal RNA is to generate multigene, i.e., polycistronic, messages. IRES elements are used to regulate the ribosome activity of 5' methylated cap-dependent translation. It is possible to bypass the scanning model and initiate translation at an internal site. IRES elements from two members of the poliovirus family (polio and encephalomyocarditis), IRES elements derived from mammalian messages have also been reported. can be linked to heterologous open reading frames, each separated by an IRES. Multiple open reading frames can be transcribed together, forming polycistronic sequences. Thanks to the IRES element, each open reading frame can generate a unique message. The frame becomes accessible to ribosomes for efficient translation. Efficient expression of multiple genes using promoters / enhancers to transcribe a single message You can also do this.

[0139] Additionally, to provide for linked or co-expression of genes within the constructs provided in this disclosure, For this purpose, certain 2A sequence elements can be used. For example, To co-express genes by linking genes to form a single cistron Exemplary cleavage sequences include F2A (foot-and-mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A). 3. Replication starting point In order to propagate a vector in a host cell, the vector contains one or more origins of replication. A site (often called an "ori"), e.g., a specific nucleic acid sequence at which replication begins EBV oriP or its function in programming as described above. It may contain a nucleic acid sequence corresponding to a genetically engineered oriP that has been enhanced or Alternatively, the origin of replication of other viruses that replicate extrachromosomally, as described above, or An autonomously replicating sequence (ARS) can be used. 4. Selectable and Screenable Markers

[0140] In some embodiments, cells comprising a construct of the present disclosure may express a marker in an expression vector. Such a molecule can be identified in vitro or in vivo by including it in a matrix. The marker induces identifiable changes in cells that allow for easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selection marker is a gene whose presence allows for selection. A negative selectable marker is a marker whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0141] Drug selection markers are usually included to facilitate cloning and identification of transformants. Helped by, for example, neomycin, puromycin, hygromycin, DHFR, G Genes that confer resistance to PT, zeocin, and histidinol are useful selectable markers. Markers that confer a phenotype that allows for the identification of transformants based on the performance of the conditions. In addition, colorimetric analysis is based on screenable markers such as GFP. Other types of markers are contemplated, such as herpes simplex virus thymidine kinase. enzyme (tk) or chloramphenicol acetyltransferase (CAT), etc. A screenable enzyme can be used as a negative selection marker. If so, we would also know how to use immunological markers, possibly in conjunction with FACS analysis. The markers used will be those that are expressed simultaneously with the nucleic acid encoding the gene product. Selection markers and screening Further examples of possible 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 also useful for the delivery of antigen receptors to a given host: As such, additional methods of recombinant gene delivery into cells are contemplated in this disclosure.

[0143] The introduction of nucleic acids, such as DNA or RNA, into immune cells of the present disclosure can be carried out using methods described herein. suitable for nucleic acid delivery to transform cells, as described herein or as known to those skilled in the art. Any method may be used, including direct delivery of DNA (e.g., exogenous DNA). in vivo transfection, injection (including microinjection); electroporation precipitation; calcium phosphate precipitation; DEAE-dextran followed by polyethylene glycosylation use of steroids; direct sonication; liposome-mediated transfection and receptor Transfection mediated by microprojectile bombardment mbardment); agitation with silicon carbide fibers; Agrobacterium-mediated trait transformation; desiccation / inhibition-mediated DNA uptake, and any combination of such methods By applying these techniques, organelles, cells, etc. can be isolated. Cells, tissues or organisms can be stably or transiently transformed. V. Treatment Method

[0144] Certain aspects of this embodiment are directed to treating diseases or disorders associated with CD79b signaling (e.g., Killing of CD79b-positive cells ameliorates at least one symptom of the disease or disorder. The present invention can be used to prevent or treat various conditions, including conditions resulting from CD79b signaling. The amount of cancer cells that can be reduced by any suitable composition that prevents the proliferation of cancer cells. Such substances express anti-CD79b antibodies or anti-CD79b CARs. The cell may be a cell that is

[0145] In some embodiments, the present disclosure provides a method for the preparation of a CAR T cell comprising administering to a subject a subject the method of the present disclosure. The present invention provides a method for immunotherapy comprising administering to a patient a therapeutically effective amount of a composition comprising: In one embodiment, administration of a CAR-expressing cell population that elicits an immune response is used to treat a medical disease. In certain embodiments of the present disclosure, a C that induces an immune response is used. Administration of the AR immune cell population treats cancer. Provided herein are methods for delaying progression, the methods comprising administering an effective amount of an antigen-specific The method includes administering a cell therapy to an individual. The method is useful, by way of example, for treating immune disorders, solid cancers, and hematologic disorders. It can be applied to the treatment of B-cell acute lymphoblastic leukemia (AL) and other cancers. L), B-cell malignancies such as diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma In lymphoma, lymphoplasmacytic lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia could be.

[0146] Tumors for which this treatment method is useful include any malignant cell type, e.g., solid tumors or hematomas. Exemplary solid tumors include those cell types found in tumors of the pancreas, colon, cecum, stomach, , brain, head, neck, ovaries, kidneys, larynx, sarcoma, lung, bladder, melanoma, prostate and breast The tumors may include, but are not limited to, tumors of organs selected from the group consisting of: Liquid tumors include tumors of the bone marrow, T- or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, and Further cancers that can be treated using the methods provided herein include: Examples include 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 (gastrointestinal cancer and gastrointestinal cancer) (including ovarian 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, These include, but are not limited to, various types of head and neck cancer, and melanoma.

[0147] The cancer may specifically be, but is not limited to, cancer of the following tissue types: neoplasms , malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous carcinoma Skin cancer; lymphoid epithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gas Trinoma, malignant; Cholangiocarcinoma; Hepatocellular carcinoma; Mixed hepatocellular carcinoma and cholangiocarcinoma; Trabecular adenocarcinoma; Adenoid cyst Cancer; adenomatous adenocarcinoma in polyps; adenocarcinoma, familial adenomatous polyposis; solid tumor; carcinoid tumor , malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic Carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary / follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortex Cancer; Endometrioid carcinoma; Skin adnexal carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cyst gland Cancer; 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 Skin 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 carcinoma Lipid cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Brown 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 Chromoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumors, Malignant;Mullerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, Malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant ;Ovarian goiter, malignant;Choriocoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's carcinoma hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; chondroblastoma , malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; enamel Epithelial odontosarcoma; ameloblastoma, malignant; ameloblastoma, fibrosarcoma; pinealoma, malignant; chordoma ;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Thin line fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal ;cerebellar sarcoma;ganglioneuroblastoma;neuroblastoma;retinoblastoma;olfactory neurogenic tumor;meningioma, malignant;neurology Fibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Granuloma lateralis; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell type, diffuse; Malignant lymphoma 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 aggressive NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky disease NHL; Mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macrophage Globulinemia; 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 Myeloid sarcoma; Hairy cell leukemia; Chronic lymphocytic leukemia (CLL); Acute lymphocytic leukemia Myeloid leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia .

[0148] Certain embodiments relate to methods of treating leukemia, which is a cancer of the blood or bone marrow. characterized by an abnormal proliferation (production by division) of blood cells, usually white blood cells (leukocytes) Leukemia is part of a broad group of diseases called hematologic malignancies. Leukemia is a broad term that encompasses a range of diseases. Leukemia can be classified clinically and pathologically into acute and chronic forms. It can be divided into three categories.

[0149] In some embodiments of the methods of the present disclosure, activated CD4 T cells in an individual and / or CD8 T cells are γ-IFN-producing CD4 T cells and / or C and / or a combination thereof, characterized by increased cytolytic activity compared to before administration. γ-IFN is a key feature of cell fixation, permeabilization, and the production of antibodies against γ-IFN. Intracellular cytokine staining (ICS), including staining by cytochrome P454, is well known in the art. Cytolytic activity can be measured by any means known in the art. For example, a cell killing assay using a mixture of effector and target cells may be used. and can be measured.

[0150] In some embodiments, the subject receives non-myeloablative lymphocyte therapy prior to T cell therapy. Ablative chemotherapy may be administered. The non-myeloablative lymphodepleting chemotherapy may be administered in any suitable manner. The non-myeloablative therapy may be any suitable such therapy that can be administered by any suitable route. Lymphocyte-depleting chemotherapy is particularly useful when the cancer is melanoma, which may be metastatic, e.g., This may include the administration of cyclophosphamide and fludarabine. An exemplary route of administration for darabine is intravenous. Similarly, any suitable dose of cyclophosphamide may be administered intravenously. Famid and fludarabine may be administered. In certain embodiments, approximately 60 mg / kg Cyclophosphamide is administered for 2 days, then approximately 25 mg / m 2 Fludarabine It 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 T cell growth factors are administered to the subject simultaneously with or subsequent to the autologous T cells. The growth factor may be any suitable growth factor that promotes the proliferation and activation of autologous T cells. Examples of cell growth factors include interleukin (IL)-2, IL-7, IL-15, and and IL-12, which may 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 IL-2) may be used. IL-12 is the preferred T cell growth factor. is.

[0152] Therapeutically effective amounts of immune cells can be administered via several routes, including parenteral administration, e.g., intravenously. The drug may be administered by intraperitoneal, intramuscular, intrasternal or intraarticular injection or infusion.

