Compositions and methods for treating cancer

Abstract

The present invention provides improved compositions for adoptive cell therapy of cancers that express CD79A and / or CD20.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 62 / 991,314, filed March 18, 2020, and U.S. Provisional Application No. 62 / 861,838, filed June 14, 2019, each of which is incorporated by reference in its entirety.

[0002] SEQUENCE LISTING STATEMENT The sequence listing for this application is submitted in text format instead of paper and is incorporated herein by reference. The name of the text file containing the sequence listing is BLBD_123_02WO_ST25. The text file is 152KB, was created on June 5, 2020, and is being submitted electronically via EFS-Web simultaneously with the filing of this application.

[0003] The present invention relates to improved compositions and methods for treating cancer. More particularly, the present invention relates to fusion polypeptides encoding anti-CD79A chimeric antigen receptors (CARs) and anti-CD20 chimeric costimulatory receptors (CCRs), genetically modified immune effector cells expressing the same, optionally including one or more genome edits, and the use of these compositions to effectively treat cancer. [Background technology]

[0004] Description of related fields Cancer is a major health problem worldwide. Based on rates from 2008-2010, 40.76% of men and women born today will be diagnosed with some form of cancer at some point in their lives. 20.37% of men will develop cancer between their 50s and 70s birthdays, and 15.30% of women will develop it. On January 1, 2010, there were approximately 13,027,914 men and women alive in the United States with a history of cancer, 6,078,974 men and 6,948,940 women. It is estimated that 1,660,290 men and women (854,790 men and 805,500 women) will be diagnosed in the United States, and 580,350 men and women will die from cancer of all sites in 2013. Howlader et al. 2013.

[0005] Malignant transformation of B cells leads to cancers including, but not limited to, lymphomas, such as multiple myeloma and non-Hodgkin's lymphoma. The majority of patients with B cell malignancies, including non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), contribute significantly to cancer mortality. The response of B cell malignancies to various forms of treatment is mixed. Conventional methods of treating B cell malignancies, including chemotherapy and radiation therapy, have limited usefulness due to toxic side effects.

[0006] New therapies for relapsed / refractory diffuse large B-cell lymphoma (DLBCL) have yet to meet the unmet need for the treatment of this disease, as evidenced by several recent setbacks in clinical trials that attempted to improve upon the standard R-CHOP (rituximab [Rituxan] with cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen. The phase III PHOENIX trial, combining ibrutinib (Imbrevica) with R-CHOP, failed to meet its primary endpoint of improved relapse-free survival. Results from the CORAL and SCHOLAR-1 trials further demonstrated the high unmet need for patients with refractory DLBCL. In each of these trials, long-term overall survival (OS) was just 15%-20% for patients who relapsed or had refractory disease within 12 months of receiving a stem cell transplant.

[0007] CD19 CAR T cell therapies Yescarta and Kymriah have been approved to treat patients with relapsed / refractory DLBCL, and CD19 CAR T cell therapy JCAR017 is in progress for approval. One of the major obstacles that still limits the efficacy of such CAR T cell therapies is the recurrence of “antigen-negative” cancer. For example, while anti-CD19 CAR T cell therapy initially produces moderate response rates in relapsed and refractory acute DLBCL, a strong possibility of relapse of CD19-negative blasts exists. The moderate efficacy of CAR T cell therapy combined with a surprisingly high rate of antigen-negative relapse reaffirms the unmet medical need to provide highly effective CAR T immunotherapy to DLBCL patients. Summary of the Invention

[0008] The present invention generally provides improved adoptive cellular immunotherapy methods and methods of using same. More specifically, the present invention provides adoptive cellular therapy methods for the prevention, treatment, or amelioration of at least one symptom of a cancer expressing CD79A and / or CD20.

[0009] In various embodiments, a fusion polypeptide is provided that comprises an anti-CD79A chimeric antigen receptor (CAR), a polypeptide cleavage signal, and an anti-CD20 chimeric costimulatory receptor (CCR).

[0010] In certain embodiments, an anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof, a first transmembrane domain, a first intracellular costimulatory signaling domain, and a primary signaling domain.

[0011] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof selected from the group consisting of a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single-chain Fv protein ("scFv"), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0012] In various embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof that is an scFv.

[0013] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof comprising a variable light chain sequence comprising the CDRL1-CDRL3 sequence set forth in SEQ ID NOs: 1-3 or 9-11, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence set forth in SEQ ID NOs: 4-6 or 12-14.

[0014] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof comprising the light chain CDRs set forth in SEQ ID NOs: 1-3 and the heavy chain CDRs set forth in SEQ ID NOs: 4-6.

[0015] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof comprising the light chain CDRs set forth in SEQ ID NOs: 9-11, and the heavy chain CDRs set forth in SEQ ID NOs: 12-14.

[0016] In various embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof comprising a variable light chain sequence set forth in any one of SEQ ID NOs: 7 or 15, and a variable heavy chain sequence set forth in any one of SEQ ID NOs: 8 or 16.

[0017] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof comprising the variable light chain sequence set forth in SEQ ID NO:7, and / or the variable heavy chain sequence set forth in SEQ ID NO:8.

[0018] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof comprising the variable light chain sequence set forth in SEQ ID NO: 15, and / or the variable heavy chain sequence set forth in SEQ ID NO: 16.

[0019] In certain embodiments, the anti-CD79A CAR comprises a first transmembrane domain isolated from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1.

[0020] In some embodiments, the anti-CD79A CAR comprises the first transmembrane domain isolated from CD8α.

[0021] In various embodiments, the anti-CD79A CAR comprises a first costimulatory signaling domain isolated from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

[0022] In a particular embodiment, the anti-CD79A CAR comprises a first costimulatory signaling domain isolated from CD137.

[0023] In a specific embodiment, the anti-CD79A CAR comprises a primary signaling domain isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0024] In a further embodiment, the anti-CD79A CAR comprises a primary signaling domain isolated from CD3ζ.

[0025] In some embodiments, a fusion polypeptide is provided comprising a variable light chain sequence set forth in any one of SEQ ID NOs: 7 or 15, and a variable heavy chain sequence set forth in any one of SEQ ID NOs: 8 or 16, an anti-CD79A CAR comprising a CD8α hinge domain, a CD8α transmembrane domain, a CD137 costimulatory domain and a CD3ζ primary signaling domain, a polypeptide cleavage signal, and an anti-CD20 CCR.

[0026] In certain embodiments, the polypeptide cleavage signal is a viral auto-cleaving polypeptide.

[0027] In various embodiments, the polypeptide cleavage signal is a viral auto-cleaving 2A polypeptide.

[0028] In some embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: foot and mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, Thosea asigna virus (TaV) 2A (T2A) peptide, porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0029] In certain embodiments, a fusion polypeptide is provided comprising an anti-CD79A CAR comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17-20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR as contemplated herein.

[0030] In certain embodiments, the anti-CD20 CCR comprises an anti-CD20 antibody or antigen-binding fragment thereof, a second transmembrane domain, and a second intracellular costimulatory domain.

[0031] In additional embodiments, the anti-CD20 CCR comprises an anti-CD20 antibody or antigen-binding fragment thereof selected from the group consisting of a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single chain Fv protein ("scFv"), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0032] In various embodiments, the anti-CD20 CCR comprises an anti-CD20 antibody or antigen-binding fragment thereof that is an scFv.

[0033] In other embodiments, the anti-CD20 CCR comprises an anti-CD20 antibody or antigen-binding fragment thereof comprising a variable light chain sequence comprising the CDRL1-CDRL3 sequences set forth in SEQ ID NOs:25-27, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences set forth in SEQ ID NOs:28-30.

[0034] In certain embodiments, the anti-CD20 CCR comprises an anti-CD20 antibody or antigen-binding fragment thereof comprising the variable light chain sequence set forth in SEQ ID NO:31, and / or the variable heavy chain sequence set forth in SEQ ID NO:32.

[0035] In certain embodiments, the anti-CD20 CCR comprises a second transmembrane domain isolated from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1.

[0036] In some embodiments, the anti-CD20 CCR comprises the second transmembrane domain isolated from CD8α.

[0037] In various embodiments, the anti-CD20 CCR comprises a second costimulatory signaling domain isolated from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

[0038] In various embodiments, the anti-CD20 CCR comprises a second costimulatory signaling domain and is isolated from CD28.

[0039] In certain embodiments, the anti-CD20 CCR comprises a CD8α hinge, a CD8α transmembrane domain, and a CD28 costimulatory signaling domain.

[0040] In certain embodiments, a fusion polypeptide is provided comprising an anti-CD79A CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 17-20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR comprising the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 35.

[0041] In a further embodiment, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37 or SEQ ID NO:39.

[0042] In certain embodiments, polynucleotides encoding the anti-CD79A CARs and anti-CD20 CARs contemplated herein are provided.

[0043] In some embodiments, a polynucleotide encoding a fusion polypeptide as contemplated herein is provided.

[0044] In various embodiments, a polynucleotide comprising the sequence set forth in SEQ ID NO:38 or SEQ ID NO:40 is provided.

[0045] In some embodiments, a vector is provided that comprises a polynucleotide as contemplated herein.

[0046] In certain embodiments, the vector is an expression vector.

[0047] In other embodiments, the vector is an episomal vector.

[0048] In certain embodiments, the vector is a viral vector.

[0049] In certain embodiments, the vector is a retroviral vector.

[0050] In various embodiments, the retroviral vector is a lentiviral vector.

[0051] In various embodiments, cells are provided that express the fusion polypeptides contemplated herein.

[0052] In certain embodiments, a cell comprising a polynucleotide encoding a fusion polypeptide as contemplated herein or a cell comprising a vector as contemplated herein is provided.

[0053] In various embodiments, cells are provided that comprise one or more polynucleotides encoding an anti-CD79A CAR comprising an anti-CD79A antibody, or antigen-binding fragment thereof; a first transmembrane domain; a first intracellular costimulatory signaling domain; and a primary signaling domain; and an anti-CD20 CCR comprising an anti-CD20 antibody, or antigen-binding fragment thereof; a second transmembrane domain; and a second intracellular costimulatory signaling domain.

[0054] In certain embodiments, the anti-CD79A antibody, or antigen-binding fragment thereof, and the anti-CD20 antibody, or antigen-binding fragment thereof, are both independently selected from the group consisting of a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single-chain Fv protein ("scFv"), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0055] In a specific embodiment, the anti-CD79A antibody, or antigen-binding fragment thereof, and the anti-CD20 antibody, or antigen-binding fragment thereof, are both scFvs.

[0056] In various embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequence set forth in SEQ ID NO:1-3 or 9-11, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence set forth in SEQ ID NO:4-6 or 12-14.

[0057] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises the light chain CDRs set forth in SEQ ID NOs:1-3, and the heavy chain CDRs set forth in SEQ ID NOs:4-6.

[0058] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises the light chain CDRs set forth in SEQ ID NOs:9-11, and the heavy chain CDRs set forth in SEQ ID NOs:12-14.

[0059] In various embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in any one of SEQ ID NOs:7 or 15, and a variable heavy chain sequence set forth in any one of SEQ ID NOs:8 or 16.

[0060] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:7 and / or the variable heavy chain sequence set forth in SEQ ID NO:8.

[0061] In additional embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:15 and / or the variable heavy chain sequence set forth in SEQ ID NO:16.

[0062] In certain embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequences set forth in SEQ ID NOs:25-27, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences set forth in SEQ ID NOs:28-30.

[0063] In various embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in SEQ ID NO:31, and a variable heavy chain sequence set forth in SEQ ID NO:32.

[0064] In certain embodiments, the first transmembrane domain and the second transmembrane domain are each independently isolated from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1.

[0065] In a further embodiment, the first transmembrane domain and the second transmembrane domain are both isolated from CD8α.

[0066] In various embodiments, the first costimulatory signaling domain and the second costimulatory domain are each independently isolated from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

[0067] In certain embodiments, the first costimulatory signaling domain is isolated from CD137.

[0068] In other embodiments, the primary signaling domain is isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0069] In certain embodiments, the primary signaling domain is isolated from CD3ζ.

[0070] In various embodiments, the second costimulatory signaling domain is isolated from CD28.

[0071] In certain embodiments, the anti-CD20 CCR comprises a CD8α hinge domain, a CD8α transmembrane domain, and a CD28 costimulatory signaling domain.

[0072] In certain embodiments, the cells express an anti-CD79A CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 17-20, and an anti-CD20 CCR comprising the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 35.

[0073] In some embodiments, the cells comprise a first polynucleotide encoding an anti-CD79A CAR and a second polynucleotide encoding an anti-CD20 CCR.

[0074] In various embodiments, the isolated polynucleotide encodes an anti-CD79A CAR and an anti-CD20 CCR.

[0075] In some embodiments, the isolated polynucleotide encodes an anti-CD79A CAR, an IRES sequence, and an anti-CD20 CCR.

[0076] In a specific embodiment, the isolated polynucleotide encodes an anti-CD79A CAR, a polypeptide cleavage signal, and an anti-CD20 CCR.

[0077] In various embodiments, the polypeptide cleavage signal is a viral auto-cleaving polypeptide.

[0078] In a particular embodiment, the polypeptide cleavage signal is a viral auto-cleaving 2A polypeptide.

[0079] In various embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: foot and mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, Thosea asigna virus (TaV) 2A (T2A) peptide, porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0080] In certain embodiments, the cells comprise an insertion or deletion of one or more nucleotides at a homing endonuclease (HE) variant cleavage target site or at a megaTAL cleavage target site within the Casitas B-lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene.

[0081] In a specific embodiment, the HE variant introduces one or more insertions or deletions into the HE target site of the CBLB gene set forth in SEQ ID NO:55.

[0082] In some embodiments, the megaTAL introduces one or more insertions or deletions into the megaTAL target site of the CBLB gene set forth in SEQ ID NO:56.

[0083] In certain embodiments, the insertion or deletion in the CBLB gene reduces CBLB expression, function and / or activity.

[0084] In a further embodiment, the cells comprise one or more altered CBLB alleles.

[0085] In additional embodiments, the cells contain one or more modified CBLB alleles that do not express or produce CBLB, or that express or produce a non-functional CBLB.

[0086] In certain embodiments, the cell comprises an insertion or deletion of one or more nucleotides at a homing endonuclease (HE) variant cleavage target site or at a megaTAL cleavage target site in the programmed cell death 1 (PDCD-1) gene.

[0087] In some embodiments, the HE variant introduces one or more insertions or deletions into the HE target site of the PDCD-1 gene set forth in SEQ ID NO:51.

[0088] In a further embodiment, the megaTAL introduces one or more insertions or deletions into the megaTAL target site of the PDCD-1 gene set forth in SEQ ID NO:52.

[0089] In certain embodiments, the insertion or deletion in the PDCD-1 gene reduces the expression, function, and / or activity of PDCD-1.

[0090] In some embodiments, the cells contain one or more altered PDCD-1 alleles.

[0091] In additional embodiments, the cells contain one or more altered PDCD-1 alleles that do not express or produce PDCD-1, or that express or produce a non-functional PDCD-1.

[0092] In a further embodiment, the cell is a hematopoietic cell.

[0093] In certain embodiments, the cells are hematopoietic stem or progenitor cells.

[0094] In other embodiments, the cells are CD34 + Hematopoietic stem or progenitor cells.

[0095] In certain embodiments, the cell is an immune effector cell.

[0096] In various embodiments, the cell is a T cell.

[0097] In certain embodiments, the cells are CD3 + , CD4+, and / or CD8 + It is a cell.

[0098] In certain embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

[0099] In some embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0100] In various embodiments, a cell population is provided that comprises a plurality of the cells contemplated herein.

[0101] In a further embodiment, a population of cells is provided that comprises one or more hematopoietic stem or progenitor cells and one or more immune effector cells as contemplated herein.

[0102] In various embodiments, one or more CD34 + Populations of cells are provided that include hematopoietic stem or progenitor cells and one or more T cells as contemplated herein.

[0103] In certain embodiments, a composition comprises a genetically modified cell as contemplated herein and a physiologically acceptable excipient.

[0104] In some embodiments, a composition comprises a cell population contemplated herein and a physiologically acceptable excipient.

[0105] In certain embodiments, a method of killing cancer cells expressing CD79A or CD20 in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0106] In certain embodiments, a method of killing cancer cells that express CD79A and CD20 in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0107] In certain embodiments, a method of killing cancer cells expressing CD79A and / or CD20 in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0108] In various embodiments, a method is provided for reducing the number of cancer cells that express CD79A and CD20 in a subject, comprising administering to the subject a therapeutically effective amount of a composition sufficient to reduce the number of cancer cells that express CD79A and CD20 compared to the number of cancer cells that express CD79A and CD20 prior to administration.

[0109] In various embodiments, methods are provided for reducing the number of cancer cells that express CD79A or CD20 in a subject, comprising administering to the subject a therapeutically effective amount of a composition sufficient to reduce the number of cancer cells that express CD79A or CD20 compared to the number of cancer cells that express CD79A or CD20 prior to administration.

[0110] In various embodiments, methods are provided for reducing the number of cancer cells that express CD79A and / or CD20 in a subject, comprising administering to the subject a therapeutically effective amount of a composition sufficient to reduce the number of cancer cells that express CD79A and / or CD20 compared to the number of cancer cells that express CD79A and / or CD20 prior to administration.

[0111] In some embodiments, a method of treating cancer in a subject in need of treatment is provided, comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0112] In certain embodiments, the cancer is a solid tumor.

[0113] In other embodiments, the solid tumor is osteosarcoma or Ewing's sarcoma.

[0114] In certain embodiments, the cancer is a liquid cancer.

[0115] In some embodiments, the cancer is a hematological malignancy.

[0116] In various embodiments, the cancer is non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

[0117] In certain embodiments, the non-Hodgkin's lymphoma is Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL).

[0118] In various embodiments, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0119] In some embodiments, the cancer is MM selected from the group consisting of overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, isolated plasmacytoma of bone, and extramedullary plasmacytoma.

[0120] In some embodiments, a method of treating a subject having DLBCL is provided that includes administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0121] In certain embodiments, methods are provided for ameliorating one or more symptoms associated with a CD79A and / or CD20 expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition contemplated herein sufficient to ameliorate at least one symptom associated with a cancer cell expressing CD79A and / or CD20.

[0122] In certain embodiments, the one or more symptoms ameliorated are selected from the group consisting of weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain, bone or joint pain, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination.

[0123] In certain embodiments, methods of generating a cell population expressing a fusion polypeptide contemplated herein include introducing into the cell population, a polynucleotide, or a vector contemplated herein.

[0124] In various embodiments, methods of generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR are provided, comprising introducing into the cell population one or more polynucleotides encoding an anti-CD79A CAR and an anti-CD20 CCR as contemplated herein.

[0125] In some embodiments, a method of generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, comprising introducing into the cell population a polynucleotide encoding a fusion polypeptide as contemplated herein.

[0126] In some embodiments, a method of generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, comprising introducing a polynucleotide encoding a fusion polypeptide as set forth in SEQ ID NO: 37 or SEQ ID NO: 39 into the cell population.

[0127] In certain embodiments, a method is provided for generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR, comprising introducing into the cell population a first polynucleotide encoding an anti-CD79A CAR set forth in any one of SEQ ID NOs: 17-20, and a second polynucleotide encoding an anti-CD20 CCR sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 35.

[0128] In certain embodiments, a method of generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, comprising introducing a polynucleotide sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 40 into the cell population.

[0129] In various embodiments, a method is provided for generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR, comprising introducing into the cell population a first polynucleotide sequence set forth in any one of SEQ ID NOs:21-24 and a first polynucleotide sequence set forth in SEQ ID NO:34 or SEQ ID NO:36.

[0130] In certain embodiments, one or more cells of the cell population comprise one or more insertions or deletions in the PDCD-1 gene of the polynucleotide sequence set forth in SEQ ID NO:51 that reduce or eliminate PDCD-1 expression and / or function.

[0131] In certain embodiments, a polynucleotide encoding an HE variant that binds and cleaves the polynucleotide sequence set forth in SEQ ID NO:51 is introduced into the cell population.

[0132] In certain embodiments, one or more cells of the cell population comprise one or more insertions or deletions in the PDCD-1 gene of the polynucleotide sequence set forth in SEQ ID NO:52 that reduce or eliminate PDCD-1 expression and / or function.

[0133] In some embodiments, a polynucleotide encoding a megaTAL that binds and cleaves the polynucleotide sequence set forth in SEQ ID NO:52 is introduced into the cell population.

[0134] In certain embodiments, one or more cells of the cell population comprise one or more insertions or deletions in the CBLB gene of the polynucleotide sequence set forth in SEQ ID NO: 55 that reduce or eliminate CBLB expression and / or function.

[0135] In certain embodiments, a polynucleotide encoding an HE variant that binds and cleaves the polynucleotide sequence set forth in SEQ ID NO:55 is introduced into the cell population.

[0136] In some embodiments, one or more cells of the cell population comprise one or more insertions or deletions in the CBLB gene of the polynucleotide sequence set forth in SEQ ID NO: 56 that reduce or eliminate CBLB expression and / or function.

[0137] In certain embodiments, a polynucleotide encoding a megaTAL that binds and cleaves the polynucleotide sequence set forth in SEQ ID NO:56 is introduced into the cell population.

[0138] In some embodiments, the cell population comprises hematopoietic stem or progenitor cells.

[0139] In certain embodiments, the cell population is CD34 + Includes hematopoietic stem or progenitor cells.

[0140] In various embodiments, the cell population comprises immune effector cells.

[0141] In certain embodiments, the cell population comprises T cells, NK cells, and / or NKT cells.

[0142] In various embodiments, the cell population comprises T cells. [Brief description of the drawings]

[0143] [Figure 1A]Figures 1A-1C show expression and activity of anti-CD79A CAR and anti-CD20 CCR. A) Expression of CD79A CAR in untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, anti-CD20 CCR fusion proteins. B) This panel shows IFNγ secretion of UTD T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR co-cultured in the absence of target cells or with RD cells (CD79A-, CD20-), RD.CD79A cells (CD79A+, CD20-), RD.CD79A.CD20 cells (CD79A+, CD20+), or Daudi cells (CD79A+, CD20+, high expression). B) This panel shows IL-2 secretion of UTD T cells, anti-CD79A CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR in the absence of target cells or co-cultured with RD cells (CD79A-, CD20-), RD.CD79A cells (CD79A+, CD20-), RD.CD79A.CD20 cells (CD79A+, CD20+), or Daudi cells (CD79A+, CD20+, high expression). [Figure 1B] Same as above. [Figure 1C] Same as above. [Diagram 2] Figures 2A and 2B show anti-CD79A CAR and anti-CD20 CCR activity on RD.CD20 cells. A) This panel shows IFNγ secretion of T cells expressing anti-CD79A CAR and anti-CD20 CCR co-cultured with UTD T cells, anti-CD79A CAR T cells, or RD cells (CD79A-, CD20-), or RD.CD20 cells (CD79A-, CD20+). C) This panel shows IL-2 secretion of T cells expressing anti-CD79A CAR and anti-CD20 CCR co-cultured with UTD T cells, anti-CD79A CAR T cells, or RD cells (CD79A-, CD20-), or RD.CD20 cells (CD79A-, CD20+). [Figure 3A]Figures 3A and 3B show expression and activity of anti-CD79A and anti-CD20 CARs. A) Expression of CD79A CAR in untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-CD28-CD3ζ fusion proteins (middle panel) or lentiviral vectors encoding anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-4-1BB-CD3ζ fusion proteins (right panel). B) This panel shows IFNγ secretion of UTD T cells or T cells expressing anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-CD28-CD3ζ fusion protein, or anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-4-1BB-CD3ζ fusion protein in the absence of target cells or co-cultured with RD cells (CD79A-, CD20-), RD.CD79A cells (CD79A+, CD20-), RD.CD20 cells (CD79A-, CD20+), or Daudi cells (CD79A+, CD20+, high expression). [Figure 3B] Same as above. [Figure 4A]Figures 4A-4C show expression and activity of anti-CD79A CAR and anti-CD20 CCR. A) Expression of CD79A CAR in untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, and anti-CD20 CCR fusion proteins. B) This panel shows IFNγ secretion of UTD T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR co-cultured in the absence of target cells or with RD cells ((CD79A-, CD20-), RD.CD79A cells (CD79A+, CD20-), or REC-1 cells (CD79A+, CD20+, high expression). B) This panel shows IL-2 secretion of UTD T cells, anti-CD79A CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR co-cultured in the absence of target cells or with RD cells (CD79A-, CD20-), RD.CD79A cells (CD79A+, CD20-), RD.CD79A.CD20 cells (CD79A+, CD20+), or REC-1 cells (CD79A+, CD20+, high expression). [Figure 4B] Same as above. [Figure 4C] Same as above. [Diagram 5] Figures 5A and 5B show anti-CD79A CAR and anti-CD20 CCR activity on RD.CD20 cells. A) This panel shows IFNγ secretion of T cells expressing anti-CD79A CAR and anti-CD20 CCR co-cultured with UTD T cells, anti-CD79A CAR T cells, or RD cells (CD79A-, CD20-), or RD.CD20 cells (CD79A-, CD20+). C) This panel shows IL-2 secretion of T cells expressing anti-CD79A CAR and anti-CD20 CCR co-cultured with UTD T cells, anti-CD79A CAR T cells, or RD cells (CD79A-, CD20-), or RD.CD20 cells (CD79A-, CD20+). [Figure 6A]Figures 6A and 6B show expression and activity of anti-CD79A and anti-CD20 CARs. A) Expression of CD79A CAR in untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-CD28-CD3ζ fusion proteins (middle panel) or lentiviral vectors encoding anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-4-1BB-CD3ζ fusion proteins (right panel). B) This panel shows IFNγ secretion of UTD T cells or T cells expressing anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-CD28-CD3ζ fusion protein, or anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-4-1BB-CD3ζ fusion protein in the absence of target cells or co-cultured with RD cells (CD79A-, CD20-), RD.CD79A cells (CD79A+, CD20-), RD.CD20 cells (CD79A-, CD20+), or Daudi cells (CD79A+, CD20+, high expression). [Figure 6B] Same as above. [Figure 7] Figure 7 shows the cytotoxicity of T cells expressing anti-CD79A CAR and anti-CD20 CCR against cell lines engineered to express CD79A or CD20. Untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, and anti-CD20 CCR fusion proteins were co-cultured in the presence of RD cells (CD79A-, CD20-; left panel), RD.79A cells (CD79A+, CD20-; center panel), and RD.20 cells (CD79A-, CD20+; right panel). [Figure 8]Figure 8 shows the efficacy of T cells expressing anti-CD79A CAR and anti-CD20 CCR against Daudi cells (CD79A+, CD20+, high expression) in vitro and in vivo. Untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, and anti-CD20 CCR fusion proteins were co-cultured in the presence of Daudi cells at a 1:1 ratio for 24 hours, and supernatants were collected and analyzed for IFNγ using Luminex (left panel, n=3). NSG mice were intravenously injected with 2x106 luciferase-expressing Daudi cells, and 13 days later, five mice were injected with vehicle (medium), 10x106 UTD T cells, or 10x106 T cells expressing anti-CD79A CAR and anti-CD20 CCR, and the mice were monitored for 30 days (right panel). [Figure 9] FIG. 9 shows the efficacy of T cells expressing anti-CD79A CAR and anti-CD20 CCR against NU-DUL-1 cells (DLBCL tumor model) in vitro and in vivo. Untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, and anti-CD20 CCR fusion proteins were co-cultured in the presence of NU-DUL-1 cells at a 1:1 ratio for 24 hours, and supernatants were collected and analyzed for IFNγ using Luminex (left panel, n=3). NSG mice were intravenously injected with 2×106 luciferase-expressing NU-DUL-1 cells, and 15 days later, five mice were injected with vehicle (medium), 10×106 UTD T cells, or 5×106 T cells expressing anti-CD79A CAR and anti-CD20 CCR, and the mice were monitored for 30 days (right panel). [Figure 10]Figure 10 shows the efficacy of T cells expressing anti-CD79A CAR and anti-CD20 CCR against the Toledo (Germinal Center B Cell (GCB)) DLBCL tumor model in vitro and in vivo. Untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, anti-CD20 CCR fusion proteins were co-cultured in the presence of GCB DLBCL cells at a 1:1 ratio for 24 hours, and supernatants were collected and analyzed for IFNγ using Luminex (left panel, n=3). NSG mice were injected intravenously with 50x106 luciferase-expressing GCB DLBCL cells and 16 days later (100mm3 tumors) 5 mice were injected with vehicle (media), 20x106 UTD T cells or 2.5x106 T cells expressing anti-CD79A CAR and anti-CD20 CCR and mice were monitored for 30 days (right panel). [Figure 11] Figure 11 shows the efficacy of T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing against the Toledo (Germinal Center B Cell (GCB)) DLBCL tumor model in vitro and in vivo. Untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, anti-CD20 CCR fusion proteins with or without genome editing at the CBLB locus were co-cultured in the presence of GCB DLBCL cells at a 1:1 ratio for 24 hours, and supernatants were collected and analyzed for IFNγ using Luminex (left panel, n=3). NSG mice were injected intravenously with 50x106 luciferase-expressing GCB DLBCL cells and 17 days later (130mm3 tumors) 5 mice were injected with vehicle (media), 5x106 UTD T cells + / - CBLB edit, or 1x106 T cells expressing anti-CD79A CAR and anti-CD20 CCR + / - CBLB edit and mice were monitored for 21 days (right panel). [Figure 12]Figure 12 shows the efficacy of T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing against the Daudi.CD20KO (CD20 knockout) tumor model in vivo. NSG mice were intravenously injected with 2x106 luciferase-expressing Daudi.CD20KO cells, and 14 days later, 5 mice were injected with vehicle (media), 20x106 UTD T cells + / - CBLB editing, or 10x106 T cells expressing anti-CD79A CAR and anti-CD20 CCR + / - CBLB editing, and the mice were monitored for 30 days. [Figure 13] Figure 13 shows cytokine secretion of T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing and activated using anti-CD3 and anti-CD28 antibodies. 1x106 cells / mL of UTD T cells + / - CBLB editing or T cells expressing anti-CD79A CAR and anti-CD20 CCR were cultured for 24 hours in 96-well high binding plates coated with monoclonal antibodies against CD3 (titrated from 1μg / mL to 0.063μg / mL) and CD28 (5μg / mL), and Il-2 (left panel) and IFNγ (right panel) were measured by Luminex (n=3). [Figure 14] Figure 14 shows enhanced IL-2 secretion in T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing. UTD T cells + / - CBLB editing, and T cells expressing anti-CD79A CAR and anti-CD20 CCR + / - CBLB were co-cultured with Daudi (Burkitt's lymphoma; CD79+, CD20+) tumor cells at a 1:1 ratio for 24 hours, supernatants were collected, and IL-2 was analyzed using Luminex. [Figure 15]Figure 15 shows that T cells treated with CBLB megaTAL show increased proliferation in serial restimulation assays compared to T cells treated with TCRα killed megaTAL. UTD T cells or T cells transduced with a single lentiviral vector encoding an anti-CD79A CAR and an anti-CD20 CCR were treated with CBLB megaTAL or TCRa killed megaTAL and subjected to serial restimulation assays (n=4). [Figure 16] Figure 16 shows increased cytokine responses of T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing against a GCB DLBCL tumor model. PBMCs from three healthy donors (DH) and three DLBCL donors (DL) transduced with lentiviral vectors encoding anti-CD79A CAR and anti-CD20 CCR were co-cultured with Toledo GCB DLBCL tumor cells at a 1:1 ratio for 24 hours, and supernatants were collected and analyzed for IFNγ using Luminex. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0144] A brief explanation of sequence numbers SEQ ID NOs:1-16 set forth exemplary light chain CDR sequences, heavy chain CDR sequences, variable domain light chain, and variable domain heavy chain amino acid sequences for the anti-CD79A CARs contemplated herein. SEQ ID NOs:17-20 set forth the amino acid sequences of exemplary anti-CD79A CARs. SEQ ID NOs:21-24 set forth the nucleic acid sequences of exemplary anti-CD79A CARs. SEQ ID NOs:25-32 set forth exemplary light chain CDR sequences, heavy chain CDR sequences, variable domain light chains, and variable domain heavy chain amino acid sequences for the anti-CD20 CCRs contemplated herein. SEQ ID NOs:33 and 35 set forth the amino acid sequences of exemplary anti-CD20 CCRs. SEQ ID NOs:34 and 36 set forth the nucleic acid sequences of exemplary anti-CD20 CCRs. SEQ ID NOs:37 and 39 set forth the amino acid sequences of exemplary anti-CD79A CAR-T2A anti-CD20 CCR fusion proteins. SEQ ID NOs:38 and 40 set forth the nucleic acid sequences of exemplary anti-CD79A CAR-T2A anti-CD20 CCR fusion proteins. SEQ ID NOs: 41 and 43 set forth the amino acid sequences of exemplary anti-CD79A CAR-T2A anti-CD20 CD28z CAR fusion proteins. SEQ ID NOs: 42 and 44 set forth the nucleic acid sequences of exemplary anti-CD79A CAR-T2A anti-CD20 CD28z CAR fusion proteins. SEQ ID NOs: 45 and 47 set forth the amino acid sequences of exemplary anti-CD79A CAR-T2A anti-CD20 BBz CAR fusion proteins. SEQ ID NOs: 46 and 48 set forth the nucleic acid sequences of exemplary anti-CD79A CAR-T2A anti-CD20 BBz CAR fusion proteins. SEQ ID NO:49 is the amino acid sequence of the I-OnuI LHE variant reprogrammed to bind to and cleave a target site in the human PDCD-1 gene. SEQ ID NO:50 is the amino acid sequence of a megaTAL that binds to and cleaves a target site in the human PDCD-1 gene. SEQ ID NO:51 is the I-OnuI LHE variant target site in the human PDCD-1 gene. SEQ ID NO:52 is the megaTAL target site in the human PDCD-1 gene. SEQ ID NO:53 is the amino acid sequence of the I-OnuI LHE variant reprogrammed to bind to and cleave a target site in the human CBLB gene. SEQ ID NO:54 is the amino acid sequence of a megaTAL that binds to and cleaves a target site in the human CBLB gene. SEQ ID NO:55 is the I-OnuI LHE variant target site in the human CBLB gene. SEQ ID NO:56 is the megaTAL target site in the human CBLB gene. SEQ ID NOs: 57-67 set forth the amino acid sequences of various linkers. SEQ ID NOs: 68-92 set forth the amino acid sequences of the protease cleavage sites and the cleavage sites of the self-cleaving polypeptide. In the foregoing sequences, X, if present, refers to any amino acid or the absence of an amino acid.

[0145] Detailed Description A. Overview Cancer is often a heterogeneous pool of cells that express different levels of various antigens.Generally, immunotherapy is initially selected to target antigens that are expressed on the majority of cancer cells and substantially lack expression on normal cells.Effective targeted immunotherapy kills the majority of cancer cells that express target antigens, resulting in partial or complete remission.However, since most cancers are inherently heterogeneous, the remaining cancer cells that do not express target antigens or express low levels of target antigens may be spared, resulting in cancer cells that are not effectively targeted by initial immunotherapy.

[0146] One of the major hindrances that still limits the efficacy of adoptive cell therapy is the recurrence of "antigen-negative" cancers. The surprisingly high rate of antigen-negative recurrence rates still represents an unmet need for adoptive cell therapy. Without intending to be bound by any particular theory, the inventors have solved the challenge of killing cancers that are heterogeneous for the expression of multiple target antigens by re-engineering immune effector cells (e.g., T cells, NK cells) to express chimeric antigen receptors (CARs) and chimeric costimulatory receptors (CCRs) that target multiple antigens, synergistically activating multiple intracellular cell signaling pathways to enhance their ability to kill tumor cells that have an inflammatory cytokine response and one or both of the target antigens, and prevent their recurrence. Surprisingly, the inventors have discovered that immune effector cells expressing CARs and CCRs against different antigens do not require the presence of both antigens on the target cell to induce an inflammatory cytokine response and kill the cell. This is surprising because CCRs do not contain a signaling domain and therefore should not signal alone in theory. Thus, the compositions and methods contemplated herein represent an important advance in T cell immunotherapy for heterologous cancers.

[0147] Another impediment limiting the efficacy of adoptive cell therapy is the hyporesponsiveness of immune effector cells due to exhaustion mediated by the tumor microenvironment. For example, exhausted T cells have a unique molecular signature that is significantly different from naive, effector, or memory T cells. They are defined as immune effector cells with reduced cytokine expression and effector function. Programmed cell death 1 (PDCD-1) is a T cell exhaustion marker, and increased PD-1 expression is associated with decreased T cell proliferation and reduced production of IL-2, TNF, and IFN-γ. Casitas B lineage (Cbl) lymphoma proto-oncogene B (CBLB) is a member of the RING finger family or E3 ubiquitin ligase involved in the negative regulation of effector T cell activity and persistence. T cells from CBLB knockout mice are proliferative, produce high levels of IL2 and IFNγ in response to antigen stimulation, are resistant to TGFβ-mediated inhibition, and have a lower activation threshold, indicating that CBLB plays a role in negatively regulating T cell activation. Without intending to be bound by any particular theory, it is contemplated that disruption of the PDCD-1 and / or CBLB genes in immune effector cells expressing a CAR directed against a first antigen and a CCR directed against a second antigen will result in more effective and sustained adoptive cell therapy.

[0148] The present invention generally relates to improved compositions and methods for preventing or treating cancers expressing CD79A and / or CD20, or for preventing, treating, or ameliorating at least one symptom associated with cancers expressing CD79A and / or CD20. In various embodiments, the present invention relates to improved adoptive cell therapy of cancers expressing CD79A and / or CD20 using genetically modified immune effector cells, which optionally contain one or more genomic edits to reduce or eliminate expression and / or function of PDCD1 and / or CBLB. Genetic approaches provide a potential means to enhance immune recognition and elimination of cancer cells. Immune effector cells modified to express chimeric antigen receptors (CARs) and chimeric costimulatory receptors (CCRs) are contemplated in certain embodiments to direct cytotoxicity to cancer cells expressing either the CAR-targeted antigen or the CCR-targeted antigen, synergistically enhancing immune effector cell responses against cancer. In particularly preferred embodiments, the immune effector cells engineered to express a CAR and a CCR further comprise one or more genomic edits that reduce or eliminate expression and / or function of PDCD-1 and / or CBLB. In other particularly preferred embodiments, the immune effector cells engineered to express a CAR and a CCR further comprise one or more genomic edits that reduce or eliminate expression and / or function of CBLB.

[0149] Improved compositions and methods of adoptive cell therapy contemplated in certain embodiments herein provide genetically engineered immune effector cells that can be readily expanded, exhibit long-term persistence in vivo, and exhibit antigen-dependent cytotoxicity against cells expressing CD79A and / or CD20, as well as resistance to immunosuppressive signals in the tumor microenvironment.

[0150] CD79A is also known as B cell antigen receptor complex associated protein alpha chain, membrane-bound immunoglobulin-associated protein (MB1, MB-1), surface IgM-associated protein, and Ig alpha (IGA). Examples of polynucleotide sequences encoding CD79A include, but are not limited to, NM_001783.3, NM_021601.3, ENST00000221972 (uc002orv.3), ENST00000597454 (uc060zdj.1), ENST00000444740 (uc002oru.4), Hs.631567, and AK223371. Examples of polypeptide sequences encoding CD79a include, but are not limited to, the following: P11912-1, P11912-2, ENSP00000400605, ENSP00000468922, ENSP00000221972, NP_001774.1, and NP_067612.1.

[0151] CD79 consists of two proteins, CD79A and CD79B. CD79A is located on chromosome 19q13.2 and encodes a 226 amino acid glycoprotein of approximately 47 kDa. The exact molecular weight depends on the degree of glycosylation. CD79B is located on chromosome 17q23 and encodes a 229 amino acid glycoprotein of approximately 37 kDa. CD79A and CD79B share an exon-intron structure and both contain a single IgSF Ig domain (a 111-residue C-form for CD79A and a 129-residue V-form for CD79B). Each also contains a highly conserved transmembrane domain and a 61 (CD79A) or 48 (CD79B) amino acid cytoplasmic tail, which also show remarkable evolutionary conservation of amino acids. CD79A and CD79B are expressed by the earliest committed B cell precursors. CD79A / B heterodimers have also been observed on the surface of early B cell precursors in the absence of μ heavy chains, but neither protein is required for the precursor to commit to the B cell lineage. Later in development, CD79A and CD79B are coexpressed with Ig of all isotypes on the surface of B cells as mature BCR complexes. CD79 proteins are specific to the B lineage and are expressed throughout B lymphopoiesis. CD79A and CD79B can be used as markers for the identification of B cell neoplasms, including DLBCL, the majority of acute leukemias of precursor B cell type, in B cell lines, B cell lymphomas, and some myelomas.

[0152] CD20 is also known as Membrane Spanning 4-Domains A (MS4A), Membrane Spanning 4-Domains, Subfamily A, Member 1, Leukocyte Surface Antigen Leu-16 (LEU-16), B-Lymphocyte Cell Surface Antigen B1 (B1), S7, and Common Variable Immunodeficiency 5 (CVID5). Examples of polynucleotide sequences encoding CD79A include, but are not limited to, the following: NM_021950.3, NM_152866.2, AK225630.1, X07203.1, AK292168.1, X12530.1, BC002807.2, NM_023945, NM_152867, ENST00000345732, and ENST00000389939. Examples of polypeptide sequences encoding CD79a include, but are not limited to, the following: P11836, NX_P11836, ENSP00000432219, ENSP00000433519, ENSP00000432270, ENSP00000314620, ENSP00000437002, ENSP00000374589, ENSP00000433179, ENSP00000433277, NP_690605.1, and NP_068769.2.

[0153] CD20 is a member of the transmembrane 4A gene family. Members of this early protein family are characterized by common structural features and similar intron / exon splice boundaries and exhibit unique expression patterns among hematopoietic cells and non-lymphoid tissues. The CD20 gene encodes a B-lymphocyte surface molecule involved in B-cell development and differentiation into plasma cells. CD20 is expressed in the majority of B-cell malignancies, including chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0154] In various embodiments, immune effector cells engineered to express CAR and CCR are highly effective, grow robustly in vivo, recognize cancer cells expressing CD79A and / or CD20, and exhibit cytotoxic activity against cancer cells expressing CD79A and / or CD20.

[0155] In various preferred embodiments, the immune effector cells engineered to express a CAR and a CCR further comprise one or more genomic edits that reduce or eliminate expression and / or function of PDCD-1 and / or CBLB.

[0156] In one embodiment, the immune effector cells are genetically modified to express an anti-CD79 CAR and an anti-CD20 CCR, and further comprise one or more genomic edits that reduce or eliminate the expression and function of a CBLB. T cells expressing a CAR and a CCR are referred to herein as CAR / CCR T cells or CAR / CCR modified T cells.

[0157] In one embodiment, a fusion polypeptide comprising an anti-CD79 CAR, a polypeptide cleavage signal, and an anti-CD20 CCR is contemplated.

[0158] In various embodiments, the genetically modified immune effector cells are administered to a subject having cancer cells that express CD79A and / or CD20, including, but not limited to, liquid tumors, hematological malignancies, and B cell malignancies. In one embodiment, immune effector cells modified to express an anti-CD79A CAR and an anti-CD20 CCR are administered to a subject having DLBCL.

[0159] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, which are cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol.I & II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols in Immunology(Edited by:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M.Shevach,Warren Strober 2001 John Wiley & Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid The Hybridization(B.Hames & S.Higgins,Eds.,1985);Transcription and Translation(B.Hames & S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press);Immobilized Cells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as research articles in journals such as, for example, Advances in Immunology.

[0160] B. Definition Before describing the present disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0161] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although similar or equivalent methods and materials to those described herein can be used to carry out or test specific embodiments, preferred compositions, methods and materials embodiments are disclosed herein.For the purposes of this disclosure, the following terms are defined below.

[0162] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0163] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0164] The term "and / or" should be understood to mean either or both of the alternatives.

[0165] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0166] In one embodiment, ranges, for example 1 to 5, about 1 to 5, or about 1 to about 5, refer to every number subsumed within the range. For example, in one non-limiting, and merely exemplary embodiment, the range "1 to 5" is equivalent to: 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0167] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about the same as a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0168] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the specified step or component or group of steps or components, but not the exclusion of any other step or component or group of steps or components. "Consisting of" means including and limited to everything that follows the word "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements that are limited to any elements recited thereafter, and other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that there are no other elements that materially affect the activity or action of the recited elements.

[0169] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Moreover, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Additionally, it should be understood that the affirmative recitation of a feature in an embodiment serves as a basis for the exclusion of that feature in certain embodiments.

[0170] As used herein, the terms "binding domain," "extracellular domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably and confer to a CAR or CCR the ability to specifically bind to a target antigen of interest. The binding domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0171] "Antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a peptide, lipid, polysaccharide, or antigenic determinant-containing nucleic acid, such as those recognized by immune cells. An "isolated antibody or antigen-binding fragment thereof" is one that has been identified and separated and / or recovered from a component of its natural environment.

[0172] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions (such as compositions that include a cancer-specific protein) that are injected into or absorbed into an animal. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. In certain embodiments, the target antigen is an epitope of a CD79A or CD20 polypeptide.

[0173] "Epitope" or "antigenic determinant" refers to a region of an antigen to which a binding agent binds. Epitopes can be formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes typically include at least 3, more commonly at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0174] As will be understood by those of skill in the art, and as described elsewhere herein, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y consists of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, with disulfide bonds (interchain) formed at the hinge. The heavy chains gamma, alpha and delta have a constant region consisting of three tandem Ig domains and a hinge domain for additional flexibility, while the heavy chains mu and epsilon have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain", respectively. Each arm of the Y contains the variable and first constant regions of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0175] Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs." The CDRs can be conventionally defined or identified by the sequences set forth in Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84:2440-2443 (1987); (see Kabat et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1991, incorporated herein by reference) or the structures set forth in Chothia et al. (Chothia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917 (1987), Chothia, C. et al, Nature, 342:877-883 (1989)).