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

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

[0155] In certain embodiments of the present disclosure, an effective amount of CD79b CAR-expressing immune cells is administered to a patient with cancer. The cells are then delivered to an individual in need thereof, such as an individual suffering from a cancer. In some embodiments, the individual's immune system is stimulated to attack immune cells. The individual is provided with two or more doses of immune cells. If so, the period between doses should be sufficient to allow time for proliferation within the individual. In certain embodiments, the period between administrations is 1, 2, 3, 4, 5, 6, 7, or more days. The number of days is as above.

[0156] In a specific embodiment, cells engineered to express a CD79b CAR are administered to an individual A therapeutically effective amount (10%) of a compound that improves at least one symptom associated with cancer cells in a 3 ~1 0 10 A 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 , 1 0 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 ~1010 cells, or 10 9 ~10 1 0 Thus, in certain embodiments, an individual with a cancer may have CD79b A therapeutically effective amount of cells expressing a CAR is provided one or more times. A. Pharmaceutical Compositions

[0157] Pharmaceutical compositions and formulations comprising CAR T cells and a pharmaceutically acceptable carrier are also provided. Provided herein.

[0158] The pharmaceutical compositions and formulations as described herein have a desired degree of purity. The active ingredient (e.g., antibody or polypeptide) may be combined with one or more optional pharmaceutically acceptable carriers. Remington's Pharmaceutical Sciences es 22 nd edition, 2012) and freeze-dried The pharmaceutical composition may be prepared in the form of a pharmaceutical preparation or an aqueous solution. are non-toxic to recipients at the dosages and concentrations used and have no carriers Buffers (e.g., phosphate, citric acid, and other organic acids); antioxidants (ascorbic acid, acetic acid, and acetic acid); Contains corbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzoate chloride) Benzalkonium chloride; Hexamethonium chloride; Benzalkonium chloride; Benzethonium chloride phenol alcohol, butyl alcohol or benzyl alcohol; alkylpara bens (e.g., methylparaben or propylparaben); catechol; resorcinol cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (approximately 10 polypeptides (less than 1000 residues); proteins (e.g., serum albumin, gelatin, or immunoglobulins); globulins); 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 dextrin); esters (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose) salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complex); and / or non-ionic surfactants (e.g., polyethylene Exemplary PEGs include, but are not limited to, PEGs. Pharmaceutically acceptable carriers for the human soluble hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 human uronidase glycoproteins, e.g., rHuPH20 (HYLENEX®), Baxter International, Inc. In such a manner, the sHASEGP may be cleaved in combination with one or more additional glycosaminoglycanases, e.g. , used in combination with chondroitinase. B. Combination Therapy

[0159] In certain embodiments, the compositions and methods of the present invention comprise at least one further The further treatment may include a T cell population in combination with a treatment comprising radiation therapy, surgery (e.g., (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy or The additional treatment may be an adjuvant or neoadjuvant therapy. This may be in the form of a steroid therapy.

[0160] In some embodiments, the additional treatment includes administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is a side effect limiting agent (e.g., a treatment Agents aimed at reducing the incidence and / or severity of side effects of, e.g., 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. Therapies include those targeting the PBK / AKT / mTOR pathway, HSP90 inhibitors, and tubulin inhibitors. The additional treatment is an anti-cancer drug, an anti-apoptotic agent, an anti-cancer drug, an anti-inflammatory drug, or a chemopreventive agent. It may be one or more chemotherapeutic agents known in the art.

[0161] Immune cell therapy can be used before, during, or after further cancer treatment, such as immune checkpoint therapy. They can be administered in various combinations, ranging from simultaneously to minutes, days, or even weeks apart. In practice, immune cell therapy is provided to patients separately from additional therapeutic agents. In some embodiments, the two compounds may still exert a beneficial combined effect on the patient. It is common to ensure that no significant period of time elapses between the points of delivery, so that In such cases, the antibody therapy and the anti-cancer therapy may be administered within about 12 to 24 or 72 hours of each other. More particularly, it is contemplated that the two may be provided to a patient within about 6 to 12 hours of each other. In such situations, the interval between each dose may be several days (2, 3, 4, 5, 6, or 7) to several If more than one week (1, 2, 3, 4, 5, 6, 7, or 8) has passed, the treatment period should be significantly extended. It may be desirable to

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

[0163] Administration of any compound or treatment of the present embodiments to a patient may be toxic to those agents. Follow general protocols for administering such compounds, taking into account any Thus, in some embodiments, a step of monitoring toxicity that may result from the combination therapy is performed. exists. 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., For example, benzodopa, carboquone, meturedopa pa) and uredopa; ethyleneimine and methylameramine ( methylamelamines) (altretamine, triethylenemelamine, triethylene Triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine (including trimethylolomelamine); acetogenins (especially blatta camptothecin (including the synthetic analog topotecan); Bryostatin; kallistatin; CC-1065 (and its adzelesin, carzelesin (c arzelesin and bizelesin synthetic analogs); Cryptophycins (especially cryptophycin 1 and cryptophycin 8); Dolastatins duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin atistatin; sarcodictyin; spongista spongistatin; nitrogen mustard (e.g., chlorambucil) chlornaphazine, colofosfamide ophosphamide), estramustine, ifosfamide, mechlorethamine, salts mechlorethamine oxide, melphalan, novembichin, Phenesterine, prednimustine, trophosfamide (trofosfamide) and uracil mustard); nitrosoureas (e.g., kaolin Lumustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnu ranimnustine; antibiotics (e.g., enediyne antibiotics (e.g., For example, calicheamicin, particularly calicheamicin gamma 11 and calicheamicin omega 11. GaI1); dynemicin (including dynemicin A); bisphos phenates (e.g., clodronate); esperamicin; and neocarzinostatin Chromophores and related chromoproteins, enediyne antibiotic chromophores, acra Sinomycin, actinomycin, autarrhinicin, azaserine, bleomycin, Cutinomycin, carabicin, carmin omycin, carzinophilin, chromomycin, Dactinomycin, daunorubicin, detorubicin, 6-di Azo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, Cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin including sorbicin, epirubicin, esorubicin, idarubicin marcellomycin, mitomycin (e.g., Itomycin C), mycophenolic acid, nogalarnicin, olivomycin, peplomycin Potfiromycin, puromycin, querramycin Quelamycin, rodorubicin, strepto Nigrin, streptozocin, tubercidin, ubenimex, zinostatin and zorbi antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU) folic acid analogs (e.g., denopterin, pteropterin, and and trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopropanol, phosphorus, thiamiprine and thioguanine; pyrimidine ana steroids (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytara) flucloxin, dideoxyuridine, doxifluridine, enocitabine and floxuridine) androgens (e.g., calcitonin, dromostanolone propionate, epithiostaphylococcus aureus, ol, mepitiostane and testolactone; anti-adrenals ) (e.g., mitotane and trilostane); folic acid supplements (e.g., fro linic acid); aceglatone; aldophosphamide glycoside (aldop hosphamide glycoside); aminolevulinic acid; eniluracil; Musacrine; Bestrabucil; Bisantrene rene; edatraxate; defofamine mine; demecolcine; diazicon; elformithine ); elliptinium acetate; epothilone; Toglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin (lonida inine; maytansinoids (e.g., maytansin ansamitocins; mitoguazone; mitoxant lon; mopidanmol; nitraelin; pentostatin; phen Namet (phenamet); pirarubicin; losoxantrone one); podophyllinic acid; 2-ethyl hydroxybenzoate Razid; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2, 2',2"-Trichlorotriethylamine; Trichothecines (especially T-2 toxin, Vera Culin A, roridin A and anguidine ;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobromitol;Mitholactone Thor; Pipobroman; Gacytosine; Arabinoside ("Ara -C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel 6-thioguanine; mercaptopurine; platinum coordination complexes (e.g., cisplatin) vinblastine; platinum; etoposide (VP-16);Ifosfamide;Mitoxantrone;Vincristine;Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xe Loda (xeloda); ibandronate; irinotecan (e.g., CPT-11); Polyisomeric enzyme inhibitor RFS2000; difluoromethylornithine (DMFO); steroids (e.g., retinoic acid); capecitabine; carboplatin, procarbazine, Ricomycin, Gemcitabine, Navelbine, Farnesyl-Protein Tansferol enzyme inhibitors, transplatinum, and any of the above Pharmaceutically acceptable salts, acids or derivatives of the compound of formula (I) are included. 2. Radiation therapy

[0165] Other agents that have been widely used to cause DNA damage include gamma rays, X-rays and / or or what is 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 being considered. All of these factors are involved in the synthesis of DNA, the precursors of DNA, and the replication and repair of DNA. and most likely to cause widespread damage to chromosome assembly and maintenance. X-ray doses range from 50 to 200 roentgens over a prolonged period (3 to 4 weeks) These range from a daily dose of 1000 to a single dose of 2000-6000 roentgens. The range of doses for radioisotopes varies widely and depends on the half-life of the isotope, the strength of the radiation emitted, and The effect depends on the type and size of the tumor and on the uptake by the tumor cells. 3. Immunotherapy

[0166] Those skilled in the art will appreciate that immunotherapy may be used in conjunction or in conjunction with the methods of the above embodiments. Understand: In the context of cancer treatment, immunotherapeutics typically target and destroy cancer cells. It relies on the use of immune effector cells and molecules to achieve this. Cimab (RITUXAN®) is such an example. For example, it can be an antibody specific to some marker on the surface of a tumor cell. They can function alone as effectors of therapy or recruit other cells to The antibody may also affect cell killing by inducing drugs or toxins (chemotherapeutic agents, radionuclides, etc.). can be conjugated to a variety of antigen-specific antigens (e.g., ricin A chain, cholera toxin, pertussis toxin, etc.) to serve as targeting agents. Alternatively, the effector may interact directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and lymphocytes with surface molecules that bind to the immune system. These include inflammatory cells and NK cells.