[0176] Illustrative examples of rules for predicting light chain CDRs include: CDR-L1 starts at about residue 24, is preceded by a Cys, is about 10-17 residues, followed by Trp (typically Trp-Tyr-Gln, also Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 starts about 16 residues after the end of CDR-L1, is generally preceded by Ile-Tyr, also Val-Tyr, Ile-Lys, Ile-Phe, and is 7 residues; CDR-L3 starts about 33 residues after the end of CDR-L2, is preceded by a Cys, is 7-11 residues, and is followed by Phe-Gly-XXX-Gly (SEQ ID NO: 97) (XXX is any amino acid).

[0177] Exemplary rules for predicting heavy chain CDRs include: CDR-H1 begins at about residue 26, is preceded by Cys-XXX-XXX-XXX (SEQ ID NO: 94), is 10-12 residues long, and is Trp (typically Trp-Val, also Trp-Ile, Trp-Ala); CDR-H2 begins about 15 residues after the end of CDR-H1, and is generally Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 95), and is is preceded by several variations, is 16-19 residues long and followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala; CDR-H3 starts approximately 33 residues after the end of CDR-H2, is preceded by Cys-XXX-XXX (typically, Cys-Ala-Arg), is 3-25 residues long and followed by Trp-Gly-XXX-Gly (SEQ ID NO: 96).

[0178] In one embodiment, the light chain and heavy chain CDRs are determined according to the Kabat method.

[0179] In one embodiment, the light chain CDR and the heavy chain CDR2 and CDR3 are determined according to the Kabat method, and the heavy chain CDR1 is determined according to the AbM method, which is included between the Kabat method and the Clothia method, see, for example, Whitelegg N & Rees AR, Protein Eng. 2000 Dec; 13 (12): 819-24 and Methods Mol Biol. 2004; 248: 51-91. Programs for predicting CDRs are, for example, published in AbYsis (www.bioinf.org.uk / abysis / ).

[0180] References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain.

[0181] References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain.

[0182] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0183] A "chimeric antibody" has framework residues derived from one species, such as human, and CDRs (which generally confer antigen binding) derived from another species, such as mouse. In certain preferred embodiments, the CAR comprises an antigen-binding domain that is a chimeric antibody or an antigen-binding fragment thereof.

[0184] Human antibodies can be constructed by combining Fv clone variable domain sequence(s) selected from a human-derived phage display library with known human constant domain sequence(s) as described above. Alternatively, human monoclonal antibodies can be produced by hybridoma methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described, for example, in Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147:86 (1991). In addition, transgenic animals (e.g., mice) can be used to produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993). Gene shuffling can also be used to derive human antibodies from non-human, e.g., rodent, antibodies, but the human antibodies have similar affinities and specificities as the starting non-human antibody. (See PCT WO 93 / 06213, published April 1, 1993). Unlike conventional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies that have no FR or CDR residues of non-human origin.

[0185] A humanized antibody is an immunoglobulin that includes a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more. Thus, all parts of a humanized immunoglobulin are substantially identical to the corresponding parts of a native human immunoglobulin sequence, except possibly for the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions, which do not substantially affect antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0186] Antibodies include antigen-binding fragments thereof, such as, for example, camelid Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins ("dsFv"), and single domain antibodies (sdAb, nanobodies), as well as the portion of a full length antibody that contributes to antigen binding. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.

[0187] "Heavy chain antibodies" consist of two VH It refers to an antibody that contains two V domains and does not contain a light chain (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). H "Humanized VHH" or "humanized camelid-like antibody" refers to a non-human VHH or camelid-like antibody that has undergone humanization to reduce potential immunogenicity of the antibody in a human recipient.

[0188] "IgNAR" for "immunoglobulin novel antigen receptor" refers to a class of antibodies from the shark immune repertoire consisting of a homodimer of one variable novel antigen receptor (VNAR) domain and five constant novel antigen receptor (CNAR) domains. IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and are highly stable and have efficient binding properties. The inherent stability can be attributed to both (i) the underlying Ig scaffold, which displays a significant number of charged and hydrophilic surface-exposed residues compared to traditional antibody VH and VL domains found in mouse antibodies, and (ii) structural features in the complementarity determining region (CDR) loops, including interloop disulfide bridges, and stabilizing patterns of intraloop hydrogen bonds.

[0189] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0190] An "Fv" is the smallest antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to the two-chain Fv species. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, but with a lower affinity than the entire binding site.

[0191] Fab fragments contain the heavy and light chain variable domains, and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge domain. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domain bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known. A bispecific Fab dimer (Fab2) has two Fab' fragments, each of which binds a different antigen. A trispecific Fab trimer (Fab3) has three Fab' fragments, each of which binds a different antigen.

[0192] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain to create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0193] A "single domain antibody" or "sdAb" or "nanobody" refers to an antibody fragment consisting of the variable region of the antibody heavy chain (VH domain) or the variable region of the antibody light chain (VL domain) (Holt, L., et al, Trends in Biotechnology, 21(11):484-490).

[0194] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, which are present in a single polypeptide chain and in any orientation (e.g., VL-VH or VH-VL). Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0195] A "linker" is an amino acid sequence that connects adjacent domains of a polypeptide or fusion polypeptide. A linker sequence is an amino acid sequence that includes a "variable region binding sequence" and connects the VH and VL domains of an antibody or antigen-binding fragment thereof, providing a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. Illustrative examples of linkers include glycine polymers (G)n, glycine-serine polymers (G1-5S1-5)n, where n is an integer of at least one, two, three, four, or five, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can function as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine has access to much more phi-psi space than alanine and is much less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Linkers can be 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 or more amino acids in length. Other exemplary linkers include, but are not limited to, the following amino acid sequences:DGGGS (SEQ ID NO:57); TGEKP (SEQ ID NO:58) (e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO:59) (Pomerantz et al. 1995, supra); (GGGGS)n, n=1, 2, 3, 4 or 5 (SEQ ID NO:60) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO:61) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSSEQLAQFRSLD (SEQ ID NO:62) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO:63); LQRDGERP (SEQ ID NO:64); LRQKDGGGSERP (SEQ ID NO:65); LRQKD(GGGS)2 ERP (SEQ ID NO:66). Alternatively, flexible linkers can be rationally designed using computer programs that can model both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994) or by phage display methods. The linker may comprise the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO:67) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0196] The spacer domain distances the antigen binding domain from the surface of the effector cell, allowing proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The spacer domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The spacer domain may be an immunoglobulin portion that includes one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may include the amino acid sequence of a natural immunoglobulin hinge domain or a modified immunoglobulin hinge domain.

[0197] "Hinge domain" refers to a type of spacer domain that positions the antigen binding domain away from the surface of an effector cell and plays an important role in proper cell / cell contact, antigen binding, and activation. The hinge domain is located between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a natural immunoglobulin hinge domain or a modified immunoglobulin hinge domain.

[0198] A "modified hinge domain" refers to (a) a naturally occurring hinge domain having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge domain that is at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids) having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) a naturally occurring hinge domain that includes a core hinge domain (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). The hinge domain may be altered by substituting one or more cysteine ​​and / or proline residues in a naturally occurring immunoglobulin hinge domain with one or more other amino acid residues (eg, one or more serine residues).

[0199] "Transmembrane domain" refers to a portion of a polypeptide that fuses the extracellular domain to the intracellular domain and anchors the polypeptide to the plasma membrane of a cell. TM domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0200] "Intracellular signaling domain" refers to a polypeptide that is involved in transducing a message inside the immune system for effective binding of a target antigen by a receptor expressed on an immune effector cell to induce effector cell functions, e.g., activation, cytokine production, proliferation, cytotoxic activity, including release of cytotoxic factors or other cellular responses elicited by antigen binding to a receptor expressed on an immune effector cell.

[0201] The term "effector function" refers to a specialized function of an immune effector cell. The effector function of a T cell may be, for example, cytolytic activity or help or activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and instructs the cell to perform a specialized function. Although the entire intracellular signaling domain can usually be employed, it is often not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transduces an effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0202] It is known that signals generated through the TCR alone are insufficient for the complete activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains that act in an antigen-independent manner to provide secondary or costimulatory signals.

[0203] "Primary signaling domain" refers to a signaling domain that controls the primary activation of the TCR complex, either stimulatory or inhibitory. Primary signaling domains that act stimulatory may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0204] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or an Fc receptor, that upon binding to an antigen generates a secondary signal required for efficient activation and function of T lymphocytes.

[0205] The terms "selectively bind" or "selectively bound" or "selective binding" or "selectively target" describe the preferential binding of one molecule to a target molecule in the presence of multiple off-target molecules (on-target binding). In certain embodiments, a megaTAL targeting HE or PDCD1 or CBLB genes selectively binds to a DNA binding site on the target at about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold, or 1000-fold greater frequency than it binds to an off-target DNA target binding site.

[0206] "Site on target" refers to the target site sequence.

[0207] "Off-target" refers to a sequence that is similar, but not identical, to the target site sequence.

[0208] A "target site" or "target sequence" is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule binds and / or cleaves, provided that sufficient conditions for binding and / or cleavage exist. When referring to a polynucleotide sequence or SEQ ID NO: that refers to only one strand of a target site or target sequence, it is understood that the target site or target sequence that is bound and / or cleaved by a nuclease variant is double-stranded and includes the reference sequence and its complementary strand. In a preferred embodiment, the target site is a sequence of the human PDCD1 gene. In a preferred embodiment, the target site is a sequence of the human CBLB gene.

[0209] "Recombination" refers to the process of exchange of genetic information between two polynucleotides, including, but not limited to, non-homologous end joining (NHEJ) and donor capture by homologous recombination. For purposes of this disclosure, "homologous recombination (HR)" refers to a specific form of such exchange that occurs, for example, during the repair of double-strand breaks in cells via the homology-directed repair (HDR) mechanism. This process is known variously as "non-crossover gene conversion" or "short tract gene conversion" because it requires nucleotide sequence homology and uses the "donor" molecule as a template to repair the "target" molecule (i.e., the one that experienced the double-strand break), leading to the transfer of genetic information from the donor to the target. Without intending to be bound by any particular theory, such transfer may involve mismatch correction of the heteroduplex DNA formed between the destroyed target and the donor, and / or "synthesis-dependent strand annealing," in which the donor is used to resynthesize the genetic information that will become part of the target, and / or related processes. Such specified HR often results in an alteration of the sequence of the target molecule such that some or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0210] "NHEJ" or "non-homologous end joining" refers to the resolution of double-stranded breaks in the absence of a donor repair template or homologous sequence. NHEJ can result in insertions and deletions at the break site. NHEJ is mediated by several sub-pathways, each of which has distinct mutational consequences. The classical NHEJ pathway (cNHEJ) requires the KU / DNA-PKcs / Lig4 / XRCC4 complex, which finally religates with minimal processing, often leading to accurate repair of the break. Alternative NHEJ pathways (altNHEJ) are also effective in resolving dsDNA breaks, but these pathways are highly mutagenic and result in imprecise repair of breaks marked by insertions and deletions. Without intending to be bound by any particular theory, it is contemplated that modification of dsDNA breaks by end-processing enzymes such as exonucleases, e.g., Trex2, can increase the likelihood of imprecise repair.

[0211] "Cleavage" refers to the destruction of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible. Double-strand cleavage can result from two separate single-strand cleavage events. DNA cleavage can result in the generation of either blunt ends or staggered ends. In certain embodiments, the polypeptides and nuclease variants contemplated herein, such as homing endonuclease variants, megaTALs, etc., are used for targeted double-strand DNA cleavage. The endonuclease cleavage recognition site can be on either DNA strand.

[0212] A "foreign" molecule is a molecule that is not normally present in a cell, but is introduced into a cell by one or more genetic, biochemical or other methods. Exemplary foreign molecules include, but are not limited to, small organic molecules, proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complex containing one or more of the above molecules. Methods for introducing foreign molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, biolistics, biopolymer nanoparticles, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0213] An "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. Additional endogenous molecules can include proteins.

[0214] "Gene" refers to a DNA region that codes for a gene product, as well as all DNA regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequence. Genes include, but are not limited to, promoter sequences, enhancers, silencers, insulators, border regions, terminators, polyadenylation sequences, post-transcriptional response elements, translation control sequences such as ribosome binding sites and internal ribosome entry sites, origins of replication, substrate binding sites, and locus control regions.

[0215] "Gene expression" refers to the conversion of information contained within a gene into a gene product. A gene product can be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNA that is modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0216] As used herein, the term "genome editing" refers to the replacement, deletion, and / or introduction of genetic material at a target site in the genome of a cell, which restores, corrects, disrupts, and / or alters the expression of a gene or gene product. In certain embodiments, genome editing contemplated includes introducing one or more nuclease variants, including but not limited to homing endonuclease variants or megaTALs, into a cell to generate a DNA lesion at or near a target site in the genome of the cell, optionally in the presence of a donor repair template.

[0217] Additional definitions are set forth throughout this disclosure.

[0218] C. Chimeric antigen receptor In various embodiments, the immune effector cells are engineered to express a chimeric antigen receptor (CAR) that targets CD79A and a chimeric costimulatory receptor that targets CD20 to direct the cytotoxicity of the immune effector cells to cancer cells that express CD79A and / or CD20. In various embodiments, the immune effector cells are engineered to express an anti-CD79 CAR and an anti-CD20 CCR, and the cells also have one or more genomic edits that reduce CBLB and / or PDCD-1 function and expression.

[0219] Chimeric antigen receptors (CARs) are molecules that combine antibody-based specificity for a desired antigen with T cell receptor-activated intracellular domains to produce chimeric proteins that exhibit antigen-specific cellular immune activity. As used herein, the term "chimera" refers to something that is composed of different protein or DNA parts from different sources. In certain embodiments, CARs include an extracellular domain that binds to CD79A (also called a binding domain or an antigen-specific binding domain), a transmembrane domain, a costimulatory signaling domain, and a primary signaling domain. The main feature of CARs is their ability to redirect the specificity of immune effector cells, which induces the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner, eliciting the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific coreceptors.

[0220] In various embodiments, the CAR comprises an extracellular binding domain comprising a CD79A-specific binding domain, a transmembrane domain, a costimulatory signaling domain, and / or a primary signaling domain.

[0221] In certain embodiments, a CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof, an extracellular binding domain comprising one or more hinge or spacer domains, a transmembrane domain, and a costimulatory signaling domain and / or a primary signaling domain.

[0222] In certain embodiments, the CAR comprises an extracellular binding domain that comprises an anti-CD79A antibody or antigen-binding fragment thereof that specifically binds to a human CD79A polypeptide expressed on a target cell, e.g., a cancer cell.

[0223] In certain embodiments, a CD79A-specific binding domain comprises a light chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to a light chain CDR sequence set forth in SEQ ID NOs: 1-3 or 9-11. In certain embodiments, a CD79A-specific binding domain comprises a light chain CDR sequence set forth in SEQ ID NOs: 1-3 or 9-11.

[0224] In certain embodiments, a CD79A-specific binding domain comprises a heavy chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to a heavy chain CDR sequence set forth in SEQ ID NO: 4-6 or 12-14. In one embodiment, a CD79A-specific binding domain comprises a heavy chain CDR sequence set forth in SEQ ID NO: 4-6 or 12-14.

[0225] In a specific embodiment, the antigen-specific binding domain is an scFv that binds to a human CD79A polypeptide.

[0226] In a particular embodiment, the antigen-specific binding domain is a humanized camelid-like VHH that binds to a human CD79A polypeptide.

[0227] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising a CDRL1-CDRL3 sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NOs: 1-3 or 9-11, and / or a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NOs: 4-6 or 12-14.

[0228] In various embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequence set forth in SEQ ID NO:1-3 or 9-11, and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence set forth in SEQ ID NO:4-6 or 12-14.

[0229] In a preferred embodiment, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in any one of SEQ ID NOs:7 or 15, and a variable heavy chain sequence set forth in any one of SEQ ID NOs:8 or 16.

[0230] In certain embodiments, the anti-CD79A CAR comprises linker residues between the various domains, e.g., added for proper spacing and structure of the molecule. In certain embodiments, the linker is a sequence that connects the variable regions. The anti-CD79A CAR may comprise one, two, three, four, or five or more linkers. In certain embodiments, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 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 or more amino acids long. Exemplary linkers include, but are not limited to, linkers encoded by SEQ ID NOs: 57-67.

[0231] In certain embodiments, the binding domain of the anti-CD79A CAR is followed by one or more spacer domains. In a preferred embodiment, the spacer domain is between the antigen binding domain and the transmembrane domain. In one embodiment, the spacer domain contains the CH2 and CH3 of IgG1, IgG4 or IgD.

[0232] In some embodiments, the binding domain of an anti-CD79A CAR is followed by one or more "hinge domains" that serve to position the antigen binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation. Exemplary hinge domains suitable for use in the CARs described herein include hinge domains derived from the extracellular regions of type 1 membrane proteins, such as CD8α and CD4, which may be the wild-type hinge domains of these molecules or may be modified. In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge. In a preferred embodiment, the hinge domain contains a CD8α hinge domain.

[0233] In preferred embodiments, the anti-CD79A CAR is derived from, or may comprise, at least the transmembrane region of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In certain embodiments, the TM domain is synthetic and comprises primarily hydrophobic residues such as leucine and valine.

[0234] In one embodiment, the anti-CD79A CAR comprises a TM domain from PD1, CD152, CD28, or CD8α. In another embodiment, the anti-CD79A CAR contains a TM domain from PD1, CD152, CD28, or CD8α, and a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, which links the TM domain to the intracellular signaling domain of the CAR. In a preferred embodiment, the TM domain is derived from CD8α.

[0235] In certain embodiments, the anti-CD79A CAR comprises one or more intracellular signaling domains. In a particularly preferred embodiment, the anti-CD79A CAR comprises a costimulatory signaling domain and a primary signaling domain. The intracellular primary signaling domain and the costimulatory signaling domain can be linked in tandem in any order to the carboxyl terminus of the transmembrane domain.

[0236] In certain embodiments, the anti-CD79A CAR comprises a costimulatory domain isolated from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), C D137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0237] In a preferred embodiment, the anti-CD79A CAR comprises a CD28, CD137, or CD134 costimulatory signaling domain.

[0238] In a specific embodiment, the anti-CD79A CAR comprises an ITAM containing a primary signaling domain isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0239] In a preferred embodiment, the anti-CD79A CAR comprises a CD3ζ primary signaling domain.

[0240] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof that specifically binds to a human CD79A polypeptide expressed on a cancer cell.

[0241] In one embodiment, an anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment that binds to a CD79A polypeptide and comprises a transmembrane domain derived from a polypeptide selected from the group consisting of: the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN1, and PD1; and one or more intracellular costimulatory signaling domains derived from a costimulatory molecule selected from the group consisting of: TLR1, and primary signaling domains from TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNRFS25, and ZAP70; and primary signaling domains from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0242] In one embodiment, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide and comprises a hinge domain selected from the group consisting of: IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, PD1 hinge, CD152 hinge, and CD8α hinge; and comprises a transmembrane domain derived from a polypeptide selected from the group consisting of: the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN1, and PD1; comprising one or more intracellular costimulatory signaling domains derived from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), C D83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNRFS25, and ZAP70; and primary signaling domains from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0243] In one embodiment, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide and comprises a hinge domain selected from the group consisting of: IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, PD1 hinge, CD152 hinge, and CD8α hinge; and a transmembrane domain derived from a polypeptide selected from the group consisting of: the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN1, and PD1; comprising a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the TM domain to the intracellular signaling domain of the CAR; comprising one or more intracellular costimulatory signaling domains derived from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CLR11, CLR12, CLR13, CLR14, CLR15, CLR16, CLR17, CLR18, CLR19, CLR20, CLR21, CLR22, CLR23, CLR24, CLR25, CLR26, CLR27, CLR28, CLR29, CLR30, CLR31, CLR32, CLR33, CLR34, CLR35, CLR36, CLR37, CLR38, CLR39, CLR40, CLR41, CLR42, CLR43, CLR44, CLR45, CLR46, CLR47, CLR48, CLR49, CLR50, CLR51, CLR52, CLR53, CLR54, CLR55, CLR56, CLR57, CLR58, CLR59, CLR60, CLR61, CLR62, CLR63, CLR64, CLR65, CLR66, CLR67, CLR68, CLR69, CLR70, CLR71, CLR72, CLR73, CLR74, CLR75, CLR76, CLR77, CLR78, CLR79, CLR ARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNRFS25, and ZAP70; and primary signaling domains from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0244] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide, comprises a hinge domain comprising an IgG1 hinge / CH2 / CH3 polypeptide and a CD8α hinge domain, comprises a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids, comprises a CD137 intracellular costimulatory signaling domain, and comprises a CD3ζ primary signaling domain.

[0245] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide, comprises a CD8α hinge domain, comprises a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids, comprises a CD134 intracellular costimulatory signaling domain, and comprises a CD3ζ primary signaling domain.

[0246] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide, comprises a CD8α hinge domain, comprises a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids, comprises a CD28 intracellular costimulatory signaling domain, and comprises a CD3ζ primary signaling domain.

[0247] In certain embodiments, an anti-CD79A CAR comprises one or more anti-CD79A VHHs that bind to a CD79A polypeptide, comprises a CD8α hinge domain, comprises a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids, comprises a CD28 intracellular costimulatory signaling domain, and comprises a CD3ζ primary signaling domain.

[0248] D. Chimeric Costimulatory Receptors In various embodiments, the immune effector cells are modified to express anti-CD79A and anti-CD20 CCRs to direct the cytotoxicity of the immune effector cells to cancer cells expressing CD79A and / or CD20, and synergistically enhance the efficacy of immune effector cell therapy. In various embodiments, the immune effector cells are modified to express anti-CD79A and anti-CD20 CCRs to direct the cytotoxicity of the immune effector cells to cancer cells expressing CD79A and / or CD20, and further include one or more gene edits that disrupt or eliminate the function and / or activity of PDCD-1 and / or CBLB, and synergistically enhance the efficacy of immune effector cell therapy.

[0249] Chimeric costimulatory receptors (CCRs) are molecules that combine antibody-based specificity for a desired antigen with a T cell receptor costimulatory domain, but lack a primary signaling domain. In certain embodiments, CCRs include an extracellular domain (also called a binding domain or an antigen-specific binding domain) that binds to CD20, a transmembrane domain, and a costimulatory signaling domain and lacks a primary signaling domain. The main feature of CCRs is their ability to redirect the specificity of immune effector cells in an MHC-independent manner and enhance immune effector cell responses in the presence of CAR.

[0250] In various embodiments, the CCR comprises a CD20-specific binding domain, a transmembrane domain, and an extracellular binding domain that includes a costimulatory signaling domain, but does not include a primary signaling domain.

[0251] In certain embodiments, the CCR comprises an extracellular binding domain comprising an anti-CD20 antibody or antigen-binding fragment thereof, one or more hinge or spacer domains, a transmembrane domain, and a costimulatory signaling domain, but not a primary signaling domain.

[0252] In certain embodiments, the CCR comprises an extracellular binding domain that comprises an anti-CD20 antibody or antigen-binding fragment thereof that specifically binds to a human CD20 polypeptide expressed on a target cell, e.g., a cancer cell.

[0253] In certain embodiments, the CD20-specific binding domain comprises a light chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to a light chain CDR sequence set forth in SEQ ID NOs: 25-27. In certain embodiments, the CD20-specific binding domain comprises a light chain CDR sequence set forth in SEQ ID NOs: 25-27.

[0254] In certain embodiments, the CD20-specific binding domain comprises a heavy chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the heavy chain CDR sequences set forth in SEQ ID NOs: 28-30. In one embodiment, the CD20-specific binding domain comprises a heavy chain CDR sequence set forth in SEQ ID NOs: 28-30.

[0255] In a specific embodiment, the antigen-specific binding domain is an scFv that binds to a human CD20 polypeptide.

[0256] In a particular embodiment, the antigen-specific binding domain is a humanized camelid-like VHH that binds to a human CD20 polypeptide.

[0257] In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising a CDRL1-CDRL3 sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NOs:25-27, and / or a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NOs:28-30.

[0258] In various embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequences set forth in SEQ ID NOs:25-27, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences set forth in SEQ ID NOs:28-30.

[0259] In a preferred embodiment, the anti-CD20 antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:31 and / or the variable heavy chain sequence set forth in SEQ ID NO:32.

[0260] In certain embodiments, the anti-CD20 CCRs include linker residues between the various domains, e.g., added for proper spacing and structure of the molecule. In certain embodiments, the linker is a sequence that joins the variable regions. The anti-CD20 CCRs can include one, two, three, four, or five or more linkers. In certain embodiments, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids.

[0261] In certain embodiments, the binding domain of the anti-CD20 CCR is followed by one or more spacer domains. In preferred embodiments, the spacer domain is between the antigen binding domain and the transmembrane domain. In one embodiment, the spacer domain contains the CH2 and CH3 of IgG1, IgG4 or IgD.

[0262] In some embodiments, the binding domain of an anti-CD20 CCR is followed by one or more "hinge domains" that serve to position the antigen binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding and activation. Exemplary hinge domains suitable for use in the CCRs described herein include hinge domains derived from the extracellular regions of type 1 membrane proteins, such as CD8α and CD4, which may be the wild-type hinge domains of these molecules or may be modified. In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge. In a preferred embodiment, the hinge domain contains a CD8α hinge domain.

[0263] Anti-CD20 CCRs may be derived from, or comprise, at least the transmembrane regions of the alpha or beta chains of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In certain embodiments, the TM domain is synthetic and comprises primarily hydrophobic residues such as leucine and valine.

[0264] In one embodiment, the anti-CD20 CCR comprises a TM domain derived from PD1, CD152, CD28, or CD8α. In another embodiment, the anti-CD20 CCR comprises a TM domain derived from PD1, CD152, CD28, or CD8α, the linker comprising a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, which links the TM domain to the intracellular signaling domain of the CCR. In a preferred embodiment, the TM domain is derived from CD8α.

[0265] In certain embodiments, the anti-CD20 CCR comprises one or more intracellular signaling domains. In particularly preferred embodiments, the anti-CD20 CCR comprises one or more costimulatory signaling domains but lacks a primary signaling domain. The costimulatory signaling domains may be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0266] In certain embodiments, the anti-CD20 CCR comprises a costimulatory domain isolated from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), C D137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), linker for activation of T-cell family member 1 (LAT), SH2 domain-containing leukocyte protein of 76 kD (SLP76), T-cell receptor-associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70).

[0267] In a preferred embodiment, the anti-CD79A CAR comprises a CD28, CD137, or CD134 costimulatory signaling domain and the anti-CD20 CCR comprises a different costimulatory domain.

[0268] In a preferred embodiment, the anti-CD79A CAR comprises a CD137 or CD134 costimulatory signaling domain and the anti-CD20 CCR comprises a CD28 costimulatory signaling domain.

[0269] In a preferred embodiment, the anti-CD79A CAR comprises a CD137 costimulatory signaling domain and the anti-CD20 CCR comprises a CD28 costimulatory signaling domain.

[0270] In certain embodiments, the anti-CD20 CCR comprises an anti-CD20 antibody or antigen-binding fragment thereof that specifically binds to a CD20 polypeptide expressed on cancer cells.

[0271] In one embodiment, the anti-CD20 CCR comprises an anti-CD20 scFv that binds to a CD20 polypeptide and comprises a transmembrane domain derived from a polypeptide selected from the group consisting of: the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN1, and PD1; Contains stimulatory signaling domains: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNRFS25, and ZAP70.

[0272] In one embodiment, the anti-CD20 CCR comprises an anti-CD20 scFv that binds to a CD20 polypeptide and comprises a hinge domain selected from the group consisting of: IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, PD1 hinge, CD152 hinge, and CD8α hinge; and comprises a transmembrane domain derived from a polypeptide selected from the group consisting of: the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN1, and PD1; comprising a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the TM domain to the intracellular signaling domain of the CAR; comprising one or more intracellular costimulatory signaling domains derived from a costimulatory molecule selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD94, CD1 34(OX40), CD137(4-1BB), CD278(ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNRFS25, and ZAP70.

[0273] In certain embodiments, the anti-CD20 CCR comprises an anti-CD20 scFv that binds to a CD20 polypeptide, comprises a hinge domain comprising an IgG1 hinge / CH2 / CH3 polypeptide and a CD8α hinge domain, comprises a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids, and comprises a CD28 intracellular costimulatory signaling domain.

[0274] In certain embodiments, the anti-CD20 CCR comprises an anti-CD20 scFv that binds to a CD20 polypeptide, comprises a CD8α hinge domain, comprises a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids, and comprises a CD28 intracellular costimulatory signaling domain.

[0275] E. Nuclease variants In various embodiments, immune effector cells engineered to express anti-CD79A CAR and anti-CD20 CCR are also genetically engineered to reduce or eliminate expression and / or function of PDCD-1 and / or CBLB, for example, using nuclease variants such as homing endonuclease (meganuclease) variants or megaTALs. The nuclease variants contemplated in certain embodiments herein are suitable for genome editing of target sites in the human PDCD-1 gene or human CBLB gene and include one or more DNA binding domains and one or more DNA cleavage domains (e.g., one or more endonuclease domains and / or exonuclease domains), and optionally one or more linkers as contemplated herein. The terms "reprogrammed nuclease," "engineered nuclease," or "nuclease variant" are used interchangeably and refer to a nuclease that contains one or more DNA binding domains and one or more DNA cleavage domains, where the nuclease has been designed and / or modified from a parent or naturally occurring nuclease to bind and cleave a double-stranded DNA target sequence.

[0276] In a particular embodiment, the nuclease variant binds and cleaves a target sequence in exon 1 of the PDCD-1 gene, preferably in SEQ ID NO: 51 in exon 1 of the PDCD-1 gene, more preferably in the sequence "ATCC" in SEQ ID NO: 51 in exon 1 of the PDCD-1 gene.

[0277] In a preferred embodiment, the nuclease variant binds and cleaves the target sequence "ATCC" in exon 6 of the CBLB gene, preferably in SEQ ID NO: 55 in exon 6 of the CBLB gene, more preferably in SEQ ID NO: 55 in exon 6 of the CBLB gene.

[0278] Nuclease variants may be designed and / or engineered from naturally occurring nucleases or from previous nuclease variants. In certain embodiments, contemplated nuclease variants may further comprise one or more additional functional domains, such as a 5' to 3' exonuclease, a 5' to 3' alkaline exonuclease, a 3' to 5' exonuclease (e.g., Trex2), a 5' flap endonuclease, a helicase, an endoprocessing enzyme domain of an endoprocessing enzyme exhibiting template-dependent DNA polymerase or template-independent DNA polymerase activity.

[0279] Examples of nuclease variants that bind to and cleave target sequences in the PDCD-1 or CBLB genes include, but are not limited to, homing endonuclease (meganuclease) variants and megaTALs.

[0280] 1. Homing endonuclease (meganuclease) variants In various embodiments, the homing endonuclease or meganuclease is reprogrammed to introduce a double-stranded break (DSB) at a target site in the PDCD-1 gene or the CBLB gene. In various embodiments, the homing endonuclease or meganuclease is reprogrammed to introduce a double-stranded break (DSB) at a target site in the CBLB gene.

[0281] In a particular embodiment, the homing endonuclease variant introduces a double-stranded break in exon 1 of the PDCD-1 gene, preferably at SEQ ID NO: 51 in exon 1 of the PDCD-1 gene, more preferably at the sequence "ATCC" at SEQ ID NO: 51 in exon 1 of the PDCD-1 gene.

[0282] In a preferred embodiment, the homing endonuclease variant introduces a double-stranded break in exon 6 of the CBLB gene, preferably at SEQ ID NO: 55 in exon 6 of the CBLB gene, more preferably at the sequence "ATCC" at SEQ ID NO: 55 in exon 6 of the CBLB gene.

[0283] "Homing endonucleases" and "meganucleases" are used interchangeably and refer to naturally occurring homing endonucleases that recognize cleavage sites of 12 to 45 base pairs and are commonly grouped into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK.

[0284] A "reference homing endonuclease" or "reference meganuclease" refers to a wild-type homing endonuclease or homing endonuclease found in nature. In one embodiment, a "reference homing endonuclease" refers to a wild-type homing endonuclease that has been modified to increase basal activity.

[0285] "Engineered homing endonuclease," "reprogrammed homing endonuclease," "homing endonuclease variant," "engineered meganuclease," "reprogrammed meganuclease," or "meganuclease variant" refers to a homing endonuclease that contains one or more DNA binding domains and one or more DNA cleavage domains, and which is designed and / or modified from a parent or naturally occurring homing endonuclease to cleave and bind to and cleave a DNA target sequence. A homing endonuclease variant may be designed and / or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant. Homing endonuclease variants contemplated in certain embodiments may further comprise one or more additional functional domains, such as an endo-processing enzyme domain of an endo-processing enzyme that exhibits 5' to 3' exonuclease, 5' to 3' alkaline exonuclease, 3' to 5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerase activity.

[0286] Homing endonuclease (HE) variants do not exist in nature and can be obtained by recombinant DNA technology or by random mutagenesis. HE variants may be obtained by making one or more amino acid changes in naturally occurring HE or HE variants, for example, by mutating, substituting, adding, or deleting one or more amino acids. In certain embodiments, HE variants include one or more amino acid changes to the DNA recognition interface.

[0287] In a specific embodiment, the homing endonuclease is an I-OnuI HE variant that binds to and cleaves the human PDCD-1 gene set forth in SEQ ID NO:51 and comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:49, or a biologically active fragment thereof.

[0288] In a preferred embodiment, the homing endonuclease is an I-OnuI HE variant that binds to and cleaves the human CBLB gene set forth in SEQ ID NO:55 and comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:53, or a biologically active fragment thereof.

[0289] 2. MegaTAL In various embodiments, the megaTALs containing the homing endonuclease variants are reprogrammed to introduce a double-stranded break (DSB) at a target site in the PDCD-1 gene or the CBLB gene. In various embodiments, the megaTALs containing the homing endonuclease variants are reprogrammed to introduce a double-stranded break (DSB) at a target site in the CBLB gene.

[0290] In a particular embodiment, the megaTAL introduces a DSB into exon 1 of the PDCD-1 gene, preferably into sequence number 52 in exon 1 of the PDCD-1 gene, more preferably into the sequence "ATCC" of sequence number 52 in exon 1 of the PDCD-1 gene.

[0291] In a preferred embodiment, the megaTAL introduces a DSB in exon 6 of the CBLB gene, preferably at SEQ ID NO: 56 in exon 6 of the CBLB gene, more preferably at the sequence "ATCC" in SEQ ID NO: 56 in exon 6 of the CBLB gene.

[0292] "megaTAL" refers to a polypeptide comprising a TALE DNA binding domain and a homing endonuclease variant that binds to and cleaves a DNA target sequence in a gene and optionally comprises one or more linkers and / or additional functional domains, e.g., a 5' to 3' exonuclease, a 5' to 3' alkaline exonuclease, a 3' to 5' exonuclease (e.g., Trex2), a 5' flap endonuclease, a helicase or an endo-processing enzyme domain that exhibits template-independent DNA polymerase activity.

[0293] A "TALE DNA-binding domain" is the DNA-binding portion of a transcription activator-like effector (TALE or TAL effector) that mimics plant transcription activators and manipulates the plant transcriptome (see, e.g., Kay et al., 2007, Science 318:648-651). In certain embodiments, TALE DNA binding domains contemplated are engineered from novel or naturally occurring TALEs, such as AvrBs3 from Xanthomonas spot blight, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae, and brg11 and hpx17 from Ralstonia solanacearum. Examples of TALE protein deriving and engineering DNA binding domains are disclosed in U.S. Patent No. 9,017,967, and the references cited therein, all of which are incorporated by reference in their entirety herein.

[0294] In a specific embodiment, the megaTAL binds to and cleaves the target site of the human PDCD-1 gene set forth in SEQ ID NO:52, and comprises the amino acid sequence set forth in SEQ ID NO:50.

[0295] In a preferred embodiment, the megaTAL binds to and cleaves the target site of the human CBLB gene of SEQ ID NO:56 and comprises the amino acid sequence set forth in SEQ ID NO:54.

[0296] F. Polypeptides A variety of polypeptides, fusion polypeptides, and polypeptide variants are contemplated herein, including, but not limited to, CAR polypeptides, CCR polypeptides, CAR-2A-CCR fusion polypeptides, CAR-2A-CAR fusion polypeptides and fragments thereof, homing endonucleases, and megaTALs. In certain embodiments, exemplary polypeptides contemplated herein include polypeptides comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-20, 25-33, 35, 37, 39, 41, 43, 45, and 47.

[0297] "Polypeptide," "peptide," and "protein" are used interchangeably and follow their conventional meanings unless specified to the contrary, i.e., as amino acid sequences. A polypeptide is not limited to a particular length, e.g., a polypeptide may contain a full-length polypeptide or a polypeptide fragment, and a polypeptide may include one or more post-translational modifications of a polypeptide, e.g., glycosylation, acetylation, phosphorylation, and other modifications, both natural and non-natural, known in the art.

[0298] As used herein, "isolated polypeptide" and the like refers to the in vitro synthesis, isolation and / or purification of a peptide or polypeptide molecule from the cellular environment and from association with other components of a cell, i.e., not substantially associated with in vivo substances. In certain embodiments, an isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0299] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above-mentioned polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of the CAR and / or CCR by introducing one or more substitutions, deletions, additions, and / or insertions into the binding domain, hinge, TM domain, costimulatory signaling domain, or, if present, the primary signaling domain. In certain embodiments, the polypeptide comprises a polypeptide having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, and typically the variant maintains at least one biological activity of the reference sequence. In certain embodiments, the biological activity is binding affinity. In certain embodiments, the biological activity is cytolytic activity.

[0300] Polypeptides include "polypeptide fragments." A polypeptide fragment may be monomeric or multimeric and refers to a biologically active polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. Examples of biologically active polypeptide fragments include antibody fragments. As used herein, the term "biologically active fragment" or "minimal biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In certain embodiments, a polypeptide fragment may contain an amino acid chain of at least 5 to about 500 amino acids in length. In certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 or more amino acids in length. Particularly useful polypeptide fragments, in certain embodiments, contain functional domains, including antigen binding domains or fragments of antibodies.

[0301] The polypeptides may also be fused in frame or attached to linkers or other sequences for ease of synthesis, purification, or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.

[0302] As mentioned above, in certain embodiments, polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of reference polypeptides can be generated by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, JD et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0303] In some embodiments, the polypeptide variant contains one or more conservative substitutions. A "conservative substitution" is a substitution in which an amino acid is replaced with another amino acid having similar properties, and one skilled in the art of peptide chemistry would expect that such a substitution will not substantially change the secondary structure and hydrophobicity of the polypeptide. In certain embodiments, modifications may be made to the expected polynucleotide and polypeptide structures, and still obtain functional molecules that code for variant or derivative polypeptides with desired characteristics. When it is desired to change the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, one skilled in the art can change one or more of the codons of the coding DNA sequence, for example according to Table 1. [Table 1]

[0304] Guidance for determining which amino acid residues can be substituted, inserted or deleted without abolishing biological activity can be found using computer programs known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. The amino acid changes in the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of a family of amino acids that are related at their side chains. Natural amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified jointly as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without changing the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly change biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.Co., p.224).

[0305] As discussed above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0306] Polypeptide variants also include glycosylated forms, aggregate conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules).Covalent variants can be prepared by linking functional groups to groups present in amino acid chains or in N- or C-terminal residues, as known in the art.Variants also include allelic variants, species variants, and mutant proteins.Truncation or deletion of regions that do not affect the functional activity of protein are also variants.

[0307] In certain embodiments, expression of the CAR and CCR in the same cell is desired. The polynucleotide sequences encoding the CAR and CCR may be separated by an IRES sequence, as discussed elsewhere herein.

[0308] In a preferred embodiment, fusion polypeptides are contemplated herein.

[0309] In certain preferred embodiments, the CAR and CCR may be expressed as a fusion polypeptide comprising one or more self-cleaving polypeptide sequences separating the CAR and CCR.

[0310] Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polypeptide segments. Fusion polypeptides are typically linked C-terminus to N-terminus, but can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or M-terminus to C-terminus. The polypeptides of a fusion protein can be in any order or specified order. In one embodiment, the fusion protein comprises a CAR, a polypeptide cleavage signal, and a CCR. In another embodiment, the fusion protein comprises a CCR, a polypeptide cleavage signal, and a CAR.

[0311] In a preferred embodiment, the fusion protein comprises an anti-CD79A CAR, a polypeptide cleavage signal, and an anti-CD20 CCR. In a preferred embodiment, the fusion protein comprises an anti-CD20 CCR, a polypeptide cleavage signal, and an anti-CD79A CCR.

[0312] In certain embodiments, the fusion protein comprises an anti-CD79A CAR comprising a variable light chain sequence comprising the CDRL1-CDRL3 sequence set forth in SEQ ID NOs: 1-3 or 9-11, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence set forth in SEQ ID NOs: 4-6 or 12-14; a polypeptide cleavage signal; and an anti-CD20 CCR comprising a variable light chain sequence comprising the CDRL1-CDRL3 sequence set forth in SEQ ID NOs: 25-27, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence set forth in SEQ ID NOs: 28-30.

[0313] In a particular embodiment, the fusion polypeptide comprises an anti-CD79A CAR comprising the variable light chain sequence set forth in SEQ ID NO:7, and / or the variable heavy chain sequence set forth in SEQ ID NO:8; a polypeptide cleavage signal; and an anti-CD20 CCR comprising the variable light chain sequence set forth in SEQ ID NO:31 and the variable heavy chain sequence set forth in SEQ ID NO:32.

[0314] In certain embodiments, the fusion protein comprises an anti-CD79A CAR comprising a variable light chain sequence comprising a CDRL1-CDRL3 sequence as set forth in SEQ ID NOs: 1-3 or 9-11, and a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence as set forth in SEQ ID NOs: 4-6 or 12-14; a CD8α hinge and transmembrane domain, a CD137 costimulatory domain and a CD3ζ primary signaling domain; a polypeptide cleavage signal, and an anti-CD20 CCR comprising a variable light chain sequence comprising a CDRL1-CDRL3 sequence as set forth in SEQ ID NOs: 25-27, and a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence as set forth in SEQ ID NOs: 28-30, a CD8α hinge and transmembrane domain, and a 4-1BB costimulatory domain.

[0315] In certain embodiments, the fusion polypeptide comprises an anti-CD79A CAR comprising the variable light chain sequence set forth in SEQ ID NO:7, and / or the variable heavy chain sequence set forth in SEQ ID NO:8; a CD8α hinge and transmembrane domain, a CD137 costimulatory domain and a CD3ζ primary signaling domain; a polypeptide cleavage signal, and an anti-CD20 CCR comprising the variable light chain sequence set forth in SEQ ID NO:31 and the variable heavy chain sequence set forth in SEQ ID NO:32, a CD8α hinge domain; a CD8α transmembrane domain, and a 4-1BB costimulatory domain.

[0316] Examples of polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8);616-26).

[0317] Suitable protease cleavage sites and autocleaving peptides are known to those of skill in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to their high cleavage stringency, in one embodiment TEV (Tobacco Etch Virus) protease cleavage sites are preferred, e.g., EXXYXQ(G / S) (SEQ ID NO:68), e.g., ENLYFQG (SEQ ID NO:69) and ENLYFQS (SEQ ID NO:70), where X represents any amino acid (cleavage by TEV occurs between Q and G or between Q and S).

[0318] In certain embodiments, the polypeptide cleavage signal is a viral autocleaving peptide or a ribosomal skipping sequence.

[0319] Examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (see Donnelly et al., 2001, J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0320] In one embodiment, the viral 2A peptide is selected from the group consisting of: foot and mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, Thosea asigna virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, theirovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0321] Examples of 2A sites are provided in Table 2. [Table 2]

[0322] In a preferred embodiment, the fusion polypeptide comprises an anti-CD79A CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 17 to 20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR comprising the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 35.

[0323] In certain of these preferred embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37 or SEQ ID NO:39.

[0324] G. Polynucleotides In a preferred embodiment, a polynucleotide is provided that encodes one or more CAR polypeptides, CCR polypeptides, or a fusion polypeptide that includes CAR, 2A peptide, and CCR. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded, and can be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotide refers to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, including ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, "intermediate length" will be readily understood to mean any length between the cited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, a polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 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% sequence identity to a reference sequence.

[0325] Illustrative examples of polynucleotides include, but are not limited to, polynucleotides that encode SEQ ID NOs: 21-24, 34, 36, 38, 40, 42, 44, 46, and 48, as well as SEQ ID NOs: 1-20, 25-33, 35, 37, 39, 41, 43, 45, and 47.

[0326] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from adjacent sequences in nature, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. In certain embodiments, an "isolated polynucleotide" also refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that does not occur in nature and is produced by man. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0327] In various embodiments, the polynucleotide comprises an mRNA that encodes a polypeptide contemplated herein, hi some embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0328] In certain embodiments, a polynucleotide may be codon-optimized. As used herein, the term "codon optimization" refers to the substitution of codons in a polynucleotide encoding a polypeptide to increase the expression, stability and / or activity of the polypeptide. Factors that affect codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene or set of genes, (iii) systematic variation of codons with their context, (iv) variation of codons with their decoding tRNA, (v) variation of codons with GC% either across the triplet or at one position of the triplet, (vi) variation in similarity to a reference sequence, e.g., a natural sequence, (vii) variation in codon frequency cutoff, (viii) structural properties of mRNA transcribed from the DNA sequence, (ix) prior knowledge of the function of the DNA sequence that is the basis for the design of the codon substitution set, (x) synthetic variation of the codon set for each amino acid, and / or (xi) isolated removal of incorrect translation start positions.

[0329] As used herein, terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits a comparable sequence identity with a reference polynucleotide sequence, or hybridizes with a reference sequence under stringent conditions as defined herein.These terms include polynucleotides in which one or more nucleotides are added or deleted or replaced with different nucleotides compared to the reference polynucleotide.In this regard, it is understood in the art that certain modifications, including mutations, additions, deletions and substitutions, can be made to the reference polynucleotide, and the modified polynucleotide can retain the biological function or biological activity of the reference polynucleotide.