[0167] Antibody-drug conjugates (ADCs) are compounds covalently linked to cytotoxic drugs for use in combination therapy. This approach involves the use of monoclonal antibodies (MAbs) against antigenic targets. The high specificity of MAbs combined with highly potent cytotoxic drugs allows for the production of a wide range of It also develops "armed" MAbs that deliver payloads (drugs) to tumor cells bearing the antigen at the same level. Targeted drug delivery also minimizes exposure to normal tissues, reducing toxicity. Exemplary ADC drugs include ADCETRI S® (brentuximab vedotin) and KADCYLA® (trastuzumab) Examples include stuzumab emtansine or T-DM1.

[0168] In one embodiment of immunotherapy, tumor cells are infected with any marker amenable to targeting. The cells must have some marker that is not present on most other cells. There are many tumor markers, and any of these may be used as a target in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosine kinase inhibitor, and tyrosine kinase inhibitor. Enzyme (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen , MucA, MucB, PLAP, laminin receptor, erb B, erb b2 and and p155. An alternative mode of immunotherapy combines anti-cancer and immune stimulatory effects. IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, etc. Which cytokines, chemokines such as MIP-1, MCP-1, IL-8, and FLT There are also immune stimulatory molecules, including growth factors such as IL-3 ligand.

[0169] Examples of immunotherapy include immunoadjuvants, such as Mycobacterium b ovis, Plasmodium falciparum, dinitrochlorobenzene and and aromatic compounds); cytokine therapy, e.g., interferon alpha, beta and gamma, IL-1 -1, GM-CSF and TNF; gene therapy, e.g., TNF, IL-1, IL-2 and p53; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2 and anti-p185. One or more anti-cancer therapies described herein It is contemplated that it may be used in conjunction with antibody therapy.

[0170] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either enhance or block signals (e.g., costimulatory molecules). Inhibitory effects that can be targeted by immune checkpoint blockade Immune checkpoints include adenosine A2A receptor (A2AR) and 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 (K IR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cells Immunoglobulin and mucin domain 3 (TIM-3) and T cell activation In particular, V-domain Ig suppressor of immune checkpoint inhibitors (VISTA) Antibody inhibitors target the PD-1 axis and / or CTLA-4.

[0171] Immune checkpoint inhibitors include small molecules, recombinant ligands, or receptors. It may be a drug or, in particular, an antibody, such as a human antibody. Known inhibitors of proteins or analogs thereof may be used, particularly chimeric and humanized forms. Alternatively, humanized antibodies may be used. As those skilled in the art will be aware, 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, lambrolizumab is known by the alternative and equivalent names MK-3475 and penicillin-resistant Staphylococcus aureus (PEN). It is known to be also known as brolizumab.

[0172] In some embodiments, the PD-1 binding antagonist is a PD-1 binding antagonist that binds to one of its ligands. In a specific embodiment, the PD-1 The ligand binding partner is PDL1 and / or PDL2. In this context, PDL1 binding antagonists inhibit PDL1 from binding to its binding partners. In a specific embodiment, the PDL1 binding partner is a molecule that inhibits PD-1 and In another embodiment, the PDL2 binding antagonist is a PDL2 inhibitor. In a specific embodiment, the IL-11 polypeptide is a molecule that inhibits L2 from binding to its binding partner. The PDL2 binding partner is PD-1. Antagonists include antibodies, their antigen-binding fragments, and The antibody may be a fragment, 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., For example, a human antibody, a humanized antibody, or a chimeric antibody. The PD-1 antibody will be selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin. a PDL1 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence) or an immunoadhesin containing the extracellular portion of PDL2 or the PD-1-binding portion In some embodiments, the PD-1 binding antagonist is AMP-224. . MDX-1106-04, MDX-1106, ONO-4538, BMS-93655 8 and nivolumab, also known as OPDIVO®, may be used as anti-PD-L1 inhibitors. -1 antibody. MK-3475, Merck3475, lambrolizumab, KEYTR Pembrolizumab, also known as UDA® and SCH-900475, An exemplary anti-PD-1 antibody is CT-0, also known as hBAT or hBAT-1. 11 is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, DL2-Fc fusion soluble receptor.

[0174] Another immune checkpoint that can be targeted in the methods provided herein is C Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as D152 The complete cDNA sequence of human CTLA-4 is available under Genbank accession number L15 CTLA-4 is found on the surface of T cells and binds to C on the surface of antigen-presenting cells. When bound to CD80 or CD86, CTLA4 acts as an "off" switch. A member of the immunoglobulin superfamily expressed on the surface of T cells , which transmits inhibitory signals to T cells. CTLA4 is a T cell costimulatory protein, CD4. 28, and both molecules bind to CD80 and CD86 (B7- and B7-respectively) on antigen-presenting cells. CTLA4 binds to the cytotoxic T cells (also known as B7-1 and B7-2). CD28 transmits stimulatory signals, whereas intracellular CTLA4 transmits regulatory signals. It is also found on T cells and may be important for the function of those cells. Activation of T cells via CD28 and CD38 results in the activation of C, an inhibitory receptor for B7 molecules. TLA-4 is highly expressed.

[0175] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody ( For example, human antibodies, humanized antibodies, or chimeric antibodies), antigen-binding fragments thereof, It may be a munoadhesin, a fusion protein or an oligopeptide.

[0176] Anti-human CTLA-4 antibodies (or VH and / or VH derived therefrom) suitable for use in the method or VL domains) can be produced using methods well known in the art. Any art-recognized anti-CTLA-4 antibody can be used. The A-4 antibody is a combination of ipilimumab (10D1, MDX-010, MDX-101, and Yer voy®) or antigen-binding fragments and variants thereof In other embodiments, the antibody comprises the heavy chain CDRs and light chain CDRs of ipilimumab. DR or heavy chain VR and light chain VR. Thus, in one embodiment, The antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ipilimumab and The VL region of ipilimumab contains the CDR1, CDR2, and CDR3 domains. In this embodiment, the antibody binds to the same epitope on CTLA-4 as the above-mentioned antibody. competes with and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In embodiments, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above. amino acid sequence identity (e.g., at least about 90%, 95%, or 99% with ipilimumab) It has a variable region identity. 4.Surgery

[0177] Approximately 60% of people with cancer undergo preventative, diagnostic or staging surgery, or curative surgery. undergoing some type of surgery, including radical and palliative surgery. This includes excision, which involves the physical removal, resection, and / or destruction of all or part of the cancerous tissue. , other treatments (e.g., the treatment of this embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy) Tumor removal is the removal of tumors. The term refers to the physical removal of at least part of a tumor. Surgical treatment includes tumor removal and Other techniques include laser surgery, cryosurgery, electrosurgery and microsurgery (Mohs surgery).

[0178] When part or all of the cancerous cells, tissue, or tumor is removed, a cavity may be formed in the body. Treatment is achieved by perfusion, direct injection, or local application of the area with additional anti-cancer therapy. Such treatments can be administered, for example, every 1, 2, 3, 4, 5, 6, or 7 days. or every 1, 2, 3, 4 and 5 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9 These treatments may be repeated every 10, 11, or 12 months. It is also possible. 5. Other agents

[0179] Other actions may be combined with certain aspects of the present invention to improve the therapeutic efficacy of treatment. It is contemplated that agents that inhibit the growth of cells may be used. These additional agents include cell surface Agents that affect receptor and gap junction upregulation, cell division Inhibitors and differentiation agents, inhibitors of cell adhesion, and inducers of apoptosis in hyperproliferative cells These include agents that increase sensitivity or other biological agents. The increased signaling between cells due to the addition of ATP to adjacent hyperproliferative cell populations In other embodiments, to improve the anti-hyperproliferative efficacy of the treatment, To this end, cytostatic or differentiating agents may be used in combination with certain aspects of this embodiment. To improve the effectiveness of this embodiment, inhibitors of cell adhesion are contemplated. Examples of adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. VI. Products or Kits