[0330] Polynucleotide variants include polynucleotide fragments that encode biologically active polypeptide fragments or variants. As used herein, the term "polynucleotide fragment" refers to a polynucleotide fragment that encodes a polypeptide that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of a naturally occurring polypeptide, including at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, It refers to a polynucleotide fragment having a length of 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides. A polynucleotide fragment refers to a polynucleotide that encodes a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, a carboxy-terminal deletion, and / or an internal deletion or substitution of one or more amino acids of a naturally-occurring or recombinantly produced polypeptide.

[0331] "Sequence identity", or phrases such as "50% identical sequence to", as used herein, refer to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window.Thus, "percentage of sequence identity" may be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) are present in both sequences to calculate the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size); and multiplying the result by 100 to calculate the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein, and typically the polypeptide variants retain at least one biological activity of the reference polypeptide.

[0332] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" comprises nucleotides and amino acid residues that are at least 12 monomeric units in length, and often 15-18 monomeric units, and often at least 25 monomeric units in length. Two polynucleotides may each contain (1) sequences that are similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) sequences that diverge between the two polynucleotides, and sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides in a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more commonly about 100 to about 150, and a sequence is compared to a reference sequence in the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may contain about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for optimal alignment of two sequences. Optimal alignment of sequences for aligning the comparison window can be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, or by inspection and best alignment (i.e., resulting in the highest homology over the comparison window) generated by any of the various methods selected. For example, see the BLAST family of programs disclosed by Altschul et al., 1997, Nucl.Acids Res.25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15, Unit 19.3.

[0333] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). A polynucleotide sequence may be annotated in a 5' to 3' or 3' to 5' direction. For DNA and mRNA, the 5' to 3' strand is designated the "sense", "plus", or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated as the "template", "antisense", "minus", or "non-coding" strand. As used herein, the term "reverse" refers to a 5' to 3' sequence written in a 3' to 5' direction or a 3' to 5' sequence written in a 5' to 3' direction.

[0334] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complementary strand of the DNA sequence 5' AGTCATG 3' is 3' TCAGTAC 5'. The latter sequence is often written as the reverse complement, 5' CATGACT 3', with the 5' end on the left and the 3' end on the right. A sequence equivalent to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," where only a portion of the nucleic acid bases match according to the base-pairing rules. Alternatively, there is "complete" or "total" complementarity between the nucleic acids.

[0335] Furthermore, those skilled in the art will recognize that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the polypeptides described herein or variant fragments thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are contemplated, and in certain embodiments, polynucleotides that are optimized for, for example, human and / or primate codon preferences are contemplated. Furthermore, alleles of genes that comprise the polynucleotide sequences provided herein may also be used. An allele is an endogenous gene that is altered as a result of one or more mutations, such as deletion, addition and / or substitution of nucleotides.

[0336] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence in a vector capable of expressing an RNA and subsequently expressing a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassettes are positionally and sequentially oriented within the vector, such that the nucleic acid in the cassette can be transcribed into RNA, translated into a protein or polypeptide if necessary, subjected to appropriate post-translational modifications required for activity in the transformed cell, targeted to an appropriate intracellular compartment for translocation to a compartment suitable for biological activity, or secreted into an extracellular compartment. The cassette preferably has a 3' and 5' end adapted for immediate insertion into the vector, e.g., a restriction endonuclease site at each end. In a preferred embodiment, the nucleic acid cassette encodes an anti-CD79A CAR and an anti-CD20 CCR. The cassette can be extracted and inserted as a single unit into a plasmid or viral vector.

[0337] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide, a polypeptide variant, or a fusion polypeptide. A vector may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In certain embodiments, the polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or injury. Polynucleotides of interest, and the polypeptides encoded therefrom, include both polynucleotides that encode wild-type polypeptides, as well as functional variants and fragments. In certain embodiments, functional variants have at least 80%, at least 90%, at least 95%, or at least 99% identity with the corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, functional variants or fragments have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the corresponding wild-type polypeptide.

[0338] The polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., disclosed elsewhere herein or known in the art, so that their overall length may vary significantly. Thus, polynucleotide fragments of almost any length may be used in certain embodiments, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0339] Polynucleotides may be prepared, manipulated, and / or expressed using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide may be inserted into an appropriate vector.

[0340] Examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as piggyBac, Sleeping Beauty, Mos1, Tc1 / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince and derivatives thereof.

[0341] Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses.

[0342] Examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40).

[0343] Exemplary expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0344] In certain embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without being integrated into the chromosomal DNA of the host, and does not decay over time with the division of the host cell, which also means that the vector replicates extrachromosomally or episomally.

[0345] "Control elements," "control sequences," present in an expression vector are non-translated regions of the vector, including origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5' and 3' non-translated regions, which interact with host cellular proteins to effect transcription and translation. Such factors may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation factors may be used, including ubiquitous and inducible promoters.

[0346] In certain embodiments, vectors, including but not limited to expression vectors and viral vectors, contain exogenous, endogenous, or heterologous control sequences, such as promoters and / or enhancers. An "endogenous" control sequence is a sequence that is naturally linked to a given gene in the genome. An "exogenous" control sequence is one that is placed in juxtaposition to a gene by means of genetic engineering (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" control sequence is an exogenous sequence that originates from a species different from the cell being genetically engineered.

[0347] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates transcription of a polynucleotide operably linked to the promoter. In certain embodiments, promoters that operate in mammalian cells contain an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or a separate sequence, a CNCAAT region, located 70-80 bases upstream from the transcription start site, where N can be any nucleotide.

[0348] The term "enhancer" refers to a DNA segment that contains a sequence that can provide enhanced transcription, and in some cases can function regardless of orientation relative to another control sequence. Enhancers can function in concert with or additively with promoter elements and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment that contains a sequence that can provide both promoter and enhancer functions.

[0349] The term "operably linked" refers to a juxtaposition where the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0350] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that allows for continuous or sequential transcription of an operably linked sequence. A structural expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows for expression in a variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that allows for expression in restricted cell and tissue types, respectively.

[0351] Exemplary ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), Moloney Murine Leukemia Virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5 and P11 promoters from vaccinia virus, elongation factor 1 alpha (EF1a) promoter, early growth response protein 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde triphosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa, beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477 - 1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, beta-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al. Journal of Virology. 2003; 77 (17): 9439-9450).

[0352] In one embodiment, the vector contains the MNDU3 promoter.

[0353] In one embodiment, the vector contains the EF1a promoter including the first intron of the human EF1a gene.

[0354] In one embodiment, the vector contains the EF1a promoter lacking the first intron of the human EF1a gene.

[0355] As used herein, "conditional expression" may refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, expression in cells or tissues in a particular physiological, biological or disease state. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, for example, expression is controlled by exposing a cell, tissue or organism to a treatment or condition that causes the polynucleotide to be expressed or that increases or decreases the expression of a polynucleotide encoded by the polynucleotide of interest.

[0356] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulated system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0357] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the vector comprises at least one (typically two) site for recombination mediated by a site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes excisive or integrative proteins, enzymes, cofactors or associated proteins involved in a recombination reaction that includes one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), and may be a wild-type protein (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or a mutant, derivative (e.g., a fusion protein comprising a recombination protein sequence or a fragment thereof), fragment and variants thereof. Exemplary recombinases suitable for use in certain embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0358] A vector may contain one or more recombination sites for any of a wide range of site-specific recombinases. It should be understood that the target site for the site-specific recombinase is in addition to any site(s) required for integration of the vector, e.g., a retroviral or lentiviral vector. As used herein, the terms "recombination sequence", "recombination site", or "site-specific recombination site" refer to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0359] For example, one recombination site for Cre recombinase is loxP, a 34 base pair sequence that contains two 13 base pair inverted repeats (which remain as recombinase binding sites) flanking an 8 base pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0360] Suitable recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod, et al., 1996), F 1、 F 2、 F3 (Schlake and Bode, 1994), F 4、 F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).

[0361] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme lambda integrase, e.g., phi-c31. φC31 SSR mediates recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000), where attB and att are named for the binding sites for phage integrase on the bacterial and phage genomes, respectively, and both contain imperfect inverted repeats that can be bound by φC31 homodimers (Groth et al., 2000). The product sites, attL and attR, are also effectively inactive to φC31-mediated recombination (Belteki et al., 2003), rendering the reaction irreversible. To catalyze insertion, it has been found that attB-bearing DNA inserts into genomic attP sites more easily than attP sites insert into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, a typical strategy is to position an attP-bearing "docking site" at a defined locus by homologous recombination, which is then joined with an attB-bearing incoming sequence for insertion.

[0362] As used herein, "internal ribosome entry site" or "IRES" refers to a factor that promotes direct entry of an internal ribosome into an initiation codon, such as ATG, of a cistron (protein coding region), resulting in cap-independent gene translation. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In certain embodiments, a vector comprises one or more polynucleotides of interest that encode one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequence may be separated by one or more IRES sequences or polynucleotide sequences that encode self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0363] As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes initial binding of mRNA to the small ribosomal subunit and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO:93), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). In certain embodiments, the vector comprises a polynucleotide having a consensus Kozak sequence that encodes a desired polypeptide, such as a CAR.

[0364] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase expression of heterologous genes. Transcription termination signals are generally present downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that induces both termination and polyadenylation of the initial RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNA has two recognition elements adjacent to the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable factor that is rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements, adding up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a polyA sequence of choice (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is a polyA sequence of SV40, bovine growth hormone polyA sequence (BGHpA), rabbit β-globin polyA sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.

[0365] In some embodiments, the polynucleotide or the cell carrying the polynucleotide utilizes a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled amplification. In certain aspects, the suicide gene is not immunogenic to the host or the cell carrying the polynucleotide. Examples of suicide genes that can be used are caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0366] In certain embodiments, one or more polynucleotides encoding an anti-CD79A CAR and an anti-CD20 CCR are introduced into a cell (e.g., an immune effector cell) by a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encoding an anti-CD79A CAR and an anti-CD20 CCR is introduced into a cell by a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encoding a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR is introduced into a cell by a non-viral or viral vector.

[0367] The term "vector" is used herein to refer to a nucleic acid molecule that can transmit or transport another nucleic acid molecule. The transmitted nucleic acid is generally inserted, for example, into a vector nucleic acid molecule. The vector may contain sequences that direct self-replication in a cell, or may contain sequences sufficient to allow integration into host cell DNA. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides contemplated herein to T cells. In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA that encodes a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR.

[0368] In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR.

[0369] Examples of non-viral vectors include, but are not limited to, mRNA, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0370] Exemplary non-viral methods of delivery of polynucleotides contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistec, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0371] Examples of polynucleotide delivery systems suitable for use in certain embodiments contemplated in certain embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted delivery, bacteria-guided delivery, and non-biological nanocell-based delivery are also contemplated in certain embodiments.

[0372] In various embodiments, the polynucleotide is the mRNA that is introduced into cell to transiently express desired polypeptide.As used herein, "transient" refers to the expression of non-integrated transgene over a period of hours, days, or weeks, and the expression period is shorter than the expression period of polynucleotide when it is integrated into the genome of cell or contained in stable plasmid replicon.

[0373] In certain embodiments, the mRNA encoding the polypeptide is an in vitro transcribed mRNA.As used herein, "in vitro transcribed RNA" refers to an RNA, preferably an mRNA, synthesized in vitro.Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector.The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.

[0374] In certain embodiments, an mRNA may further comprise a 5' cap or modified 5' cap, and / or a poly(A) sequence. As used herein, a 5' cap (also referred to as an RNA cap, an RNA 7-methylguanosine cap, or an RNA m 7G A 5' cap (also called a cap) is a modified guanine nucleotide added to the "pre" or 5' end of a eukaryotic messenger RNA immediately after the initiation of transcription. The 5' cap is linked to the first transcribed nucleotide and contains a terminal group that is recognized by the ribosome and protected from RNases. The capping moiety can be modified to modulate the function of the mRNA, such as translation stability or efficiency. In certain embodiments, the mRNA comprises a poly(A) sequence of about 50 to about 5000 adenines. In one embodiment, the mRNA comprises a poly(A) sequence of about 100 to about 1000 bases, about 200 to about 500 bases, or about 300 to about 400 bases. In one embodiment, the mRNA comprises a poly(A) sequence of about 65 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1000 bases. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functions such as localization, stability, or efficiency of translation.

[0375] In certain embodiments, viral vectors containing contemplated polynucleotides may be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or local application as described below. Alternatively, vectors may be delivered ex vivo to cells, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsies, etc.) or hematopoietic stem cells of a universal donor, followed by reimplantation of the cells into the patient.

[0376] In one embodiment, a viral vector comprising a polynucleotide encoding an anti-CD79A CAR and an anti-CD20 CCR is administered directly to an organism, and transduces cells in vivo. In one embodiment, a viral vector comprising a polynucleotide encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR is administered directly to an organism, and transduces cells in vivo. Alternatively, naked DNA may be administered. Administration may be by any of the routes normally used to introduce molecules to ultimately contact blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and known to those skilled in the art, but multiple routes may be used to administer a particular composition, and a particular route can often provide a more immediate and effective response than another route.

[0377] Examples of viral vector systems suitable for use in certain embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0378] In various embodiments, one or more polynucleotides encoding a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR, are introduced into immune effector cells, optionally including one or more genome edits to reduce or eliminate expression and / or function of PDCD-1 or CBLB, e.g., in T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) comprising the one or more polynucleotides.

[0379] AAV is a small (about 26 nm), replication-incompetent, mainly episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may integrate its genome into the genome of a host cell. Recombinant AAV (rAAV) typically consists of a minimal transgene and its control sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are about 145 bp in length. In certain embodiments, rAAV contains the ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0380] In some embodiments, chimeric rAAVs are used. The ITR sequences are isolated from one AAV serotype, and the capsid sequences are isolated from another AAV serotype. For example, a rAAV containing ITR sequences from AAV2 and capsid sequences from AAV6 is called AAV2 / AAV6. In certain embodiments, a rAAV vector may contain ITRs from AAV2 and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV contains ITR sequences from AAV2 and capsid sequences from AAV6. In a preferred embodiment, the rAAV contains ITR sequences from AAV2 and capsid sequences from AAV2.

[0381] In some embodiments, methods of engineering and selection may be performed on AAV capsids to increase the probability that they will transduce cells of interest.

[0382] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Patent Nos. 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058, and 8,784,799, which are incorporated by reference herein in their entireties.

[0383] In various embodiments, one or more polynucleotides encoding a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR, are introduced into immune effector cells, optionally including one or more genome edits to reduce or eliminate expression and / or function of PDCD-1 or CBLB by transducing the cells with a retrovirus, e.g., a lentivirus, comprising the one or more polynucleotides.

[0384] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, and then covalently integrates its genomic DNA into the host genome.Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentivirus.

[0385] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2), Visna-Maedi virus (VMV), Caprine arthritis-encephalitis virus (CAEV), Equine infectious anemia virus (EIAV), Feline immunodeficiency virus (FIV), Bovine immunodeficiency virus (BIV), and Simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are preferred.

[0386] In various embodiments, lentiviral vectors contemplated herein contain one or more LTRs, and one or more or all of the following accessory elements: cPPT / FLAP, Psi (Ψ) packaging signal, export element, poly(A) sequence, and may optionally contain a WPRE or HPRE, an insulator element, a selection marker, and a cell suicide gene, as otherwise discussed herein.

[0387] In certain embodiments, the lentivirus vector contemplated herein can be integrated, or non-integrated, or integration-defective lentivirus.As used herein, the term "integration-defective lentivirus" or "IDLV" refers to the lentivirus that has an integrase that is deficient in making viral genome integrate into host cell genome.Integration-defective viral vectors are described in patent application WO2006 / 010834, which is incorporated herein by reference in its entirety.

[0388] Examples of mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to, the following: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A. , K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0389] In one embodiment, the HIV-1 integrase defective pol gene comprises a D64V, D116I, D116A, E152G, or E152A mutation, a D64V, D116I, and E152G mutation, or a D64V, D116A, and E152A mutation.

[0390] In one embodiment, the HIV-1 integrase defective pol gene comprises a D64V mutation.

[0391] The term "long terminal repeat (LTR)" refers to the domains of base pairs located at the ends of retroviral DNA; in the native sequence, the LTRs are direct repeats and contain the U3, R and U5 regions.

[0392] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of retroviruses, such as HIV-1 and HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. In another embodiment, the lentiviral vector contains a FLAP element having one or more mutations in the cPPT and / or CTS elements. In yet another embodiment, the lentiviral vector contains either the cPPT or CTS elements. In yet another embodiment, the lentiviral vector does not contain a cPPT or CTS element.

[0393] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located in the retroviral genome, which is required for inserting viral RNA into the viral capsid or viral particle.See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0394] The term "export factor" refers to a cis-acting post-transcriptional regulator that controls the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export factors include, but are not limited to, the rev-responsive factor (RRE) of human immunodeficiency virus (HIV) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the post-transcriptional regulator (HPRE) of hepatitis B virus.

[0395] In certain embodiments, the expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulators, efficient polyadenylation sites, and optionally transcription termination signals into the vector.Various post-transcriptional regulators can increase the expression of heterologous nucleic acid in protein.For example, Woodchuck Hepatitis Virus post-transcriptional regulator (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); Post-transcriptional regulator (HPRE) present in Hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); etc. (Liu et al., 1995, Genes Dev., 9:1766).

[0396] Lentiviral vectors preferably contain several safety enhancements as a result of LTR modification. "Self-inactivating" (SIN) vectors refer to vectors that lack replication capacity, for example, the right (3') LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to inhibit viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5'LTR with a heterologous promoter that induces transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, Simian Virus 40 (SV40) (e.g., early or late), Cytomegalovirus (CMV) (e.g., immediate early), Moloney Murine Leukemia Virus (MoMLV), Rous Sarcoma Virus (RSV), and Herpes Simplex Virus (HSV) (thymidine kinase) promoters.

[0397] As used herein, the term "pseudotype" or "pseudotyped" refers to a virus that has a viral envelope protein replaced with an envelope protein of another virus that has favorable properties. For example, the HIV envelope protein (encoded by the env gene) is normally encoded by the CD4 +Although targeting the virus to presentation cells, pseudotyping HIV with the vesicular stomatitis virus G-protein (VSV-G) envelope protein allows HIV to infect a broad range of cells.

[0398] In one embodiment, lentiviral vectors are produced by known methods, see, e.g., Kutner et al., BMC Biotechnol. 2009;9:10.doi:10.1186 / 1472-6750-9-10; Kutner et al. Nat. Protoc. 2009;4(4):495-505.doi:10.1038 / nprot.2009.22.

[0399] According to certain embodiments contemplated herein, most or all of the backbone sequences of viral vectors are derived from lentiviruses, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, or a number of combined substitutions and modifications of a particular lentiviral sequence can be accommodated without compromising the ability of the transfer vector to perform the functions described herein. In addition, various lentiviral vectors are known in the art. See Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136. Many of them can be adapted to produce viral vectors or transfer plasmids contemplated herein.

[0400] In various embodiments, one or more polynucleotides encoding a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR, are introduced into immune effector cells, optionally including one or more genome edits that reduce or eliminate expression and / or function of PDCD-1 or CBLB, by transducing the cells with an adenovirus containing the one or more polynucleotides.

[0401] Adenoviral vectors can transduce many cell types with high efficiency and do not require cell division. Such vectors can be used to obtain high titers and high levels of expression. The vectors can be produced in large quantities in a relatively simple system. Most adenoviral vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes. The resulting replication-deficient vectors are amplified in human 293 cells, which supply the missing gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing and differentiated cells, such as those found in the liver, kidney, and muscle. Traditional Ad vectors have a large carrying capacity.

[0402] The creation and amplification of current replication-deficient adenovirus vectors may utilize a unique helper cell line called 293. This cell line was transformed with Ad5 DNA fragments from human embryonic kidney cells and constitutively expresses E1 protein (Graham et al., 1977). Because the E3 region of the adenovirus genome is not essential (Jones & Shenk, 1978), current adenovirus vectors carry foreign DNA in the E1, D3, or both regions with the help of 293 cells (Graham & Prevec, 1991). Adenovirus vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Experiments in administering recombinant adenoviruses to various tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). Examples of the use of Ad vectors in clinical trials include polynucleotide therapy for antitumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0403] In various embodiments, one or more polynucleotides encoding an anti-CD79A CAR and an anti-CD20 CCR or fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and a polycistronic message encoding an anti-CD20 CCR are introduced into an immune effector cell, optionally including one or more genome edits to reduce or eliminate expression and / or function of PDCD-1 or CBLB by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising the one or more polynucleotides.

[0404] Mature HSV virions consist of an enveloped icosahedral capsid containing a viral genome consisting of a linear double-stranded DNA molecule of 152 kb. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-defective. Most replication-defective HSV vectors contain deletions to remove one or more intermediate-early, early, or late HSV genes, inhibiting replication. For example, the HSV vector may lack an immediate-early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantage of HSV vectors is their ability to enter late stages and produce long-term DNA expression, and their large viral DNA genome, which can accommodate exogenous DNA inserts up to 25 kb. HSV-based vectors are described, for example, in U.S. Pat. Nos. 5,837,532, 5,846,782 and 5,804,413, and International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637 and WO99 / 06583, which are incorporated herein by reference in their entireties.

[0405] H. Genetically Modified Cells In various embodiments, cells genetically modified to express the anti-CD79A CAR and anti-CD20 CCR contemplated herein are provided for use in the treatment of cancer. In various preferred embodiments, immune effector cells genetically modified to express the anti-CD79A CAR and anti-CD20 CCR and containing one or more genomic edits that reduce or eliminate the function and / or expression of PDCD-1 and / or CBLB are used to treat cancer. In further preferred embodiments, T cells or NK cells genetically modified to express the anti-CD79A CAR and anti-CD20 CCR and containing one or more genomic edits that reduce or eliminate the function and / or expression of CBLB are used to treat cancer.

[0406] In certain embodiments, anti-CD79A CAR and anti-CD20 CCR or fusion proteins encoding anti-CD79A CAR, 2A self-cleaving polypeptides, and anti-CD20 CCRs contemplated herein are introduced and expressed in immune effector cells to redirect their specificity to a target antigen of interest, e.g., CD79A or CD20. In a preferred embodiment, anti-CD79A CAR and anti-CD20 CCR or fusion proteins encoding anti-CD79A CAR, 2A self-cleaving polypeptides, and anti-CD20 CCRs are introduced and expressed in immune effector cells comprising one or more genomes in the CBLB to redirect their specificity to a target antigen of interest, CD79A or CD20. An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC, etc.). Exemplary immune effector cells contemplated herein include cytotoxic T cells (CTL; ​​CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + In certain embodiments, the cells comprise αβ T cells. In certain embodiments, the cells comprise γδ T cells. In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0407] Immune effector cells may be self or non-self (e.g., allogeneic, syngeneic or xenogeneic). As used herein, "self" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the compared cells. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the compared cells. As used herein, "xenogeneic" refers to cells of a different species from the compared cells. In a preferred embodiment, the cells are autologous.

[0408] Exemplary immune effector cells for use with anti-CD79A CAR and anti-CD20 CCR contemplated in certain embodiments include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, such as T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells may be helper T cells (HTL; CD4 + T cell) CD4 + T cells, cytotoxic T cells (CTL:CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells (T N ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), and effector T cells (T EFF ) are mentioned.

[0409] As will be understood by those skilled in the art, other cells can also be used as immune effector cells with anti-CD79A CAR and anti-CD20 CCR as contemplated herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include precursors of effector cells, and such precursors may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs), contained within the CD34+ population of cells derived from umbilical cord blood, bone marrow or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject, or may be induced to differentiate into mature immune effector cells in vitro.

[0410] As used herein, the term "CD34+ cell" refers to a cell that expresses CD34 protein on its cell surface.As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes.CD34+ cell population contains hematopoietic stem cells (HSCs), which differentiate when administered to patients and give rise to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of monocyte / macrophage lineage.

[0411] Methods of generating immune effector cells expressing the anti-CD79A CAR and anti-CD20 CCR contemplated herein are provided in certain embodiments. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express an anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR contemplated herein.

[0412] In a preferred embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express a fusion protein encoding an anti-CD79A CAR and an anti-CD20 CCR or an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR as contemplated herein. The method further comprises introducing into the cells a polynucleotide encoding an HE variant or a megaTAL that binds and cleaves a target site in the PDCD-1 gene or the CBLB gene, preferably in the CBLB gene. In certain embodiments, the transduced and edited cells are then cultured for growth before being administered to a subject.

[0413] In an embodiment, immune effector cells are isolated from an individual and genetically modified and / or edited without further manipulation in vitro. Such cells may then be directly re-administered to the individual. In a further embodiment, immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express anti-CD79A CAR and anti-CD20 CCR, and then edited using HE variants or megaTALs targeting PDCD-1 or CBLB, preferably CBLB. In this regard, immune effector cells may be cultured before and / or after genetic modification and / or genome editing (i.e., transduced or transfected to express anti-CD79A CAR and anti-CD20 CCR as contemplated herein).

[0414] In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of immune effector cells as described herein. In certain embodiments, the modified immune effector cells contain T cells.

[0415] In certain embodiments, PBMCs may be directly genetically modified using the methods contemplated herein to express a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR. In certain embodiments, following isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be preserved into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0416] Immune effector cells such as T cells can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of progenitor cells) in vitro before being genetically modified and / or genome edited. In certain embodiments, immune effector cells such as T cells are activated and stimulated for proliferation, then genetically modified with a chimeric antigen receptor contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding an anti-CD79A CAR and an anti-CD20 CCR or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and a polycistronic message encoding an anti-CD20 CCR), and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification using, for example, the methods described in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0417] In one embodiment, CD34 + The cells are transduced with a nucleic acid construct as contemplated herein. In certain embodiments, the transduced CD34 + The cells are differentiated in vivo into mature immune effector cells, generally after administration to a subject from whom the cells were originally isolated. + Cells may be stimulated in vitro prior to exposure to, or after genetic modification with, one or more of the following cytokines: Flt-3 ligand (FLT3), stem cell factor (SCF), megakaryocyte growth and differentiation factor (TPO), IL-3, and IL-6, as previously described (Asheuer et al., 2004; Imren, et al., 2004).

[0418] In certain embodiments, the population of modified immune effector cells for the treatment of cancer comprises the CAR and CCR contemplated herein.For example, the population of modified immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient (autologous donor) diagnosed with a B-cell malignancy as described herein.PBMCs form a heterogeneous population of T lymphocytes that may be CD4+, CD8+, or CD4+ and CD8+.

[0419] PBMCs may also contain other cytotoxic lymphocytes such as NK cells or NKT cells. In certain embodiments, expression vectors carrying coding sequences for CARs and CCRs contemplated are introduced into human donor T cell, NK cell, or NKT cell populations. In certain embodiments, successfully transduced T cells carrying expression vectors can be sorted using flow cytometry to isolate CD3 positive T cells, which can then be further expanded to increase the number of these CAR and CCR expressing T cells in addition to cell activation using anti-CD3 and / or anti-CD28 antibodies, and IL-2, or any other method known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of non-human animal derived products such as fetal calf serum and fetal bovine serum. A heterogeneous population of PBMCs is genetically modified such that the resulting transduced cells are a heterogeneous population of modified cells comprising a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR, or a polynucleotide encoding a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR as contemplated herein. In certain embodiments, a heterogeneous population of PBMCs is genetically modified and genome edited such that the resulting cells are a heterogeneous population of modified cells comprising a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR, or a polynucleotide encoding a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR, and further comprises one or more genome edits that reduce or eliminate the function and expression of PDCD-1 and / or CBLB.

[0420] In further embodiments, a mixture of, for example, one, two, three, four, five, or more different expression vectors can be used in genetically modifying a donor population of immune effector cells, each vector encoding a different chimeric antigen receptor protein as contemplated herein, and the resulting modified immune effector cells form a mixed population of modified cells.

[0421] IT cell manufacturing method In various embodiments, the genetically modified T cells are expanded by contacting them with an agent that stimulates a CD3 TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.

[0422] In certain embodiments, PBMCs, or isolated T cells, are contacted with stimulatory and costimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies or antibodies bound to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2, IL-7 and / or IL-15.

[0423] In certain embodiments, PBMCs, or isolated T cells are contacted with stimulatory and costimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies or antibodies bound to beads or other surfaces, in culture medium with appropriate cytokines, such as IL-2, IL-7 and / or IL-15, and / or a PI3K inhibitor.

[0424] In certain embodiments, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell manufacturing methods contemplated herein. PBMCs are CD4 + , CD8 + , or CD4 + and CD8 +The modified T cells form a heterogeneous population of T lymphocytes that may be resistant to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-15, IL-2, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-15, IL-15, IL-2, IL-15, IL-15, IL-15, IL-15, IL-2, IL-15, IL-15, IL-15, IL-15, IL-15, IL-15, IL-14 ...

[0425] To obtain a sufficient therapeutic dose of a T cell composition, T cells often undergo one or more rounds of stimulation, activation and / or expansion. T cells can generally be activated and expanded using methods described, for example, in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041. These patents are incorporated herein by reference in their entireties.

[0426] In a preferred embodiment, the T cells produced by the methods contemplated herein provide improved adoptive immunotherapy compositions.Without intending to be bound by any particular theory, it is believed that the T cell compositions produced by the methods of certain embodiments contemplated herein have superior properties, including increased survival, proliferation in the relative absence of differentiation, and persistence in vivo.In one embodiment, the method of producing T cells includes contacting cells with one or more agents that modulate the PI3K cell signaling pathway.

[0427] In certain embodiments, T cells are produced by stimulating T cells to activation and proliferation in the presence of one or more stimulatory signals and a PI3K inhibitor.

[0428] The T cells can then be modified to express a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR. In one embodiment, the T cells are modified by transducing the T cells with a viral vector comprising a polycistronic message encoding an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR as contemplated herein. In a particular embodiment, the T cells are modified in the presence of an inhibitor of the PI3K cell signaling pathway prior to stimulation and activation. In another embodiment, the T cells are modified in the presence of an inhibitor of the PI3K cell signaling pathway after stimulation and activation. In another embodiment, the T cells are modified in the presence of an inhibitor of the PI3K cell signaling pathway within 12 hours, 24 hours, 36 hours, or 48 hours of stimulation and activation. In a particular embodiment, the T cells are modified in the presence of a PI3K inhibitor.

[0429] In certain embodiments, after immune effector cells are transduced with a viral vector comprising a polynucleotide encoding an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR, the cells are edited by introducing into the cells a polynucleotide encoding a HE variant or a megaTAL that binds and cleaves a target site in the PDCD-1 gene or the CBLB gene, preferably the CBLB gene.

[0430] After the T cells are transduced and / or edited, the cells are cultured and expanded. The T cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more, and for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion. In certain embodiments, the T cells are modified to expand in the presence of a PI3K inhibitor.

[0431] In various embodiments, the T cell compositions are manufactured in the presence of a PI3K inhibitor. Without intending to be bound by any particular theory, treating or contacting T cells with one or more inhibitors of the PI3K pathway during the stimulation, activation, and / or expansion phases of the manufacturing process will preferentially expand young T cells, thereby producing a superior therapeutic T cell composition.

[0432] As used herein, the term "PI3K inhibitor" refers to a nucleic acid, peptide, or small organic molecule that binds to and inhibits at least one activity of PI3K. PI3K proteins can be divided into three classes: class 1 PI3K, class 2 PI3K, and class 3 PI3K. Class 1 PI3K exists as a heterodimer consisting of one of four p110 catalytic subunits (p110α, p110β, p110δ, and p110γ) and one of two regulatory subunit families. PI3K inhibitors preferably target class 1 PI3K inhibitors. In one embodiment, the PI3K inhibitor exhibits selectivity for one or more isoforms of class 1 PI3K inhibitors (i.e., selectivity for p110α, p110β, p110δ, and p110γ or p110α, p110β, p110δ, and p110γ). In another embodiment, the PI3K inhibitor does not exhibit isoform selectivity and is considered a “pan-PI3K inhibitor.” In one embodiment, the PI3K inhibitor competes for binding with ATP to the PI3K catalytic domain.

[0433] In certain embodiments, PI3K inhibitors can target, for example, PI3K and additional proteins in the PI3K-AKT-mTOR pathway. In certain embodiments, PI3K inhibitors that target both mTOR and PI3K can be referred to as either mTOR inhibitors or PI3K inhibitors. PI3K inhibitors that target only PI3K can be referred to as selective PI3K inhibitors. In one embodiment, selective PI3K inhibitors can be understood to refer to agents that show a 50% inhibitory concentration for PI3K that is at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 1000 times, or more lower than the IC50 of the inhibitor for mTOR and / or other proteins in the pathway.

[0434] In certain embodiments, exemplary PI3K inhibitors inhibit PI3K with an IC50 (concentration that inhibits 50% of activity) that is about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM or less. In one embodiment, the PI3K inhibitor inhibits PI3K with an IC50 of about 2 nM to about 100 nM, more preferably about 2 nM to about 50 nM, even more preferably about 2 nM to about 15 nM.

[0435] Examples of PI3K inhibitors suitable for use in the T cell manufacturing methods contemplated in certain embodiments include, but are not limited to, BKM120 (Class 1 PI3K inhibitor, Novartis), XL147 (Class 1 PI3K inhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline), and PX-866 (Class 1 PI3K inhibitor, p110α, p110β, and p110γ isoforms, Oncotherion).

[0436] Other illustrative examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145.

[0437] Further illustrative examples of pan-PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

[0438] In a preferred embodiment, the PI3K inhibitor is ZSTK474.

[0439] In certain embodiments, methods are provided for increasing the proliferation of T cells expressing engineered T cell receptors, such methods include, for example, harvesting a source of T cells from a subject, stimulating and activating the T cells, modifying the T cells to express an anti-CD79A CAR and an anti-CD20 CCR, editing the genome of the cells with HE variants or megaTALs, and expanding the T cells in culture, where the T cells are produced in the presence of one or more inhibitors of the PI3K pathway.

[0440] The manufacturing methods contemplated herein may further include cryopreservation of the modified T cells for storage and / or preparation for use in human subjects. In one embodiment, the method of preserving genetically modified immune effector cells includes cryopreserving the immune effector cells such that the cells remain viable upon thawing. The T cells are cryopreserved such that the cells remain viable upon thawing. If desired, the cryopreserved, transformed cells can be thawed, expanded, and expanded to a larger number of such cells. As used herein, "cryopreservation" refers to preserving cells by cooling to a temperature below zero, such as (typically) 77K or -196°C (the boiling point of liquid nitrogen). Cryopreservatives are often used at temperatures below zero so that the cells being preserved are not damaged by freezing at low temperatures or heating to room temperature. Cryopreservatives and optimal cooling rates can protect against cell damage. Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959;183:1394-1395; Ashwood-Smith, Nature, 1961;190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960;85:576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962;196:48). The preferred cooling rate is 1°-3°C / min. After at least 2 hours, the T cells will have reached a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage, such as in a long-term cryogenic storage vessel.

[0441] J. Compositions and Formulations The compositions contemplated herein may include one or more CAR polypeptides, CCR polypeptides, polynucleotides, vectors comprising same, genetically modified immune effector cells, etc. contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. In a preferred embodiment, the compositions include one or more cells modified to express an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR. In a preferred embodiment, the compositions include one or more cells modified to express an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR, and the cells have been genome-edited to reduce or eliminate the expression and function of CBLB.

[0442] "Pharmaceutical composition" refers to a composition formulated in a pharma- ceutically or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. If desired, it should also be understood that the composition may be administered in combination with other agents as well, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharma- ceutical active agents. There is virtually no limit to the other components that may be included in the composition, provided that the added agents do not adversely affect the ability of the composition to deliver the intended therapy. In a preferred embodiment, the pharmaceutical composition comprises a pharma- ceutical acceptable carrier, diluent, or excipient, and one or more genome-edited cells that have been modified to express a fusion protein encoding an anti-CD79A CAR and an anti-CD20 CCR, or an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR.

[0443] As used herein, the phrase "pharmacologically acceptable" is employed to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0444] As used herein, a "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, an adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine. Exemplary pharma- ceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicones, bentonite, silicic acid, zinc oxide, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol; phosphate buffers, and other compatible substances employed in pharmaceutical formulations.

[0445] In certain embodiments, the composition comprises an amount of anti-CD79A CAR and anti-CD20 CCR expressing immune effector cells as contemplated herein. In a preferred embodiment, the composition comprises an amount of genome-edited immune effector cells expressing anti-CD79A CAR and anti-CD20 CCR, wherein the genome edit(s) reduces or eliminates the expression and function of PDCD-1 and / or CBLB, preferably CBLB. As used herein, the term "amount" refers to an "effective amount" or "effective amount" of genetically modified therapeutic cells, such as T cells, to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0446] "Prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactic dose is used before or in subjects at an early stage of disease, so the prophylactically effective amount is less than the therapeutically effective amount.

[0447] The "therapeutically effective amount" of genetically modified therapeutic cells may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition.

[0448] Generally, the pharmaceutical compositions comprising the T cells described herein are 2 ~10 10 Cells / kg body weight, preferably 10 5 ~10 6It can be said that a dose of cells / kg body weight can be administered, including all integer values ​​within the range. The number of cells depends on the intended end use of the composition and the type of cells contained in the composition. For the uses presented herein, the cells are generally in a volume of liters or less, and can be 500mL or less, or even 250mL or 100mL or less. Thus, the desired cell density is often 10 6 cells / ml, typically greater than 10 7 >10 cells / ml, typically 8 Clinically relevant immune cell counts may be divided into multiple infusions, with a cumulative total of 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, more than 10 cells are injected, specifically because all cells injected are redirected to a specific target antigen. 6 / kilogram (10 per patient) 6 ~10 11 ) may be administered. The compositions may be administered multiple times at doses within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance induction of an immune response.

[0449] In general, compositions comprising cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases occurring in immunocompromised individuals. In certain embodiments, compositions comprising immune effector cells modified to express an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR as contemplated herein, and comprising one or more genomic edits that reduce or eliminate the expression and function of PDCD-1 and / or CBLB, preferably CBLB, are used to treat cancer. The modified immune effector cells may be administered alone or as a pharmaceutical composition in combination with carriers, diluents, excipients, and / or other components, such as, for example, IL-2 or other cytokines or other cell populations. In certain embodiments, the pharmaceutical composition contains an amount of genetically modified T cells in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients.

[0450] The pharmaceutical composition comprising the anti-CD79A CAR and anti-CD20 CCR or fusion protein encoding the anti-CD79A CAR, the 2A self-cleaving polypeptide, and the genome-edited immune effector cell population engineered to express the anti-CD20 CCR, such as T cells, may comprise a buffer, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol, proteins: polypeptides or amino acids, such as glycine, antioxidants, chelating agents, such as EDTA or glutathione (e.g., aluminum hydroxide), and preservatives. The composition is preferably formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

[0451] Liquid pharmaceutical compositions, whether in solution, suspension or other similar form, may contain one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that may be provided as a solvent or suspending medium, polyethylene glycol, glycerin, propylene glycol or other solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0452] In one embodiment, the T cell composition contemplated herein is formulated in a pharmaceutically acceptable cell culture medium. Such a composition is suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0453] Serum-free media have several advantages over serum-containing media, including a simpler and more transparent composition, lower amounts of contaminants, elimination of potential sources of infectious entities, and lower costs. In various embodiments, serum-free media may be animal-free and optionally protein-free. Optionally, the media may contain biopharmaceutical acceptable recombinant proteins. "Animal-free" media refers to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, whose nutrients are obtained from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0454] Examples of serum-free media for use in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0455] In a preferred embodiment, a composition comprising immune effector cells as contemplated herein is formulated in a solution comprising PlasmaLyte A.

[0456] In another preferred embodiment, the composition comprising immune effector cells contemplated herein is formulated in a solution comprising a cryopreservation medium.For example, a cryopreservation medium comprising a cryopreservative may be used to maintain high cell activity after thawing.Exemplary cryopreservation media used in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0457] In a more preferred embodiment, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.

[0458] In certain embodiments, the composition comprises an effective amount of genome-edited immune effector cells engineered to express an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR, alone or in combination with one or more therapeutic agents. Thus, the CAR-expressing immune effector cell composition may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc. The composition may be administered in combination with an antibiotic. Such therapeutic agents may be accepted in the art as standard treatment for certain disease conditions described herein, such as, for example, certain cancers. Examples of contemplated therapeutic agents include, in certain embodiments, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active adjunctive agents.

[0459] In certain embodiments, the composition comprising a genome-edited immune effector cell engineered to express an anti-CD79A CAR and an anti-CD20 CCR, or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR, may be administered in conjunction with any number of chemotherapeutic agents.

[0460] A variety of other therapeutic agents may be used in conjunction with the compositions described herein. In one embodiment, the composition comprising the genome-edited immune effector cells, anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR is administered with an anti-inflammatory agent.

[0461] In one embodiment, the composition comprising the genome-edited immune effector cells, anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR is administered with a therapeutic antibody. Exemplary therapeutic antibodies suitable for use with CAR-modified T cells contemplated in certain embodiments include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (avastin), bivatuzumab, blinatumomab, conatumumab, crizotinib, daratumumab, duligotumab, dacetuzumab, dalotuzumab, dutuzumab, teufeliz ... These include ruvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0462] K. Target Cells and Antigens In certain embodiments, genetically modified immune effector cells are provided that are redirected to target cells, such as cancer cells, express anti-CD79A CAR and anti-CD20 CCR, and contain PDCD-1 and / or CBLB, preferably CBLB. As used herein, the term "cancer" generally refers to a type of disease or condition in which abnormal cells divide uncontrollably and can invade nearby tissues.

[0463] As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of the following: uncontrolled proliferation (i.e., dividing beyond normal limits), invasion (i.e., invading and destroying adjacent tissues), and metastasis (i.e., spreading to other parts of the body via lymph or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. A tumor formed by the spread cells is called a "metastatic tumor" or "metastatic cancer." A metastatic tumor contains cells similar to those of the original tumor (primary tumor).

[0464] As used herein, the terms "benign" or "non-malignant" refer to tumors that may grow large but do not spread to other parts of the body. Benign tumors are self-limited and often do not invade or metastasize.

[0465] "Cancer cell" refers to an individual cell of a cancerous growth or tissue. Cancer cells include both solid and liquid cancers. "Tumor" or "tumor cell" generally refers to a swelling or lesion formed by the abnormal proliferation of cells, which may be benign, precancerous, or malignant. Most cancers form tumors, but liquid cancers, such as leukemia, do not necessarily form tumors. For cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors.

[0466] The term "recurrence" refers to the diagnosis of a return of cancer, or the diagnosis of returning signs and symptoms of cancer, after a period of improvement or remission.

[0467] "Remission", also called "clinical remission", includes both partial and complete remission. In a partial remission, some but not all signs and symptoms of cancer disappear. In a complete remission, all signs and symptoms of cancer disappear, but cancer may still be present in the body.

[0468] "Refractory" refers to a cancer that is resistant or unresponsive to therapy with a particular therapeutic agent. A cancer may be refractory from the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent) or may become refractory as a result of developing resistance to the therapeutic agent either during the initial treatment period or during subsequent treatment periods.

[0469] In one embodiment, the target cell expresses a target antigen, for example, that is substantially not found on the surface of other normal (desired) cells.

[0470] In one embodiment, the target cell is an osteosarcoma cell or a Ewing's sarcoma cell.

[0471] In one embodiment, the target cell is a hematopoietic cell, a lymphoid cell, or a bone marrow cell.

[0472] In certain embodiments, the target cell is part of blood, lymphoid tissue, or bone marrow tissue.

[0473] In certain embodiments, the target cell is a cancer cell or a cancer stem cell that expresses CD79A and / or CD20. In certain embodiments, the target cell is a cancer cell or a cancer stem cell that expresses CD79A and CD20. In certain embodiments, the target cell is a cancer cell or a cancer stem cell that expresses CD79A or CD20.

[0474] In certain embodiments, the target cell is a liquid or blood cancer cell that expresses CD79A and / or CD20. In certain embodiments, the target cell is a liquid or blood cancer cell that expresses CD79A and CD20. In certain embodiments, the target cell is a liquid or blood cancer cell that expresses CD79A or CD20.

[0475] Illustrative examples of liquid or hematological cancers that may be prevented, treated, or ameliorated with the compositions contemplated in certain embodiments include, but are not limited to, leukemia, lymphoma, and multiple myeloma.

[0476] Illustrative examples of cells that may be targeted by immune effector cells expressing anti-CD79A CAR and anti-CD20 CCR as contemplated in certain embodiments include, but are not limited to, leukemias: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera.

[0477] Illustrative examples of cells that may be targeted by compositions comprising immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR and methods contemplated in certain embodiments include, but are not limited to, those of the following lymphomas: Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, and non-Hodgkin lymphoma, including but not limited to B-cell non-Hodgkin lymphomas: Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma, and T-cell non-Hodgkin lymphomas: mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, and precursor T-lymphoblastic lymphoma.

[0478] Illustrative examples of cells that may be targeted by compositions comprising immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR, and methods contemplated in certain embodiments, include, but are not limited to, multiple myeloma: overt multiple myeloma, smoldering multiple myeloma (MGUS), plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, monocytic plasmacytoma of bone, and ectoplasmocytoma.

[0479] In a preferred embodiment, the CD79A and / or CD20 expressing target cell is a DLBCL cancer cell. In a preferred embodiment, the CD79A and CD20 expressing target cell is a DLBCL cancer cell. In a preferred embodiment, the CD79A or CD20 expressing target cell is a DLBCL cancer cell.

[0480] L. Treatment method

[0481] The genetically engineered immune effector cells expressing anti-CD79A CARs and anti-CD20 CCRs contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment and amelioration of CD79A and / or CD20 expressing cancers, or for use in the prevention, treatment or amelioration of at least one symptom associated with CD79A and / or CD20 expressing cancers.