[0180] Also included are products or kits containing immune cells, antibodies, reagents, buffers, or combinations thereof. The article of manufacture or kit is provided herein for treating cancer in an individual or for administering a The use of immune cells to slow the progression of cancer or to enhance immune function in individuals with cancer The antigen-specific antibodies described herein may further comprise a package insert containing instructions for use. Any of the immune cells may be included in the product or kit. Examples include bottles, vials, bags and syringes. The containers may be glass, plastic, plastics (e.g., polyvinyl chloride or polyolefins) or metal alloys (e.g., The ferrule may be made from a variety of materials, such as stainless steel or Hastelloy. In this embodiment, the container holds the formulation and a label, the label being attached to the container. The container may contain or be associated with a product, and instructions for use may be printed on the container. The product or kit may further contain other materials that are desirable from a commercial and user standpoint. These materials may include other buffers, diluents, filters, needles, syringes, etc. and a package insert containing instructions for use. The product contains one or more additional agents (e.g., chemotherapeutic and antineoplastic agents). Suitable containers for the one or more agents include, for example, bottles, vials, etc. , bags and syringes. VII. SEQUENCES USED IN CERTAIN EMBODIMENTS Clone T26 VH region: nucleic acid GAGGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAAGC CTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTA CACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGG CCTGGACCAGGCCTTGAGTGGATCGGAGCAATTGATCCTT CAGATAGTTATACTGGCTACAATCAAAAGTTCAAGGGCAA GGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTAC ATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCT ATTTCTGTACAAGAAGCTACTATGGTAACTCCTGGTTTGC TTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (3 57 nt) (SEQ ID NO: 8) Amino acid sequence EVQLQESGAELVKPGASVKMSCKASGYTFTSYWMHWVKQR PGPGLEWIGAIDPSDSYTGYNQKFKGKATLTVDTSSSTAY MHLSSLTSEDSAVYFCTRSYYGNSWFAYWGQGTLVTVSA (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 ACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGT GTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGC CAAAGTGTTGATTATGATGGTGATAGTTATATAAACTGGT ACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTA TGCTGCATCCAATCTAGAATCTGGAATCCCAGCCAGGTTT AGTGCCAGTGGGTCTGGGACAGACTTCACCCTCAACATCC ATCCTGTGGAGGAGGAGGATGTTGCAGCCTATTACTGTCA GCAAAGTAATGAGGACCCATTCACGTTCGGCTCGGGGACA AGGTTGGAAATAAAAC (330 nt) (SEQ ID NO: 1 0) Amino acid sequence IVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYINWYQ QKPGQPPKLLIYAASNLESGIPARFSASGSGTDFTLNIHP VEEEDVAAYYCQQSNEDPFTFGSGTRLEIK (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 GAGGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAAGC CTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTA CACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGG CCTGGACAAGGCCTTGAGTGGATCGGAGCAATTGATCCTT CAGATAGTTATACTGGCTACAATCAAAAGTTCAAGGGCAA GGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTAC ​​​​​​​​​​​​​ MHLSSLTSEDSAVYFCTRSYYGNSWFDYWGQGTLVTVSA (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 GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTTGGT CTCTAGGGCAGAGGGCACCATCTCCTGCAAGGCCAGCCA AAGTGTTGATTATGAAGGTGATAGTTATATGAACTGGTAC CAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATG CTGCATCCAATCTAGAATCTGGAATCCCAGCCAGGTTTAG TGGCAGTGGTCTGGGACAGACTTCACCCTCAACATCCAT CCTGTGGAGGAGGATGCTGCAACCTATCACTGTCAGC AAAGTAATGAGGACCGTTCACGTTCGGAGGGGGGACCAA GTTGGAAATAAAAA (333 nt) (SEQ ID NO: 20) amino acid sequence DIVLTQSPASLAVSLGQRATISCKASQSVDYEGDSYMNWY QQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIH PVEEEDAATYHCQQSNEDPFTFGGGTKLEIK (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 GAGGTGCAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAAGC CTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTA CACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGG CCTGGACAAGGCCTTGAGTGGATCGGAGCAATTGATCCTT CAGATAGTTATACTGGCTACAATCAAAAGTTCAAGGGCAA GGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTAC ATGCACCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCT ATTTCTGTACAAGAAGCTACTATGGTAACTCCTGGTTTGC TTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (3 57 nt) (SEQ ID NO: 28) Amino acid sequence EVQLQESGAELVKPGASVKMSCKASGYTFTSYWMHWVKQR​​​​​​​​​​28B VH CDR2: DPSDSYT (SEQ ID NO: 22) 28B VH CDR3: SWFAYWGQGTLV (SEQ ID NO: 23 ) Clone 28B VL region: nucleic acid GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTTGGT CTCTAGGGCAGAGGGCACCATCTCCTGCAAGGCCAGCCA AAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTAC CAACAGAAACCAGGACAGCCACCCCAAACTCCTCATTTATG TTGCATCCAATCTAGAATCTGGAATCCCAGCCAGGTTTAG TGGCAGTGGTCTGGGACAGACTTCACCCTCAACATCCAT CCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGC AAAGTAATGAGACCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAC (333 nt) (SEQ ID NO: 30) amino acid sequence DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWY QQKPGQPPKLLIYVASNLESGIPARFSGSGSGTDFTLNIH PVEEEDAATYYCQQSNEDPFTFGSGTKLEIN (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 CGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAG ACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAA AAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCG GTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTC TAGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGA AATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAAC AGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATAC GCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGT CGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTC AAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACA AAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACT GTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAAC AGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCC ATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCC CAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGG GAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAA GGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCC AGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGA CGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTG ACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCAT GGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCC GGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACG GATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGG GCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCC CTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCT TCATGCGAAGG SEQ ID NO: 41 EGFRIII-IV RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILP VAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPEN RTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSL KEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISN RGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGR ECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTG RGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADA GHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGA LLLLLVVALGIGLFMRR SEQ ID NO: 42 CD8Leader ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTC TGCTGCTGCATGCCGCTAGACCC 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 GGAGGGTGGTAGTGGTGGGGGGGGGGG GAAGT SEQ ID NO: 49 リンカー3 GGGGSGGGGSGGGGS SEQ ID NO: 50 リンカー4 GGAGGGTGGTAGTGGTGGAGGAGGAGGTGGCGGAG GTTCTGGAGGTGGAGGTTCC SEQ ID NO: 51 リンカー4 GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 52 CD8 Link ACAACTACTCCAGCACCACGACCACACACCTGCTCCAA CTATCGCATCTCAACCACTTTCTCTACGTCCAGAAGCATG CCGACCAGCTGCAGGAGGTGCAGTTCATACGAGGTCTA GATTTCGCATGTGAT SEQ ID NO: 53 CD8 Hinge 1 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACD SEQ ID NO: 54 CD8 Hinge 2 AAGCCCACAACTACTCCAGCACCACGACCACCAACACCTG CTCCAACTATCGCATCTCAACCACTTTCTCTACGTCCAGA AGCATGCCGACCAGCTGCAGGAGGTGCAGTTCATACGAGA GGTCTAGATTTCGCATGTGAT SEQ ID NO: 55 CD8 Hinge 2 KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACD SEQ ID NO: 56 CD8 Hinge 3 TTCAGCCACTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCA CAACTACTCCAGCACCACGACCACCAACACCTGCTCAAC TATCGCATCTCAACCACTTTCTCTACGTCCAGAAGCATGC CGACCAGCTGCAGGAGGTGCAGTTCATACGAGAGGTCTAG ATTTCGCATGTGAT SEQ ID NO: 57 CD8 Hinge 3 FSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACD SEQ ID NO: 58 CD28 Hinge ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGA AGAGCAATGGAACCATTATCCATGTGAAAGGG SEQ ID NO: 59 CD28 ヒンジ IEVMYPPPYLDNEKSNGTIIHVKG SEQ ID NO: 60 IgG4 ヒンジ GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCA SEQ ID NO: 61 IgG4 ヒンジ ESKYGPPCPSCP SEQ ID NO: 62 IgG4 CH2 GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCAC CTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCC AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAG GTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCG AGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCA TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA GCCAAAGGG SEQ ID NO: 63 IgG4 CH2 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKG SEQ ID NO: 64 IgG4 CH2CH3 GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCAC CTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCC AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAG GTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCG AGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCA TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGC CCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAG GAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTC TGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCC GGGTAAA SEQ ID NO: 65 IgG4 CH2CH3 ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSCEDPEWQFNWYVDGVEVHNACTKPREEQFQS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSIECTICS AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYCTTTPPVLDSDGFFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 66 IgG4 CH1CH2CH3 GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCT GCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACC TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGG TGGACAAGAGGTGAGTCCAAATATGGTCCCCCATGCCC ATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTC TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCT CCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAG CCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG AGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCAC CGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACA GGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACC CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACA AGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGC CTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 67 IgG4 CH1CH2CH3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO: 68 CD8 TM 1 ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCC TTCTCCTATCACTGGTTATCACCCTTTACTGC SEQ ID NO: 69 CD8 TM 1 IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 70 CD8 TM 2 ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCC TTCTCCTATCACTGGTTATCACCCTTTACTGCAACCACAG GAAC SEQ ID NO: 71 CD8 TM 2 IYIWAPLAGTCGVLLLSLVITLYCNHRN SEQ ID NO: 72 CD28 TM AAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTA AGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGC TTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC SEQ ID NO: 73 CD28 TM KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF SEQ ID NO: 74 CD28 AGAAGTAAAAGAAGTAGGCTACTTCATAGTGATTACATGA ATATGACTCCTCGACGACCTGGTCCCACCCGTAAGCATTA TCAGCCCTATGCACCACCACGAGATTTCGCAGCCTATCGC TCC SEQ ID NO: 75 CD28 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR S SEQ ID NO: 76 4-1BB AAACGAGGTAGAAAAAAACTTCTTTATATATTCAAACAAC CATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGG ATGTAGTTGTCGATTTCCAGAAGAAGAAGAAGGAGGATGT GAACTG SEQ ID NO: 77 4-1BB RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 78 OX-40 AGGCGCGACCAGCGGCTGCCACCTGATGCACACAAGCCAC CAGGAGGAGGCTCTTTCCGGACCCCAATCCAGGAGGAGCA GGCAGACGCACACAGCACACTGGCCAAGATC SEQ ID NO: 79 OX-40 RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO: 80 CD3ζ intracellular AGAGTTAAATTTAGCAGAAGTGCAGATGCTCCTGCGTATA AACAGGGTCAAAACCAACTATATAATGAACTAAATCTAGG ACGAAGAGAAGAATATGATGTTTTAGATAAAAGACGTGGT CGAGATCCTGAAATGGGAGGAAAACCTAGAAGAAAAAATC CTCAAGAAGGCCTATATAATGAACTACAAAAAGATAAGAT GGCAGAAGCTTATAGTGAAATTGGAATGAAAGGAGAACGT CGTAGAGGTAAAGGTCATGATGGTCTTTATCAAGGTCTTA GTACAGCAACAAAAGATACATATGATGCACTTCATATGCA AGCACTTCCACCTCGT SEQ ID NO: 81 CDζ intracellular RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 82 Enhanced GFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGC CCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAA GTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGC TGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTA CGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAG CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAA CAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACG AGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGC CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG SEQ ID NO: 83 Enhanced GFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATY GKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMK QHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 84 T2A GAGGGCAGAGGCAGTCTGCTGACATGCGGTGACGTGGAAG AGAATCCCGGCCCT SEQ ID NO: 85 T2A EGRGSLLTCGDVEENPGP SEQ ID NO: 86 CD8α TM (nucleic acid) ATCTACATCTGGGCACCATTGGCTGGGACTTGTGGTGTCC TTCTCCTATCACTGGTTATCACC SEQ ID NO: 87 CD8α TM (amino acid sequence) IYIWAPLAGTCGVLLLSLVIT [Example]