[0482] In various embodiments, the genetically modified immune effector cells contemplated herein provide improved methods of adoptive immunotherapy used to increase cytotoxicity in cancer cells expressing CD79A and / or CD20 in a subject, or to reduce the number of cancer cells expressing CD79A and / or CD20 in a subject.

[0483] In certain embodiments, the specificity of the primary immune effector cells is redirected to cells expressing CD79A and / or CD20, e.g., cancer cells, by genetically modifying the primary immune effector cells with a CAR as contemplated herein. In various embodiments, viral vectors are used to genetically modify the immune effector cells with specific polynucleotides encoding an anti-CD79A CAR and an anti-CD20 CCR.

[0484] In one embodiment, a kind of cell therapy is provided in which T cells are genetically modified to express anti-CD79A CAR and anti-CD20 CCR that targets CD79A and / or CD20 expressing cancer cells, and the T cells are infused into recipients in need thereof.The infused cells can kill disease-causing cells in recipients.Unlike antibody therapy, T cell therapy can replicate in vivo, resulting in long-term persistence that can provide sustained cancer treatment.

[0485] In one embodiment, T cells expressing an anti-CD79A CAR and an anti-CD20 CCR can undergo robust in vivo T cell expansion and can persist for extended periods of time, hi another embodiment, T cells expressing an anti-CD79A CAR and an anti-CD20 CCR evolve into specific memory T cells or stem cell memory T cells that can be reactivated to inhibit any additional tumor formation or growth.

[0486] In certain embodiments, compositions comprising immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR as contemplated herein are used to treat conditions associated with CD79A- and / or CD20-expressing cancer cells or cancer stem cells.

[0487] Illustrative examples of conditions that may be treated, prevented, or ameliorated using immune effector cells expressing anti-CD79A CAR and anti-CD20 CCR as contemplated in certain embodiments.

[0488] In certain embodiments, compositions comprising T cells expressing an anti-CD79A CAR and an anti-CD20 CCR as contemplated herein are used to treat osteosarcoma or Ewing's sarcoma.

[0489] In certain embodiments, compositions comprising T cells expressing an anti-CD79A CAR and an anti-CD20 CCR as contemplated herein are used to treat liquid or hematological cancers.

[0490] In certain embodiments, the liquid or hematological cancer is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.

[0491] In certain embodiments, the liquid or blood cancer is selected from the group consisting of: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma. (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0492] In certain embodiments, the liquid or blood cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

[0493] In a preferred embodiment, the liquid or hematological cancer is DLBCL.

[0494] In a preferred embodiment, the liquid or hematological cancer is relapsed / refractory DLBCL.

[0495] In certain embodiments, methods are provided that include administering a therapeutically effective amount of immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR as contemplated herein, or a composition comprising the same, to a patient in need thereof, alone or in combination with one or more therapeutic agents. In certain embodiments, the cells are used to treat a patient at risk of developing a pathology associated with cancer cells expressing CD79A and / or CD20. Thus, certain embodiments provide a method of treating or preventing or ameliorating at least one symptom of cancer, comprising administering to a subject in need thereof a therapeutically effective amount of engineered T cells expressing an anti-CD79A CAR and an anti-CD20 CCR as contemplated herein.

[0496] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal exhibiting symptoms of a disease, injury, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein. In a preferred embodiment, a subject includes any animal exhibiting symptoms of a cancer-related disease, injury, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, domestic animals, or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included. Exemplary subjects include human patients with cancers that express CD79A and / or CD20, human patients diagnosed with cancers that express CD79A and / or CD20, or human patients at risk for or with cancers that express CD79A and / or CD20, such as DLBCL.

[0497] As used herein, the term "patient" refers to a subject who has been diagnosed with a particular disease, injury or condition that can be treated using the gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein.

[0498] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally include either a reduction in the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0499] As used herein, "prevent" and similar terms, such as "prevented" and "preventing", refer to an approach aimed at preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or condition. It also refers to delaying the occurrence or recurrence of a disease or condition, or delaying the occurrence or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition before the occurrence or recurrence of the disease or condition.

[0500] As used herein, "amelioration of at least one symptom of" refers to a reduction in one or more symptoms of the disease or condition that the subject is being treated for. In certain embodiments, the disease or condition that is being treated is cancer, in which case the one or more symptoms that are improved include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (caused by distended abdominal organs), bone or joint pain, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0501] "Enhance" or "promote" or "increase" or "expand" generally refers to the ability of the compositions contemplated herein, such as genetically modified T cells expressing anti-CD79A CAR and anti-CD20 CCR, to produce, elicit, or generate a greater physiological response (i.e., downstream effects) compared to the response from a vehicle or control molecule / composition. Measurable physiological responses include, among others, increased expansion, activation, persistence, and / or cancer cell killing capacity of T cells, as will be apparent from an understanding of the art and the present description. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500-fold, 1000-fold) (including all integers and decimals in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response provided by the vehicle or control composition.

[0502] "Reduce" or "lower" or "lower" or "reduce" or "attenuate" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a physiological response (i.e., downstream effect) that is smaller than the response caused by a vehicle or control molecule / composition. The amount of "reduction" or "reduction" is typically a "statistically significant" amount and may include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500-fold, 1000-fold) (including all integers and decimals between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response caused by a vehicle, a control composition, or a response in a particular cell line.

[0503] "Maintain" or "preserve" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refer to the ability of the compositions contemplated herein to produce, elicit, or produce a similar or equivalent physiological response (i.e., downstream effect) in a cell compared to the response produced by a vehicle, a control molecule / composition, or in a particular cell line. An equivalent response is one that is not substantially different or measurably different from the reference response.

[0504] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising a genetically modified immune effector cell as contemplated herein. The amount and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, although appropriate doses may be determined by clinical trials.

[0505] In one embodiment, the amount of immune effector cells, e.g., T cells expressing an anti-CD79A CAR and an anti-CD20 CCR, in the composition administered to a subject is at least 0.1x10 5 Cells, at least 0.5x10 5 Cells, at least 1x10 5 Cells, at least 5x10 5 Cells, at least 1x10 6 Cells, at least 0.5x10 7 Cells, at least 1x10 7 Cells, at least 0.5x10 8 Cells, at least 1x10 8 Cells, at least 0.5x10 9 Cells, at least 1x10 9 Cells, at least 2x10 9 Cells, at least 3x10 9 Cells, at least 4x10 9 Cells, at least 5x10 9 cells, or at least 1x10 10 It is a cell.

[0506] In certain embodiments, about 1×10 7 T cells ~ approx. 1 x 10 9 T cells, approximately 2 x 10 7 T cells ~ approx. 0.9×10 9 T cells, approximately 3 x 10 7 T cells ~ approx. 0.8×10 9 T cells, approximately 4 x 10 7 T cells ~ approx. 0.7×10 9 T cells, approximately 5 x 10 7 T cells ~ approx. 0.6×10 9 T cells, or approximately 5 × 10 7 T cells ~ approx. 0.5×10 9 The T cells are administered to a subject.

[0507] In one embodiment, the amount of immune effector cells, e.g., T cells expressing an anti-CD79A CAR and an anti-CD20 CCR, in the composition administered to a subject is at least 0.1×10 4 At least 0.5x10 cells / kg body weight 4 At least 1x10 cells / kg body weight 4 At least 5x10 cells / kg body weight 4 At least 1x10 cells / kg body weight 5 At least 0.5x10 cells / kg body weight 6 At least 1x10 cells / kg body weight 6 At least 0.5x10 cells / kg body weight 7 At least 1x10 cells / kg body weight 7 At least 0.5x10 cells / kg body weight 8 At least 1x10 cells / kg body weight 8 At least 2x10 cells / kg body weight 8 At least 3x10 cells / kg body weight 8 At least 4x10 cells / kg body weight 8 At least 5x10 cells / kg body weight 8 At least 1x10 cells / kg or body weight 9 cells / kg.

[0508] In certain embodiments, about 1×10 61x10 T cells / kg body weight 8 T cells / kg body weight, approximately 2x10 6 0.9x10 T cells / kg body weight 8 T cells / kg body weight, 3x10 6 T cells / kg ~ approx. 0.8x10 8 T cells / kg body weight, approximately 4x10 6 Approximately 0.7x10 T cells / kg body weight 8 T cells / kg body weight, approximately 5x10 6 Approximately 0.6 x 10 T cells / kg body weight 8 T cells / kg, or approximately 5x10 6 Approximately 0.5 x 10 T cells / kg body weight 8 T cells / kg are administered to the subject.

[0509] Those skilled in the art will recognize that multiple administrations of the compositions contemplated herein may be required to achieve the desired therapeutic effect. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.

[0510] In certain embodiments, it may be desirable to administer activated immune effector cells to a subject, then draw blood (or perform apheresis) from the subject, activate T cells derived therefrom, and reinfuse these activated and expanded T cells back into the patient. This process may be performed multiple times, every few weeks. In certain embodiments, immune effector cells may be activated from a blood draw of 10cc to 400cc. In certain embodiments, immune effector cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Without being bound by theory, the use of a multiple blood draw / multiple reinfusion protocol may aid in the selection of certain populations of immune effector cells.

[0511] The administration of the compositions contemplated herein may be performed in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, transplantation, or implantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0512] In one embodiment, an effective amount of the composition is administered to a subject in need thereof to increase a cellular immune response to a B cell-related condition in the subject. The immune response may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. A humoral immune response may also be elicited that is mediated primarily by helper T cells that can activate B cells, thus resulting in antibody production. Various techniques can be used to analyze the type of immune response induced by the composition, and are well explained in the art, for example, Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0513] In one embodiment, a method is provided for treating a subject diagnosed with a CD79A and / or CD20 expressing cancer comprising removing immune effector cells from the subject, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding an anti-CD79A CAR and an anti-CD20 CCR as contemplated herein, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0514] In certain embodiments, a method of stimulating an immune effector cell-mediated immunomodulatory response against a target cell population in a subject is provided, the method comprising administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding an anti-CD79A CAR and an anti-CD20 CCR.

[0515] Methods of administering the cell compositions contemplated in certain embodiments include any method that effectively reintroduces ex vivo genetically modified immune effector cells, either directly expressing an anti-CD79A CAR and an anti-CD20 CCR in the subject, or reintroducing genetically modified precursors of immune effector cells that differentiate into mature immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR when introduced into the subject. One method includes transducing peripheral blood T cells ex vivo with a nucleic acid construct contemplated herein and returning the transduced cells to the subject.

[0516] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0517] The foregoing embodiments are described in detail in the figures and examples for clarity and understanding, but in light of the teachings anticipated herein, it will be readily apparent to those skilled in the art that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and not for limiting purposes. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce essentially similar results. EXAMPLES

[0518] Example 1 Construction of anti-CD79A CAR-anti-CD20 CCR bicistronic construct A lentiviral vector was designed, constructed, and validated, comprising a bicistronic construct comprising a humanized anti-CD79A CAR, a T2A self-cleaving polypeptide, and an anti-CD20 CCR. A construct comprising an MNDU3 promoter operably linked to an anti-CD79A CAR comprising a CD8α signal sequence, an anti-CD79A scFv, a CD8α hinge and transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain; a T2A self-cleaving polypeptide; and an anti-CD20 CCR comprising an anti-CD20scFv, a CD8α hinge and transmembrane domain, and a CD28 costimulatory domain was cloned into a lentiviral vector. Exemplary anti-CD79A CAR / T2A / anti-CD20 CCR polypeptide sequences are set forth in SEQ ID NOs: 37 and 39, and exemplary anti-CD79A CAR / T2A / anti-CD20 CCR polynucleotide sequences are set forth in SEQ ID NOs: 38 and 40.

[0519] Example 2 T cells expressing both anti-CD79A CAR and anti-CD20 CCR exhibit antigen-dependent cytokine release Peripheral blood mononuclear cells (PBMCs) were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR (SEQ ID NO: 18) and an anti-CD20 CCR (SEQ ID NO: 33). The polynucleotide is expressed as a fusion protein (SEQ ID NO: 37), in which the anti-CD79A CAR and anti-CD20 CCR are separated by a T2A viral self-cleaving polypeptide. Untransduced (UTD) cells were used as a control. After transduction, cells were expanded for 10 days in T cell growth medium containing IL-2.

[0520] After expansion, transduced and non-transduced T cell cultures were assessed by flow cytometry for anti-CD79A CAR expression using recombinant Fc-CD79A fusion protein conjugated to a PE fluorescent dye. Higher expression was observed in T cells transduced with lentiviral vectors encoding anti-CD79A CAR compared to non-transduced control T cells (Figure 1A).

[0521] The functionality of anti-CD79A CAR-anti-CD20 CCR T cells was also evaluated. Untransduced T cells or T cells transduced with lentiviral vectors encoding anti-CD79A CAR (SEQ ID NO: 18) or encoding anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO: 37) were cultured alone or co-cultured at a 1:1 ratio with tumor cells lacking the target antigen (RD; a rhabdomyosarcoma cell line), and with RD.79A cells (RD modified to express CD79A), RD.CD79A.CD20 cells (RD modified to express CD79A and CD20), and Daudi cells (Burkitt's lymphoma, with high endogenous expression of both CD79a and CD20). After 24 hours of co-culture, supernatants were collected and analyzed for IFNγ and IL-2 cytokines using a Luminex assay. Anti-CD79A CAR-anti-CD20 CCR T cells produced IFNγ cytokines in response only to cell lines expressing CD79A, but not to RD cells. Figure 1B. Anti-CD79A CAR-anti-CD20 CCR T cells showed increased IL-2 production compared to anti-CD79A CAR T cells, because activity of anti-CD20 CCR through its CD28 costimulatory domain induces the PI3K pathway and promotes IL-2 expression. Figure 1C. Graphs show mean + SEM of duplicates from a single PBMC donor.

[0522] Example 3 T cells expressing both anti-CD79A CAR and anti-CD20 CCR respond to target cells expressing CD20 PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors containing polynucleotides encoding anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 37), or anti-CD79A CAR alone (SEQ ID NO: 18). Untransduced (UTD) cells were used as controls. After transduction, cells were expanded for 10 days in T cell growth medium containing IL-2. After expansion, UTD cells, anti-CD79A CAR T cells, and anti-CD79A CAR-anti-CD20 CCR T cells were co-cultured with CD20-negative RD cells or RD.CD20 cells (RD cells modified to express CD20) at a 1:1 ratio. After 24 hours of co-culture, supernatants were collected and analyzed for IFNγ and IL-2 cytokines using Luminex.

[0523] Surprisingly, anti-CD79A CAR-anti-CD20 CCR T cells produced IFNγ and IL-2 cytokines after co-culture with RD.CD20 cells. Figures 2A and 2B. Lack of activity against the RD parental line indicates specific activity against CD20 targets. These data show CAR antigen-independent activity against CCR antigen-expressing cells (lacking CAR antigen). This finding is unexpected since CD28 signaling normally amplifies T cell receptor signaling, and therefore, in the absence of CAR activity signaling through CD3ζ, it is expected that CAR-CCR T cells would not be active against cells expressing CCR antigen alone. These data indicate that anti-CD79A CAR-anti-CD20 CCR T cells can engage in cytotoxic activity against both single CAR or CCR positive target cells as well as dual CAR and CCR positive target cells. Graphs show mean + SEM of duplicates of a single PBMC donor.

[0524] Example 4 T cells expressing both anti-CD79A and anti-CD20 CARs exhibit antigen-dependent cytokine release PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, a T2A self-cleaving polypeptide, and an anti-CD20 CD28 CD3ζ CAR (SEQ ID NO: 41), or a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, a T2A self-cleaving polypeptide, and an anti-CD20 41BB CD3ζ CAR (SEQ ID NO: 45). Untransduced (UTD) cells were used as controls. After transduction, cells were expanded for 10 days in T cell growth medium containing IL-2.

[0525] After expansion, transduced and non-transduced T cell cultures were assessed by flow cytometry for anti-CD79A CAR expression using a recombinant Fc-CD79A fusion protein conjugated to a PE fluorescent dye. Expression was observed in T cells transduced with a lentiviral vector encoding the anti-CD79A CAR compared to non-transduced control T cells (Figure 3A).

[0526] The functionality of anti-CD79A CAR-anti-CD20 CAR T cells was also evaluated. Untransduced T cells or T cells transduced with lentiviral vectors encoding anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CD28 CD3ζ CAR fusion protein (SEQ ID NO: 41) or anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 41BB CD3ζ CAR fusion protein (SEQ ID NO: 45) were cultured alone or co-cultured with tumor cells lacking the target antigen (RD; rhabdomyosarcoma cell line) as well as with RD.79A, RD.CD20 and Daudi cells at a 1:1 ratio. After 24 hours of co-culture, supernatants were collected and analyzed for IFNγ using a Luminex assay. Both dual CD79A-CD20 CAR T cells produced IFNγ cytokines in response to RD cells expressing either target antigen alone, although the response was somewhat weaker compared to the CD79A CAR-CD20 CCR T cell response to either antigen. Both dual CD79A-CD20 CAR T cells also showed a strong response to Daudi cells, but this response was again weaker compared to the CD79A CAR-CD20 CCR T cell response to Daudi cells. Figure 3B. Graph shows the mean + SEM of duplicates from a single PBMC donor.

[0527] Example 5 T cells expressing both anti-CD79A CAR and anti-CD20 CCR exhibit antigen-dependent cytokine release PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR (SEQ ID NO: 19) and an anti-CD20 CCR (SEQ ID NO: 33). The polynucleotide is expressed as a fusion protein (SEQ ID NO: 39), where the anti-CD79A CAR and anti-CD20 CCR are separated by a T2A viral self-cleaving polypeptide. Untransduced (UTD) cells were used as a control. After transduction, cells were expanded for 12 days in T cell growth medium containing IL-2.

[0528] After expansion, transduced and non-transduced T cell cultures were assessed by flow cytometry for anti-CD79A CAR expression using recombinant Fc-CD79A fusion protein conjugated to a PE fluorescent dye. Higher expression was observed in T cells transduced with lentiviral vectors encoding the anti-CD79A CAR compared to non-transduced control T cells (Figure 4A).

[0529] The functionality of anti-CD79A CAR-anti-CD20 CCR T cells was also evaluated. Untransduced T cells or T cells transduced with lentiviral vectors encoding anti-CD79A CAR (SEQ ID NO: 19) or encoding anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO: 39) were cultured alone or co-cultured with tumor cells lacking the target antigen (RD; rhabdomyosarcoma cell line) and with RD.79A cells (RD modified to express CD79A), RD.CD79A.CD20 cells (RD modified to express CD79A and CD20), and REC1 cells (mantle cell lymphoma, with high endogenous expression of both CD79a and CD20) at a 1:1 ratio. After 24 hours of co-culture, supernatants were collected and analyzed for IFNγ and IL-2 cytokines using a Luminex assay. Anti-CD79A CAR-anti-CD20 CCR T cells produced IFNγ cytokines in response only to cell lines expressing CD79A, but not to RD cells. Figure 4B. Anti-CD79A CAR-anti-CD20 CCR T cells showed increased IL-2 production compared to anti-CD79A CAR T cells, because activity of anti-CD20 CCR through its CD28 costimulatory domain induces the PI3K pathway and promotes IL-2 expression. Figure 4C. Graphs show mean + SEM of duplicates from a single PBMC donor.

[0530] Example 6 T cells expressing both anti-CD79A CAR and anti-CD20 CCR respond to target cells expressing CD20 PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors containing polynucleotides encoding anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 39) or encoding anti-CD79A CAR (SEQ ID NO: 19) alone. Untransduced (UTD) cells were used as controls. After transduction, cells were expanded for 10 days in T cell growth medium containing IL-2. After expansion, UTD cells, anti-CD79A CAR T cells, and anti-CD79A CAR-anti-CD20 CCR T cells were co-cultured with CD20-negative RD cells or RD.CD20 cells (RD cells modified to express CD20) at a 1:1 ratio. After 24 hours of co-culture, supernatants were collected and analyzed for IFNγ and IL-2 cytokines using Luminex.

[0531] Surprisingly, anti-CD79A CAR-anti-CD20 CCR T cells produced IFNγ and IL-2 cytokines after co-culture with RD.CD20 cells. Figures 5A and 5B. Lack of activity against the RD parental line indicates specific activity against CD20 targets. These data show CAR antigen-independent activity against CCR antigen-expressing cells (lacking CAR antigen). This finding is unexpected since CD28 signaling normally amplifies T cell receptor signaling, and therefore in the absence of CAR activity signaling through CD3ζ, it is expected that CAR-CCR T cells would not be active against cells expressing CCR antigen alone. These data show that anti-CD79A CAR-anti-CD20 CCR T cells can engage in cytotoxic activity against both single CAR or CCR positive target cells as well as dual CAR and CCR positive target cells. Graphs show mean + SEM of duplicates of a single PBMC donor.

[0532] Example 7 T cells expressing both anti-CD79A and anti-CD20 CARs exhibit antigen-dependent cytokine release PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, a T2A self-cleaving polypeptide, and an anti-CD20 CD28 CD3ζ CAR (SEQ ID NO: 43), or a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, a T2A self-cleaving polypeptide, and an anti-CD20 41BB CD3ζ CAR (SEQ ID NO: 47). Untransduced (UTD) cells were used as controls. After transduction, cells were expanded for 10 days in T cell growth medium containing IL-2.

[0533] After expansion, transduced and non-transduced T cell cultures were assessed by flow cytometry for anti-CD79A CAR expression using recombinant Fc-CD79A fusion protein conjugated to a PE fluorescent dye. Expression was observed in T cells transduced with a lentiviral vector encoding the anti-CD79A CAR compared to non-transduced control T cells (Figure 6A).

[0534] The functionality of anti-CD79A CAR-anti-CD20 CAR T cells was also evaluated. Untransduced T cells or T cells transduced with lentiviral vectors encoding anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CD28 CD3ζ CAR fusion protein (SEQ ID NO: 43) or anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 41BB CD3ζ CAR fusion protein (SEQ ID NO: 47) were cultured alone or co-cultured with tumor cells lacking the target antigen (RD; rhabdomyosarcoma cell line), as well as with RD.79A, RD.CD20, and Daudi cells at a 1:1 ratio. After 24 hours of co-culture, supernatants were collected and analyzed for IFNγ using a Luminex assay. Both dual CD79A-CD20 CAR T cells produced IFNγ cytokines in response to RD cells expressing either target antigen alone, although the response was somewhat weaker compared to the CD79A CAR-CD20 CCR T cell response to either antigen. Both dual CD79A-CD20 CAR T cells also showed a strong response to Daudi cells, but this response was again weaker compared to the CD79A CAR-CD20 CCR T cell response to Daudi cells. Figure 6B. Graph shows the mean + SEM of duplicates from a single PBMC donor.

[0535] Example 8 T cells expressing an anti-CD79A CAR and an anti-CD20 CCR efficiently kill cell lines expressing CD79A or CD20 PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors containing polynucleotides encoding anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion proteins (e.g., SEQ ID NO: 37 or 39). After transduction, cells were expanded for 10 days in T cell growth medium containing IL-2.

[0536] Following expansion, transduced and non-transduced T cell cultures were assessed for in vitro cytotoxic function using non-invasive electrical impedance monitoring by xCELLigence Real Time Cell Analysis (RTCA).