[0181] VIII. Working Examples The following examples are included to demonstrate certain embodiments of the disclosure. The techniques disclosed therein are techniques that the inventors have found to work well in the practice of the present invention. It will be readily apparent to those skilled in the art that the present invention may be considered a preferred form for its implementation. However, it should be recognized that those skilled in the art, in light of this disclosure, Many changes can be made in the specific embodiment, and these changes will remain within the spirit and scope of the invention. should recognize that other modifications without departing from the scope and spirit of the present invention will still produce the same or similar results. is. Example 1 - CD79b Antibody and CAR Development

[0182] CD79b expression is restricted to the B cell lineage : Using real-time PCR, CD7 9b, Mino, Daudi, HBC-1, Jeko, SUDHL6, SUDHL4 and It is expressed in a wide range of B-cell lymphoma cell lines, including U2932 and U2932, but not in Jurkat. It was found not to be expressed in the t and J76 T-cell lymphoma / leukemia cell lines (Fig. 1 A) To determine whether CD79b is expressed on normal tissues, 20 normal human FirstChoice Human Total RNA Surfactants containing tissue total RNA The Vey Panel was obtained from Applied Biosystems. Total RNA was extracted from purified B and T cells from the sample and analyzed using PCR. CD79b transcript was used as an inactive and negative control. It is present only in lymphoid tissues such as the spleen and lymph nodes and is not present in all normal non-lymphoid tissues. It was found to be absent in tissues (Fig. 1B).

[0183] Using publicly available gene expression datasets (Oncomine), CD79b However, 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 It was found to be highly expressed in tumors (e.g., thyroid cancer, thyroid cancer, and mantle cell lymphoma) (Fig. 1D). Sample numbers for tumor type are shown in parentheses.

[0184] Generation of multiple monoclonal antibodies against human CD79b and characterization of the heavy chain sequences and Identification of the DNA and light chain sequences : Mice were immunized with mouse fibroblast L cells expressing human CD79b. By using this method, anti-human CD79b monoclonal antibodies were produced using hybridoma technology. The binding to recombinant human CD79b protein was determined by ELISA (Figure 2A). Three clones with high potency were identified (Figure 2B). The compatibility was further measured by Octet Assay and was 1.44, 17.8 and 2. Three clones, 14 (IgG1), 16A (Ig), and 17A (IgG), each had a Kd value of 0 nM. G2) and 45 (IgG2) were selected for further development (Figure 2C). The monoclonal antibody clone #14 was conjugated to a fluorescent dye and was used in BD Biosciences. It showed comparable staining of B-cell lymphoma cell lines to the commercially available anti-CD79b antibody from ES. (Fig. 2D). Total RNA was extracted from the hybridomas of the above monoclonal antibodies, and cDNA was generated. The heavy chain and The light chain V gene was cloned. The protein sequence was predicted from the DNA sequence. Hybridoma culture supernatant was purified and analyzed by MD Anderson Proteomics C The protein sequences of the heavy and light chains were confirmed by mass spectrometry at the Imagine Facility. Ta.

[0185] Generation of anti-CD79b CAR T cells : Single chain fragment of variable region (scFv) Several constructs of anti-CD79b CARs were generated using specific sequences. To detect CAR expression in the T cells, CAR-enhanced green fluorescent protein ( eGFP) fusion constructs or a truncated form of the human epidermal growth factor receptor (huEGFRt) The latter was used to remove CAR T cells in the event of severe toxicity. It can also act as a safety switch for T cell activation by providing a signal for T cell activation. The CD3-zeta (CD3z) chain is incorporated into the co-stimulatory domain to provide signal 2. The antibodies were engineered to incorporate the agonists CD28 or 4-1BB (Figure 3A).

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

[0187] CAR T against Daudi cells labeled with CellTrace Far Red The cytotoxic activity of the cells was assessed by flow cytometry for 16 hours at the indicated effector:target (E:T) ratios. The ratio of 20:1 was measured by Aqua staining in a cytometry assay (Fig. 3C). Representative dot plots (top right) with the percentage of dead cells for various culture conditions at different E:T ratios. The data are presented in Figure 3D. Both 9b-CAR T cells demonstrated a significant improvement in Da compared with untransduced control T cells. These results demonstrate that IFN-γ was highly cytotoxic to HIV-1 induced Burkitt's lymphoma cells.

[0188] Anti-CD79b CAR T cells target CD19 + Lymphoma cells and CD19 - Lymphoma cells It is cytotoxic to both :CD19 negative (CD19 - ) Anti-lymphoma cells To measure the efficacy of CD79b CAR T cells, degranulation and cytotoxicity assays were performed. We used SUDHL6, a CD19-deficient diffuse large B-cell lymphoma cell line, to investigate the effect of CD19 on the proliferation of B-cell lymphoma. First, CD19 was knocked out using CRISPR-Cas9 (CD19 These lymphoma cells were then transfected with an anti-CD19 CAR construct. The exon 2, which is the binding site for the anti-CD19 antibody clone FMC63 used in The cells were transduced with a CD19 splice variant (CD19Dex2) lacking the CD19 gene.

[0189] Non-transduced primary T cells (control T), clone-14-CD79b- CD28 CAR, 14-CD79b-4-1BB CAR and FMC63-CD19 -CD28 CAR was transfected with Daudi cells or the above-mentioned SUDHL6 cells (CD19KOS T cells were co-cultured with UDHL6-CD19Dex2) at an E:T ratio of 5:1. The target cells were labeled with CellTrace Far Red and labeled with CellTrace Violet. After 2 hours, Golgi inhibitors and degranulation markers (CD107a / b) were added to the cultures. In addition, T cell degranulation was measured. Cytotoxic activity against tumor cells was measured after 4 days of co-culture. 14-CD79b-CD28 and 14-CD79b-4-1BB CARs were measured. T cells showed significantly higher degranulation and cytotoxic activity against both cell lines, but In contrast, FMC63-CD1 T cells did not show any significant changes (Figures 4A and 4B). 9-CD28 CAR, CD19 + It was cytotoxic to Daudi but CD1 It was not cytotoxic to 9KOSUDHL6-CD19Dex2 tumor cells.

[0190] After 4 days of co-culture, CountBright Absorbent for flow cytometry was used. Absolute viable tumor cell counts were also determined using lute counting beads. (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. (Figures 4A and 4B). These experiments were repeated at least three times with similar results. It was.

[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, The Mino mantle cell lymphoma cell line expressing the luciferase gene was transfected into NSG mice. to 2×10 6 The mice were then IV injected with 10 ... No primary T cells, anti-CD19-CD28 CAR T cells or clone 45 anti-CD79 b-CD28 CAR T cells, 10 × 10 6 Cars + T cells or transduced T cells / mouse were treated by tail vein injection. Bioluminescence imaging was used to The tumor burden was assessed (Figure 5A). The results showed that the tumor burden was significantly higher in mice treated with untransduced T cells than in mice treated with untransduced T cells. In contrast, the mice showed progressive tumor growth. Significant improvements (p<0.05) were observed with both anti-CD19 and anti-CD79b CAR T cells. This was observed in treated mice (Figures 5B and 5C). Each individual experiment was verified at least three times, and the in vivo results were verified twice. Anti-CD79b CAR therapy targets B-cell malignancies that express or do not express CD19. It can be used for the treatment of cancer tumors. Example 2 - Development of further CD79b antibodies and CARs

[0192] Generation of anti-CD79b CAR T cells using a new anti-CD79b antibody clone: ​​Further Single-chain fragments of the variable regions from the antibody clones T26, 5B, and 28B ( Several constructs of anti-CD79b CARs were generated using specific sequences of the CD79b antibody (scFv). To detect CAR expression in transduced T cells, a CAR-enhanced green fluorescent tag was used. Protein (eGFP) fusion construct or truncated human epidermal growth factor receptor (huEGF Rt) was used (Figure 6A). The latter allows for removal of CAR T cells in the event of severe toxicity. It can also act as a safety switch to block signal 1 for T cell activation. Incorporating CD3-zeta (CD3z) chain to provide signal 2 The costimulatory domains CD28, 4-1BB, or OX-40 were incorporated (Figure 6A ).

[0193] Anti-CD79b CAR T cells inhibited Daudi lymphoma cells in vitro. The above CAR constructs (T26 and 28B) were expressed as lentiviral vectors. The vector was cloned into pLVEG and then used to transduce primary healthy donor T cells. Anti-CD79b-CAR T cells were generated by transfecting anti-CD19-CAR T cells. Daudi cells (CellTrace Violet) were used as a control. CAR T cells (labeled with CellTrace Far Red) against The cytotoxic activity of the indicated effector:target (E:T) ratios was measured at 1 and 4 days. After co-culture for 1 h, the number of cells was measured by flow cytometry assay (Figure 6B). Dot plots are shown in Figure 6C. Data are for anti-CD19-CAR T cells and anti-CD79b Both CAR T cells were highly cytotoxic against Daudi-Burkitt lymphoma cells This indicates that it was sexual.

[0194] Anti-CD79b CAR T cells show in vivo efficacy against Daudi lymphoma xenografts Showing efficacy: Efficacy of anti-CD79b CAR T cells (clone T26) in vivo Daudi Burkitt expressing the firefly luciferase gene to test for sex Lymphoma cell lines were inoculated into NSG mice at 2 × 10 4 Tumor cells / mouse were injected IV. 10 days later , mice were either untreated, or treated with anti-CD19-CAR T cells, or clone T2 3 x 10 with 6 anti-CD79b-CAR T cells 6 Tail vein injection of CAR+T cells / mouse 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) was observed in patients treated with anti-CD19 CAR T cells. This was observed in both mice treated with anti-CD79b CAR T cells and mice treated with anti-CD79b CAR T cells. These results demonstrate that anti-CD79b CAR T cells potently stimulated the immune response in vivo (Figure 6E). It has been demonstrated to have antitumor activity. Example 3 - Further development and investigation of CD79b antibodies and CARs

[0195] Binding of anti-CD79b antibodies to human CD79b: intact CD79b isolated from hybridoma supernatants Mouse line using long anti-CD79b monoclonal antibodies (clones 5B and 28B) Binding to human CD79b expressed on fibroblastic L cells tested by ELISA (Figure 7).

[0196] Generation of CAR constructs: Single-chain Flag of variable regions derived from VH and VL of clone 28B Using the specific sequence of the scFv (shown in Figure 7), several anti-CD7 9b CAR constructs were made using either the CD8α or CD28 hinge / transmembrane domains. To detect CAR expression in transduced T cells, a CAR-sensitive green fluorescent protein (CAR-G) was used. Fluorescent protein (eGFP) fusion constructs or truncated human epidermal growth factor receptor (hGF) uEGFRt) was used (Figure 8A). Anti-CD19 CAR construct was used as a control. The latter included a safety switch to remove CAR T cells if severe toxicity occurred. It can also function as a CD4+ signal to provide signal 1 for T cell activation. It incorporates the CD3-zeta (CD3ζ) chain and a costimulatory domain to provide signal 2. These constructs were then inserted into the nucleoside-binding domains CD28, 4-1BB, or OX40 (Figure 8A). , and cloned into the lentiviral vector pLVEG containing the EF1α promoter (Figure 1 8B), which are then used to transduce T cells and perform various in vitro and in vivo assays.

[0197] Anti-CD79b CAR specifically recognizes human CD79b: To measure signaling capacity, lentivirus was used to transduce their CAR (CD7 9b) clone 28B was used for CAR in Jurkat-Lucia TM NF AT reporter cells (Fig. 9A). In this cell line, secreted Coelentella The Lucia gene, which encodes the gene-utilizing luciferase, contains six copies of the NFAT constitutive Driven by the ISG54 minimal promoter fused to the sas transcriptional response element These transduced Jurkat cells were then treated with anti-CD3 / anti-CD28 antibodies or Daud antibodies. When cultured with Burkitt's lymphoma cells for 24 hours, the lymphocytes showed stronger lymphocyte proliferation than untreated cells. Luciferase activity was induced (Figure 9B). Recognition of CD79b by these CAR molecules To establish the specificity of CD19 and CD79, we used CRISPR / Cas9. SUDHL6, a diffuse large B-cell lymphoma cell line expressing β-glucanase (GlcNAc), or wild-type (WT), CD19 knockout (CD19KO), CD79bKO, and Isogenic CD19 / CD79b double KO (CD19KO CD79bKO) (Figure 9C). CAR-transduced Jurkat-Lucia TM NFAT Report Co-culture of parenteral cells with the SUDHL6 isogenic cell line demonstrated that the CD79b CAR binds to the parental cells Recognizes both CD79bKO cells and CD19KO cells, but not CD79bKO cells or CD19KOCD When 79bKO cells were used, the reactivity was significantly reduced. In contrast, CD1 9 CAR recognized both parental and CD79bKO cells, but not CD19KO or did not recognize CD19KOCD79bKO cells (Fig. 9D).

[0198] Transduction of CAR into primary human T cells: eGFP expression, and / or As measured by recombinant human CD79b-Fc protein staining using the CAR construct (Figure 8A). Representative transduction efficiencies measured are shown in Figure 10A. Anti-CD79b CAR T cells were either untreated or treated with anti-CD 3 / CD28 antibody or recombinant human CD79b-Fc protein. 15 min later Phosphorylation of CD3ζ and ERK1 / 2 was assessed by flow cytometry. Results showed no significant difference in baseline phosphorylation between CAR+ and CAR-T cells. The lack of sustained signaling suggests that anti-CD Stimulation with CD3 / CD28 antibody induced phosphorylation of CD3ζ in both CAR+ T cells. In contrast, recombinant human CD79b-Fc Protein stimulation increased the phosphorylation of CD3ζ and ERK1 / 2 in CAR+ T cells. These CARs induced CD4+ activation in the CAR-T cells but not in the CAR-T cells, suggesting that these CARs are It was shown that 79b protein can induce signaling in primary T cells in response to its recognition. This is suggested (Figure 10B).

[0199] Proliferative activity of anti-CD79b CAR T cells: The above CAR constructs (Figure 8A) were used to Anti-CD79b CAR T cells generated from healthy donor T cells were used in CellTrace Far Red-labeled CD79b-expressing Daudi Burkitt lymphoma tumor cells and 1: The cells were co-cultured at an effector-to-target (E:T) ratio of 1. After 4 days, CAR T cell proliferation The results were evaluated by flow cytometry using the dye dilution method. Both 9b CAR T cells and CD8+ anti-CD79b CAR T cells were able to treat Daudioma The anti-CD79 T cells showed significant proliferation in response to tumor cells compared to non-transduced T cells. b CAR T cell proliferation was comparable to anti-CD19 CAR T cells (Fig. 11A and and Figure 11B).

[0200] Cytokine production by anti-CD79b CAR T cells: the above CAR constructs (Figure 8A ) to generate anti-CD79b CAR T cells from healthy donor T cells. Burkitt's lymphoma tumor cells were co-cultured at an E:T ratio of 1:1. After 24 hours, The cytokine levels were assessed by multiplex cytokine assay. Results showed that anti-CD79b CAR T cells responded to Daudi tumor cells and inhibited the proliferation of non-transduced IL-2, GM-CSF, IFN-γ, and IL-17A in comparison with T cells. Cytokine production by anti-CD79b CAR T cells was significantly higher than that by anti-C This was comparable to that of D19 CAR T cells (Figure 12).

[0201] Anti-CD79b CAR T cells degranulate in response to lymphoma cells: the CAR structure described above Anti-CD79b CAR T cells generated from healthy donor T cells using the construct (Figure 8A). were co-cultured with Daudi Burkitt lymphoma tumor cells at an E:T ratio of 1:1 for 6 hours. Degranulation was subsequently assessed by flow cytometry after staining for CD107a / b. The results showed that CD4+ anti-CD79b CAR T cells and CD8+ anti-CD79b Both CAR T cells were effective in response to Daudi tumor cells compared with non-transduced T cells. Degranulation by anti-CD79b CAR T cells was significantly suppressed by anti-CD1 9 CAR T cells. Degranulation was comparable to that of CD28 hinge / transmembrane domain-containing antibodies. Compared to CD79b CAR T cells, anti-CD7 containing the CD8α hinge / transmembrane domain 9b CAR T cells were numerically superior (Figures 13A and 13B).

[0202] Anti-CD79b CAR T cells are cytotoxic to lymphoma cells: CA R construct (Figure 8A) was generated from healthy donor T cells and analyzed by CellTrace Fa r Red-labeled anti-CD79b CAR T cells were transfected with CellTrace Vio Daudi Burkitt lymphoma tumor cells or SUDHL6 isogene labeled with lett Transgenic cell lines (parental, CD19KO, CD79bKO and CD19 / CD79b double KO) ) at an E:T ratio of 1:1. After 4 days, the absolute number of remaining viable tumor cells was by flow cytometry by measuring the number of cells and calculating the percent lysis. The results showed that CD79b CAR T cells showed that transduced T cells induced significant cytotoxicity of Daudi tumor cells compared to non-transduced T cells. The cytotoxic activity of anti-CD79b CAR T cells was similar to that of anti-CD19 CAR T cells. The cytotoxic activity was observed in the CD28 hinge / transmembrane domain-containing anti-CD79b CA. Compared with RT cells, anti-CD79b CAR T containing the CD8α hinge / transmembrane domain Furthermore, anti-CD79b CAR T cells were significantly more potent than parental cells (Figure 14A). CD19KO induced significant lysis of both SUDHL6 cells and CD7 The expression of 9bKO or CD19KOCD79bKO cells was significantly reduced. CD19 CAR T cells induced significant lysis of both parental and CD79bKO cells However, lysis was significantly reduced in CD19KO or CD19KOCD79bKO cells. (Figure 14B).

[0203] Anti-CD79b CAR T cells exert antitumor effects in vivo: Anti-CD79 To examine the efficacy of b-CAR T cells in vivo, we used the firefly luciferase gene. Daudi Burkitt lymphoma cells expressing the gene were cultured at 2 × 10 4 Tumor cells / mouse NSG mice were IV-injected with 1000 mg of IgG1-positive T cells. Eleven days later, mice were treated with either untransduced T cells or the IgG1-positive T cells described above. Anti-CD19-CAR T cells or anti-CD19 CAR T cells generated using the CAR construct (Figure 8A) 5 × 10 79b-CAR T cells 6 CAR+ T cells / mouse administered by tail vein injection Bioluminescence imaging was used to assess tumor burden (Figure 15A). Mice treated with non-transduced T cells showed rapid tumor growth. Significant tumor control and improved survival were observed in mice treated with anti-CD19 CAR T cells. This was seen in both mice treated with anti-CD79b CAR T cells and mice treated with anti-CD79b CAR T cells. Tumor control and survival time were significantly associated with anti-CD79b CA containing the CD28 hinge / transmembrane domain Compared with RT cells, anti-CD79b CAR T containing the CD8α hinge / transmembrane domain The best survival time was observed in the CD8α hinge / transmembrane domain and OX4 This was observed with anti-CD79b CAR T cells containing the 0 costimulatory domain (Figure 15B).

[0204] All of the methods disclosed and claimed herein may be practiced without undue experimentation in light of the present disclosure. The compositions and methods of the present invention can be implemented and performed without the need for the following: Although the present invention has been described in terms of its application, no particular limitation or modifications may be made thereto without departing from the concept, spirit and scope of the present invention. that variations may be applied to the methods and method steps or order of steps described therein; More specifically, any combination of chemicals may be used as long as the same or similar results are achieved. Certain biologically and physiologically relevant agents may be substituted for the agents described herein. It will be apparent that such similar substitutes and All modifications falling within the spirit, scope and concept of the invention as defined by the appended claims. is considered to be within literature The following references provide exemplary procedures or supplementary information to those described herein: To the extent that other details are provided, they are specifically incorporated herein by reference. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associ ates 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 ndedition, 2012. Sadelain et al., 2013. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Ha rbor 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: 21; Ｈ CDRs; (b) a second V comprising the amino acid sequence set forth in SEQ ID NO: 22; Ｈ CDRs; (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 23 Ｈ CDRs; (d) a first V comprising the amino acid sequence set forth in SEQ ID NO: 24; Ｌ CDRs; (e) a second V comprising the amino acid sequence set forth in SEQ ID NO: 25; Ｌ CDRs; and (f) a third V comprising the amino acid sequence set forth in SEQ ID NO: 26 Ｌ CDRs; or (II) (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 11; Ｈ CDRs; (b) a second V comprising the amino acid sequence set forth in SEQ ID NO: 12; Ｈ CDRs; (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 13; Ｈ CDRs; (d) a first V comprising the amino acid sequence set forth in SEQ ID NO: 14; Ｌ CDRs; (e) a second V comprising the amino acid sequence set forth in SEQ ID NO: 15; Ｌ CDRs; and (f) a third V comprising the amino acid sequence set forth in SEQ ID NO: 16; Ｌ CDRs; 1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising:

2. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 21; Ｈ CDRs; (b) a second V comprising the amino acid sequence set forth in SEQ ID NO: 22; Ｈ CDRs; (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 23 Ｈ CDRs; (d) a first V comprising the amino acid sequence set forth in SEQ ID NO: 24 Ｌ CDRs; (e) a second V comprising the amino acid sequence set forth in SEQ ID NO: 25; Ｌ CDRs; (f) a third V comprising the amino acid sequence set forth in SEQ ID NO: 26 Ｌ CDR.

3. The antibody or antigen-binding fragment thereof is selected from the group consisting of V of SEQ ID NO: 27 Ｈ V domain that is at least 80% identical to Ｈ domain, and V of SEQ ID NO: 29 Ｌ V domain that is at least 80% identical to Ｌ 3. The antibody or antigen-binding fragment thereof of claim 2, comprising the domain.

4. The antibody or antigen-binding fragment thereof is selected from the group consisting of V 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 3. The antibody or antigen-binding fragment thereof of claim 2, comprising the domain.

5. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 11; Ｈ CDRs; (b) a second V comprising the amino acid sequence set forth in SEQ ID NO: 12; Ｈ CDRs; (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 13 Ｈ CDRs; (d) a first V comprising the amino acid sequence set forth in SEQ ID NO: 14; Ｌ CDRs; (e) a second V comprising the amino acid sequence set forth in SEQ ID NO: 15; Ｌ CDRs; (f) a third V comprising the amino acid sequence set forth in SEQ ID NO: 16; Ｌ CDR.

6. V of SEQ ID NO: 17 Ｈ V domains that are at least 80% identical to Ｈ Domain and V of SEQ ID NO: 19 Ｌ V domains that are at least 80% identical to Ｌ 6. The antibody or antigen-binding fragment thereof of claim 5, comprising the domain.

7. V 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 6. The antibody or antigen-binding fragment thereof of claim 5, comprising the domain.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is recombinant.

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

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is a Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or single domain antibody.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which is a humanized antibody or a deimmunized antibody.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, conjugated to an imaging agent, a chemotherapeutic agent, a toxin or a radionuclide.

13. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 in a pharmaceutically acceptable carrier.

14. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 11.

15. V of the amino acid sequences shown in SEQ ID NOs: 11, 12 and 13 Ｈ Antibody V comprising CDRs 1-3 of domain Ｈ Domains and V of the amino acid sequences shown in SEQ ID NOs: 14, 15 and 16 Ｌ Antibody V comprising CDRs 1-3 of domain Ｌ A recombinant polypeptide comprising a domain.

16. V of the amino acid sequences shown in SEQ ID NOs: 21, 22 and 23 Ｈ Antibody V comprising CDRs 1-3 of domain Ｈ Domain and V of the amino acid sequences shown in SEQ ID NOs: 24, 25 and 26 Ｌ Antibody V comprising CDRs 1-3 of domain Ｌ A recombinant polypeptide comprising a domain.

17. 17. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding a recombinant polypeptide according to claim 15 or 16.

18. A host cell comprising one or more polynucleotide molecules encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 or the recombinant polypeptide of claim 15 or 16.

19. 19. The host cell of claim 18, wherein the host cell is a mammalian cell, a yeast cell, a bacterial cell, a ciliated cell, or an insect cell.

20. A composition for use in a method for treating a subject with cancer, the composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

21. 21. The composition of claim 20, wherein the cancer is a B-cell malignancy.

22. 21. The composition of claim 20, wherein the antibody or antigen-binding fragment thereof is in a pharmaceutically acceptable composition.

23. 21. The composition of claim 20, which is administered systemically to a subject.

24. 21. The composition of claim 20, wherein the composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically to a subject.

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

26. 26. The composition of claim 25, wherein the second anti-cancer treatment is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

27. 26. The composition of claim 25, wherein the second anti-cancer treatment comprises adoptive T cell therapy.

28. An engineered CD79b CAR or TCR having the following antigen binding domain: (I): (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 21; Ｈ CDRs; (b) a second V comprising the amino acid sequence set forth in SEQ ID NO: 22; Ｈ CDRs; (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 23 Ｈ CDRs; (d) a first V comprising the amino acid sequence set forth in SEQ ID NO: 24 Ｌ CDRs; (e) a second V comprising the amino acid sequence set forth in SEQ ID NO: 25; Ｌ CDRs; and (f) a third V comprising the amino acid sequence set forth in SEQ ID NO: 26 Ｌ CDRs; Including, Or, (II): (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 11; Ｈ CDRs; (b) a second V comprising the amino acid sequence set forth in SEQ ID NO: 12; Ｈ CDRs; (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 13 Ｈ CDRs; (d) a first V comprising the amino acid sequence set forth in SEQ ID NO: 14; Ｌ CDRs; (e) a second V comprising the amino acid sequence set forth in SEQ ID NO: 15; Ｌ CDRs; and (f) a third V comprising the amino acid sequence set forth in SEQ ID NO: 16; Ｌ CDRs; an engineered CD79b CAR or TCR comprising:

29. The antigen-binding domain is V 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 29. The CAR or TCR of claim 28, comprising a domain.

30. 29. The CAR or TCR of claim 28, wherein the antigen binding domain comprises: (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 21; Ｈ CDRs, (b) a second V comprising the amino acid sequence set forth in SEQ ID NO: 22; Ｈ CDRs, (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 23 Ｈ CDRs, (d) a first V comprising the amino acid sequence set forth in SEQ ID NO: 24 Ｌ CDRs, (e) a second V comprising the amino acid sequence set forth in SEQ ID NO: 25; Ｌ CDRs, and (f) a third V comprising the amino acid sequence set forth in SEQ ID NO: 26 Ｌ CDR A CAR or TCR comprising:

31. The antigen-binding domain is V of SEQ ID NO: 27 Ｈ V domains that are at least about 80% identical to Ｈ domain, and V of SEQ ID NO: 29 Ｌ V domains that are at least about 80% identical to Ｌ 31. The CAR or TCR of claim 30, comprising a domain.

32. The antigen-binding domain is V 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 31. The CAR or TCR of claim 30, comprising a domain.

33. 29. The CAR or TCR of claim 28, wherein the antibody or antigen-binding fragment thereof comprises: (a) a first V comprising the amino acid sequence set forth in SEQ ID NO: 11; Ｈ CDRs, (b) a second V comprising the amino acid sequence set forth in SEQ ID NO: 12; Ｈ CDRs, (c) a third V comprising the amino acid sequence set forth in SEQ ID NO: 13 Ｈ CDRs, (d) a first V comprising the amino acid sequence set forth in SEQ ID NO: 14; Ｌ CDRs, (e) a second V comprising the amino acid sequence set forth in SEQ ID NO: 15; Ｌ CDRs, and (f) a third V comprising the amino acid sequence set forth in SEQ ID NO: 16; Ｌ CDR A CAR or TCR comprising:

34. 34. The CAR or TCR of claim 33, wherein the antigen-binding domain comprises a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO: 17, and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:

19.

35. The CAR or TCR of claim 28, wherein the CAR comprises one or more signaling domains selected from the group consisting of CD3 ACD28, OX40 / CD134, 4-1BB / CD137, and combinations thereof.

36. The CAR or TCR of claim 28, wherein the CAR comprises a CD3 signal transduction domain and a CD28 signaling domain.

37. The CAR or TCR of claim 28, wherein the CAR comprises a CD3 signal transduction domain and a 4-1BB signal transduction domain.

38. The CAR or TCR of claim 28, wherein the CAR comprises a CD3 signal transduction domain and an OX-40 signaling domain.

39. 29. The CAR or TCR of claim 28, wherein the CAR or TCR is encoded by a viral vector.

40. 40. The CAR or TCR of claim 39, wherein the viral vector is a lentiviral vector.

41. The antigen-binding domain is V Ｈ V linked to domain Ｌ The CAR or TCR according to any one of claims 28 to 40, comprising a domain.

42. The CAR or TCR of claim 41, wherein the linker comprises linker 1 (SEQ ID NO:45) or a linker encoded by a polynucleotide of SEQ ID NO:44, linker 2 (SEQ ID NO:47) or a linker encoded by a polynucleotide of SEQ ID NO:46, linker 3 (SEQ ID NO:49) or a linker encoded by a polynucleotide of SEQ ID NO:48, or linker 4 (SEQ ID NO:51) or a linker encoded by a polynucleotide of SEQ ID NO:

50.

43. 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 43. The CAR or TCR of claim 42, comprising:

44. The CAR or TCR of any one of claims 28 to 43, wherein the CAR or TCR comprises a hinge.

45. The CAR or TCR of claim 44, 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.

46. The CAR or TCR of any one of claims 28 to 45, wherein the CAR comprises a transmembrane domain.

47. The CAR or TCR of claim 46, wherein 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 TM (SEQ ID NO: 73) or SEQ ID NO: 72, or a transmembrane domain encoded by the polynucleotide of CD8 TM (SEQ ID NO: 87) or SEQ ID NO:

86.

48. 29. The CAR or TCR of claim 28, further comprising a transduction marker and / or a safety switch.

49. 49. The CAR or TCR of claim 48, wherein the transduction marker is enhanced green fluorescent protein (eGFP).

50. 50. The CAR or TCR of claim 49, wherein the eGFP has the amino acid sequence of SEQ ID NO:

83.

51. 49. The CAR or TCR of claim 48, wherein the transduction marker and / or safety switch is a truncated epidermal growth factor receptor (EGFR).

52. 52. The CAR or TCR of claim 51 , wherein the EGFR has the amino acid sequence of SEQ ID NO:

41.

53. 49. The CAR or TCR of claim 48, wherein the transduction marker and / or safety switch is linked to the CAR by a truncation peptide.

54. 54. The CAR or TCR of claim 53, wherein the truncated peptide is a 2A peptide.

55. 55. The CAR or TCR of claim 54, wherein the 2A peptide is a T2A peptide.

56. 56. The CAR or TCR of claim 55, wherein the T2A peptide has the amino acid sequence of SEQ ID NO:

85.

57. 29. The CAR or TCR of claim 28, wherein the CAR further comprises a second antigen-binding domain.

58. 58. The CAR or TCR of claim 57, wherein the second antigen-binding domain is a CD19, CD20, or CD22 antigen-binding domain.

59. The CAR or TCR according to any one of claims 28 to 58, wherein the CAR or TCR comprises a CD8 transmembrane domain and transmembrane domain (SEQ ID NO: 87), or a transmembrane domain encoded by the polynucleotide of SEQ ID NO: 86, a human OX-40 signaling domain (SEQ ID NO: 79), or a signaling domain encoded by the polynucleotide of SEQ ID NO: 78, and a CD3 transmembrane domain (SEQ ID NO: 81), or a domain encoded by the polynucleotide of SEQ ID NO:

81.

60. An expression vector encoding the CAR or TCR according to any one of claims 28 to 59.

61. Host cells engineered to express a CD79b CAR or a CD79b TCR.

62. The cell of claim 61, wherein the cell is a cell engineered to express the CAR of any one of claims 28 to 59.

63. 62. The cell of claim 61 , wherein the host cell is an immune cell.

64. 64. The cell of claim 63, wherein the immune cell is a T cell.

65. 65. The cell of claim 64, wherein the T cell is a primary human T cell or a TIL.

66. The cell of claim 64, wherein the T cell is a CD4-positive T cell or a CD8-positive T cell.

67. 66. The cell of claim 65, wherein the primary human T cells are obtained from a healthy donor.

68. 65. The cell of claim 64, wherein the T cell is autologous.

69. 65. The cell of claim 64, wherein the T cell is allogeneic.

70. 62. The cell of claim 61, wherein the cell has been engineered using a CRISPR or transposase system.

71. A pharmaceutical composition comprising a CD79b-targeting T cell engineered to express the CAR or TCR of any one of claims 28 to 59 and a pharmaceutically acceptable carrier.

72. A composition for treating cancer in a subject, comprising an effective amount of CD79b-targeting T cells engineered to express the CAR or TCR of any one of claims 28 to 59.

73. Use of a composition for treating cancer in a subject, comprising an effective amount of CD79b-targeting T cells engineered to express the CAR or TCR of any one of claims 28 to 59.

74. 60. A composition for use in a method for treating cancer in a subject, comprising an effective amount of CD79b-targeted T cells engineered to express a CAR or TCR according to any one of claims 28 to 59.

75. 75. The composition of claim 74, wherein the cancer is a B-cell malignancy.

76. 76. The composition of claim 75, 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.

77. 75. The composition of claim 74, wherein the subject has a history of receiving a CD19 CAR therapy.

78. 78. The composition of claim 77, wherein the subject is resistant to CD19 CAR therapy.

79. 79. The composition of claim 78, wherein the subject has CD19 antigen loss.

80. 80. The composition of claim 79, wherein the subject is relapsing with a CD19-negative tumor.

81. 75. The composition of claim 74, wherein the CD79b-targeted T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

82. 75. The composition of claim 74, wherein the CD79b-targeted T cells are administered intravenously.

83. 75. The composition of claim 74, wherein the composition further comprises administering to the subject at least a second anti-cancer treatment.

84. 84. The composition of claim 83, wherein the second anti-cancer treatment is surgical treatment, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

85. 75. The composition of claim 74, wherein the cancer is a CD79b-expressing cancer.

Images