[0537] UTD T cells and T cells expressing anti-CD79A CAR and anti-CD20 CCR were co-cultured with RD (rhabdomyosarcoma) cell lines or RD cell lines engineered to express either CD79A (RD.CD79A) or CD20 (RD.CD20) at ratios of 10:1, 5:1, and 2.5:1. After 6 hours of co-culture, cell index was measured by non-invasive electrical impedance on a CELLigence RTCA MP. Cytotoxicity rates were calculated by normalizing the T cell status cell index to the tumor-alone control cell index. Absence of activity against RD parental cells indicated specific activity against CD79A CAR targets and CD20 CCR targets. Figure 7 (left panel). Transduced T cells showed cytotoxicity to both RD.CD79A cells (Figure 7, center panel) and RD.CD20 cells (Figure 7, right panel). Panels show mean + SD cytotoxicity at effector:target ratios of 10:1, 5:1, and 2.5:1 from duplicates of a single PBMC donor.

[0538] The ability of T cells expressing anti-CD79A CAR and anti-CD20 CCR to kill both CD79a and CD20 single positive targets indicates the novel dual targeting ability of these cells. Without intending to be bound by any particular theory, it is believed that CCR-mediated cytotoxicity may occur via CD20 engagement independent of the CAR target and is a unique and innovative trait of these cells.

[0539] Example 9 T cells expressing an anti-CD79A CAR and an anti-CD20 CCR are effective in a Daudi (Burkuit's lymphoma) tumor model. PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO: 37). After transduction, cells were expanded in T cell expansion medium containing IL-2 for 10 days and then cryopreserved or assessed for functionality.

[0540] In vitro. UTD T cells or T cells expressing anti-CD79A 41BB CD3ζ CAR, T2A, or anti-CD20 CCR fusion proteins were transfected with Daudi tumor cells (CD79A + , CD20 + ) at a 1:1 ratio for 24 hours, after which supernatants were collected and analyzed for IFNγ using Luminex. Figure 8 (left panel) shows the mean + SEM of IFN-cytokines from duplicate assays across three PBMC donors.

[0541] In vivo, 2 × 10 mice were cultured in NSG mice. 6 10 x 10 luciferase-expressing Daudi tumor cells were injected into the mice (5). Thirteen days later, mice (5) were injected with vehicle (medium), 10 x 10 6 UTD T cells, or 10 x 10 expressing anti-CD79A CAR and anti-CD20 CCR 6 T cells were injected into the mice. After 20 days, only CD79A CAR / CD20 CCR treated mice had cleared tumor cells and maintained clearance until the end of the study on day 30. Figure 8 (right panel) shows the mean + SEM for N=5 mice for vehicle, N=4, N=5 for UTD, and N=5 for CD79A CAR / CD20 CCR, except for the 6 day time point. * Asterisks indicate animal sacrifice due to tumor size and animal health.

[0542] Example 10 T cells expressing an anti-CD79A CAR and an anti-CD20 CCR are effective in the NU-DUL-1 ABC DLBCL tumor model. PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO: 37). After transduction, cells were expanded in T cell expansion medium containing IL-2 for 10 days and then cryopreserved or assessed for functionality.

[0543] In vitro. UTD T cells or T cells expressing anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion proteins were transfected with NU-DUL-1 ABC cells (CD79A + ,CD20 + ) at a 1:1 ratio for 24 hours, after which supernatants were collected and analyzed for IFNγ using Luminex. Figure 9 (left panel) shows the mean + SEM of IFNγ cytokines from duplicate assays across three PBMC donors.

[0544] In vivo, 10 × 10 6 luciferase-expressing NU-DUL-1 ABC tumor cells were injected into the mice (5). Fifteen days later, mice (6) were injected with vehicle (medium), 10 x 10 6 UTD T cells, or 5 x 10 expressing anti-CD79A CAR and anti-CD20 CCR 6 T cells were injected. In this model, only CD79A CAR / CD20 CCR treated mice caused tumor regression. Figure 9 (right panel) shows the mean + SEM in N = 5 mice for vehicle, N = 4, N = 5 for UTD, and N = 5 for CD79A CAR / CD20 CCR, except for the 23 day time point. * Asterisks indicate animal sacrifice due to tumor size and animal health.

[0545] Example 11 T cells expressing an anti-CD79A CAR and an anti-CD20 CCR are effective in the Toledo Germinal Center B cell (GCB) DLBCL tumor model. PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO: 37). After transduction, cells were expanded in T cell expansion medium containing IL-2 for 10 days and then cryopreserved or assessed for functionality.

[0546] In vitro. UTD T cells or T cells expressing anti-CD79A 41BB CD3ζ CAR, T2A, or anti-CD20 CCR fusion proteins were cultured with Toledo GCB DLBCL cells (CD79A low , CD20 high ) at a 1:1 ratio for 24 hours, after which supernatants were collected and analyzed for IFNγ using Luminex. Figure 10 (left panel) shows the mean + SEM of IFNγ cytokines from duplicate assays across three PBMC donors.

[0547] In vivo, 50 × 10 6 Each mouse was injected with 100 luciferase-expressing Toledo GCB DLBCL tumor cells. Sixteen days later (tumors approximately 100 mm 3 ), mouse (5) received vehicle (medium), 20 × 10 6 2.5 × 10 UTD T cells, or 2.5 × 10 expressing anti-CD79A CAR and anti-CD20 CCR 6 T cells were injected. Only mice treated with CD79A CAR / CD20 CCR cleared tumor cells and maintained clearance until the end of study day 29, with complete tumor clearance in 3 / 5 mice. Figure 10 (right panel) shows the mean + SEM across N=5 mice for each condition. * Asterisks indicate animal sacrifice due to tumor size and animal health.

[0548] Example 12 CBLB-edited T cells expressing anti-CD79A CAR and anti-CD20 CCR show increased efficacy in the Toledo GCB DLBCL tumor model PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO:37). Three days after activation, transduced cells were electroporated with mRNA encoding a megaTAL (SEQ ID NO:54) that cleaves the CBLB gene and expanded in T cell growth medium containing IL-2 for 10 days, after which they were either cryopreserved or assessed for function.

[0549] In vitro. UTD T cells (+ / - CBLB edited) or T cells expressing anti-CD79A 41BB CD3ζ CAR, T2A, and anti-CD20 CCR fusion proteins (+ / - CBLB edited) were transfected with Toledo GCB DLBCL cells (CD79A low , CD20 high ) at a 1:1 ratio for 24 hours, after which supernatants were collected and analyzed for IFNγ using Luminex. Figure 11 (left panel) shows the mean + SEM of IFNγ cytokines of duplicate assays across three PBMC donors.

[0550] In vivo, 50 × 10 6 Each mouse was injected with 1000 luciferase-expressing Toledo GCB DLBCL tumor cells. After 17 days (tumors were approximately 130 mm 3 ), mouse (5) received vehicle (medium), 5 × 10 6 1 × 10 UTD T cells (+ / -CBLB edited), or 1 × 10 expressing anti-CD79A CAR and anti-CD20 CCR (+ / -CBLB edited) 6T cells were injected into mice. Mice treated with CBLB-edited T cells expressing CD79A CAR / CD20 CCR cleared tumor cells and maintained clearance until the end of the study day, day 21, with 1 / 5 mice completely clearing the tumor. Figure 11 (right panel) shows N=5 mice for Vehicle, except for the 17 day time point, N=3, N=5 for CD79A CAR / CD20 CCR, and N=4, N=5 for CD79A CAR / CD20 CCR (+CBLB edited), except for the 21 day time point. * Asterisks indicate animal sacrifice due to tumor size and animal health.

[0551] Example 13 CBLB-edited T cells expressing anti-CD79A CAR and anti-CD20 CCR show increased efficacy in the Daudi CD20 knockout tumor model PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO: 37). Three days after activation, transduced cells were electroporated with mRNA encoding a megaTAL (SEQ ID NO: 54) that cleaves the CBLB gene and expanded in T cell growth medium containing IL-2 for 10 days before being cryopreserved or assessed for function.

[0552] In vivo, 2 × 10 6 Luciferase-expressing Daudi.CD20KO cells (CD79A + , CD20 - ) was injected into the mouse (5) after 14 days. 6 10 × 10 UTD T cells (+ / - CBLB edited) or expressing anti-CD79A CAR and anti-CD20 CCR (+ / - CBLB edited) 6T cells were injected. Mice treated with CD79A CAR / CD20 CCR expressing T cells showed anti-tumor activity. CBLB edited CD79A CAR / CD20 CCR expressing T cells showed improved anti-tumor activity compared to all other conditions. Figure 12 (right panel) shows the mean + SEM across N=5 mice for all conditions. * Asterisks indicate animal sacrifice due to tumor size and animal health.

[0553] Example 14 Cytokine secretion in CBLB-edited T cells PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO: 37). Three days after activation, transduced cells were electroporated with mRNA encoding a megaTAL (SEQ ID NO: 54) that cleaves the CBLB gene and expanded in T cell growth medium containing IL-2 for 10 days before being cryopreserved or assessed for function.

[0554] Plate UTD T cells (+ / - CBLB edited), or T cells expressing anti-CD79A CAR and anti-CD20 CCR (+ / - CBLB edited), at 1 x 10 in T cell growth medium lacking exogenous IL-2 onto 96-well anti-CD3 (+ / - CBLB edited) plates previously coated with CD3 (1 µg / mL–0.063 µg / mL) and CD28 (5 µg / mL) monoclonal antibodies. 6 Cells were seeded at a concentration of 1000 cells / mL. After 24 hours, supernatants were harvested and cytokine detection was measured via Luminex. CBLB-edited UTD T cells and CD79A CAR / CD20 CCR expressing cells showed increased IL-2 (Figure 13, left panel) and IFNγ (Figure 13, right panel) production. UTD T cell conditions were seeded in duplicate and transduced T cell conditions were seeded in quadruplicate. Figure 13 shows the mean + SEM across N=3 donors.

[0555] Example 15 CBLB-edited T cells expressing anti-CD79A CAR and anti-CD20 CCR show increased IL-2 secretion in the Daudi tumor model PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion protein (SEQ ID NO: 37). Three days after activation, transduced cells were electroporated with mRNA encoding a megaTAL (SEQ ID NO: 54) that cleaves the CBLB gene and expanded in T cell growth medium containing IL-2 for 10 days before being cryopreserved or assessed for function.

[0556] UTD T cells (+ / -CBLB edited) or T cells expressing anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion proteins (+ / -CBLB edited) were co-cultured with Daudi cells at a 1:1 ratio for 24 hours, after which supernatants were collected and analyzed for IL-2 using Luminex. CBLB edited T cells expressing CD79A CAR / CD20 CCR produced increased amounts of IL-2 compared to all other conditions tested. Figure 14 shows the mean + SEM of IL-2 cytokine production of duplicates across a single PBMC donor.

[0557] Example 16 Expansion of CBLB-edited T cells PBMCs were obtained from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors containing polynucleotides encoding anti-CD79A 41BB CD3ζ CAR, T2A, and anti-CD20 CCR fusion proteins (SEQ ID NO: 37). Three days after activation, transduced cells were transduced with mRNA encoding a megaTAL (SEQ ID NO: 54) that cleaves the CBLB gene or an inactive TCRα megaTAL (TCRα DEAD), expanded in T cell growth medium containing IL-2 for 10 days, and then cryopreserved or assessed for function.

[0558] GFP-expressing Daudi cells were seeded at a density of 50,000 per well in 96-well plates in T cell growth medium containing low levels of IL-2. Daudi cells were mixed with 50,000 UTD T cells (CBLB or TCRa DEAD ) or anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion proteins (CBLB or TCRα DEAD ) expressing T cells. After 4 days, half of the T cell growth medium was removed and replaced with fresh medium containing low levels of IL-2. After 3 days, the T cells were resuspended and counted. A total of 50,000 T cells (from each condition) were then transferred to a fresh 96-well plate containing 50,000 Daudi-GFP tumor cells. After 4 days (day 11), the medium was removed and replaced with fresh medium containing low levels of IL-2. After 3 days (day 14), the T cells were resuspended and counted. A total of 50,000 T cells (UTD or transduced (+ / - edited)) were then transferred to a fresh 96-well plate containing 50,000 Daudi-GFP tumor cells. After 3 days (day 17), the medium was removed and replaced with fresh medium containing low levels of IL-2. After 3 days (day 20), the T cells were resuspended and counted. After a third round of tumor cell stimulation, CBLB-edited T cells expressing CD79 CAR / CD20 CCR showed increased proliferation compared to TCRα death control treated cells. Figure 15 shows the mean + SD of 4 replicates per condition.

[0559] Example 17 CBLB-edited T cells expressing anti-CD79A CAR and anti-CD20 CCR show increased IFNγ in the Toledo GCB DLBCL tumor model PBMCs were collected from three healthy human donors and three DLBCL-disease donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors containing polynucleotides encoding anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO:37). Three days after activation, transduced cells were electroporated with mRNA encoding megaTAL (SEQ ID NO:54) that cleaves the CBLB gene and expanded in T cell growth medium containing IL-2 for 10 days before being cryopreserved or assessed for function.

[0560] UTD T cells or T cells expressing anti-CD79A 41BB CD3ζ CAR, T2A, and anti-CD20 CCR fusion proteins (+ / - CBLB edited) were transfected with Toledo GCB DLBCL cells (CD79A low , CD20 high ) at a 1:1 ratio for 24 hours, after which supernatants were collected and analyzed for IFNγ using Luminex. CBLB-edited T cells expressing anti-CD79A 41BB CD3ζ CAR, T2A, anti-CD20 CCR fusion proteins produced more IFNγ cytokines in response to tumor cells compared to UTD or non-edited CD79A CAR / CD20 CCR expressing T cells in all healthy and DLBCL donors. Figure 16 (left panel) shows the mean + SEM of IFNγ cytokines of two duplicate assays across all donors.

[0561] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure. In certain embodiments, for example, the following are provided: (Item 1) A fusion polypeptide comprising an anti-CD79A chimeric antigen receptor (CAR), a polypeptide cleavage signal, and an anti-CD20 chimeric costimulatory receptor (CCR). (Item 2) 2. The fusion polypeptide of item 1, wherein the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof, a first transmembrane domain, a first intracellular costimulatory signaling domain, and a primary signaling domain. (Item 3) The anti-CD79A CAR can be a Fab' fragment, F(ab') 2 Fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) 2 3. The fusion polypeptide of claim 1 or 2, comprising an anti-CD79A antibody or antigen-binding fragment thereof selected from the group consisting of: a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody). (Item 4) 4. The fusion polypeptide of any one of items 1 to 3, wherein the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof that is an scFv. (Item 5) 5. The fusion polypeptide of any one of items 1 to 4, wherein the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof comprising a variable light chain sequence comprising a CDRL1-CDRL3 sequence as set forth in SEQ ID NOs: 1-3 or 9-11, and a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence as set forth in SEQ ID NOs: 4-6 or 12-14. (Item 6) 6. The fusion polypeptide of any one of items 1 to 5, wherein the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof comprising a light chain CDR set forth in SEQ ID NOs: 1 to 3 and a heavy chain CDR set forth in SEQ ID NOs: 4 to 6. (Item 7) 6. The fusion polypeptide of any one of items 1 to 5, wherein the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof comprising a light chain CDR set forth in SEQ ID NOs: 9 to 11 and a heavy chain CDR set forth in SEQ ID NOs: 12 to 14. (Item 8) 6. The fusion polypeptide of any one of items 1 to 5, wherein the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof comprising a variable light chain sequence set forth in any one of SEQ ID NOs: 7 or 15, and a variable heavy chain sequence set forth in any one of SEQ ID NOs: 8 or 16. (Item 9) 6. The fusion polypeptide of any one of items 1 to 5, wherein the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof comprising the variable light chain sequence set forth in SEQ ID NO: 7 and / or the variable heavy chain sequence set forth in SEQ ID NO: 8. (Item 10) 6. The fusion polypeptide of any one of items 1 to 5, wherein the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof comprising the variable light chain sequence set forth in SEQ ID NO: 15 and / or the variable heavy chain sequence set forth in SEQ ID NO: 16. (Item 11) 11. The fusion polypeptide of any one of items 1 to 10, wherein the anti-CD79A CAR comprises a first transmembrane domain isolated from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. (Item 12) 12. The fusion polypeptide of any one of items 1 to 11, wherein the anti-CD79A CAR comprises a first transmembrane domain isolated from CD8α. (Item 13) 13. The fusion polypeptide of any one of items 1 to 12, wherein the anti-CD79A CAR comprises a first costimulatory signaling domain isolated from a costimulatory molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. (Item 14) 14. The fusion polypeptide of any one of items 1 to 13, wherein the anti-CD79A CAR comprises a first costimulatory signaling domain isolated from CD137. (Item 15) 15. The fusion polypeptide of any one of paragraphs 1 to 14, wherein the anti-CD79A CAR comprises a primary signaling domain isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. (Item 16) 16. The fusion polypeptide of any one of items 1 to 15, wherein the anti-CD79A CAR comprises a primary signaling domain isolated from CD3zeta. (Item 17) A fusion polypeptide comprising a variable light chain sequence set forth in any one of SEQ ID NOs: 7 or 15, and a variable heavy chain sequence set forth in any one of SEQ ID NOs: 8 or 16, an anti-CD79A CAR comprising a CD8α hinge domain, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3ζ primary signaling domain, a polypeptide cleavage signal, and an anti-CD20 CCR. (Item 18) 18. The fusion polypeptide according to any one of items 1 to 17, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide. (Item 19) 19. The fusion polypeptide according to any one of items 1 to 18, wherein the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide. (Item 20) 20. The fusion polypeptide according to any one of items 1 to 19, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of foot and mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, Thosea asigna virus (TaV) 2A (T2A) peptide, porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. (Item 21) A fusion polypeptide comprising an anti-CD79A CAR having an amino acid sequence set forth in any one of SEQ ID NOs: 17 to 20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR. (Item 22) 22. The fusion polypeptide of any one of items 1 to 21, wherein the anti-CD20 CCR comprises an anti-CD20 antibody or an antigen-binding fragment thereof, a second transmembrane domain, and a second intracellular costimulatory domain. (Item 23) The anti-CD20 CCR is a Fab' fragment, F(ab') 2 Fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) 2 23. The fusion polypeptide of any one of items 1 to 22, comprising an anti-CD20 antibody or antigen-binding fragment thereof selected from the group consisting of: a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody). (Item 24) 24. The fusion polypeptide of any one of items 1 to 23, wherein the anti-CD20 CCR comprises an anti-CD20 antibody or an antigen-binding fragment thereof that is an scFv. (Item 25) 25. The fusion polypeptide according to any one of items 1 to 24, wherein the anti-CD20 CCR comprises an anti-CD20 antibody or an antigen-binding fragment thereof comprising a variable light chain sequence comprising a CDRL1-CDRL3 sequence as set forth in SEQ ID NOs: 25 to 27 and a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence as set forth in SEQ ID NOs: 28 to 30. (Item 26) 26. The fusion polypeptide of any one of items 1 to 25, wherein the anti-CD20 CCR comprises an anti-CD20 antibody or an antigen-binding fragment thereof comprising the variable light chain sequence set forth in SEQ ID NO: 31 and the variable heavy chain sequence set forth in SEQ ID NO: 32. (Item 27) 27. The fusion polypeptide of any one of items 1 to 26, wherein the anti-CD20 CCR comprises a second transmembrane domain isolated from a polypeptide selected from the group consisting of an alpha or beta chain of the T-cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, and PD1. (Item 28) 28. The fusion polypeptide of any one of items 1 to 27, wherein the anti-CD20 CCR comprises the second transmembrane domain isolated from CD8α. (Item 29) 29. The fusion polypeptide of any one of items 1 to 28, wherein the anti-CD20 CCR comprises a second costimulatory signaling domain isolated from a costimulatory molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. (Item 30) 30. The fusion polypeptide of any one of items 1 to 29, wherein the anti-CD20 CCR comprises a second costimulatory signaling domain isolated from CD28. (Item 31) 31. The fusion polypeptide of any one of items 1 to 30, wherein the anti-CD20 CCR comprises a CD8α hinge domain, a CD8α transmembrane domain, and a CD28 costimulatory signaling domain. (Item 32) A fusion polypeptide comprising an anti-CD79A CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 17 to 20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR comprising the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 35. (Item 33) A fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO:37 or SEQ ID NO:39. (Item 34) A polynucleotide encoding the anti-CD79A CAR and anti-CD20 CCR of any one of items 1 to 33. (Item 35) A polynucleotide encoding the fusion polypeptide according to any one of items 1 to 33. (Item 36) A polynucleotide comprising the sequence set forth in SEQ ID NO:38 or SEQ ID NO:40. (Item 37) A vector comprising a polynucleotide encoding the fusion polypeptide according to any one of Items 1 to 33, or the polynucleotide according to any one of Items 34 to 36. (Item 38) 38. The vector according to item 37, wherein the vector is an expression vector. (Item 39) Item 39. The vector of item 37 or item 38, wherein the vector is an episomal vector. (Item 40) 40. The vector according to any one of items 37 to 39, wherein the vector is a viral vector. (Item 41) 41. The vector according to any one of items 37 to 40, wherein the vector is a retroviral vector. (Item 42) 42. The vector according to any one of items 37 to 41, wherein the vector is a lentiviral vector. (Item 43) A cell expressing the fusion polypeptide according to any one of items 1 to 33. (Item 44) A cell comprising a polynucleotide encoding the fusion polypeptide according to any one of items 1 to 33, a polynucleotide according to any one of items 34 to 36, or a vector according to any one of items 36 to 42. (Item 45) below, (a) an anti-CD79A CAR comprising an anti-CD79A antibody or antigen-binding fragment thereof, a first transmembrane domain, a first intracellular costimulatory signaling domain, and a primary signaling domain; and (b) a cell comprising one or more polynucleotides encoding an anti-CD20 antibody or antigen-binding fragment thereof, a second transmembrane domain, and an anti-CD20 CCR comprising a second intracellular costimulatory signaling domain. (Item 46) The anti-CD79A antibody or antigen-binding fragment thereof and the anti-CD20 antibody or antigen-binding fragment thereof may both be Fab' fragments, F(ab') 2 Fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) 2 46. ​​The cell of item 45, wherein the antibody is independently selected from the group consisting of a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), and a single domain antibody (sdAb, nanobody). (Item 47) The cell of claim 45 or 46, wherein the anti-CD79A antibody or antigen-binding fragment thereof and the anti-CD20 antibody or antigen-binding fragment thereof are both scFv. (Item 48) 48. The cell according to any one of items 45 to 47, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising a CDRL1-CDRL3 sequence set forth in SEQ ID NO: 1 to 3 or 9 to 11, and a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence set forth in SEQ ID NO: 4 to 6 or 12 to 14. (Item 49) 49. The cell according to any one of items 45 to 48, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a light chain CDR set forth in SEQ ID NOs: 1 to 3 and a heavy chain CDR set forth in SEQ ID NOs: 4 to 6. (Item 50) 49. The cell according to any one of items 45 to 48, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a light chain...

Claims

1. A fusion polypeptide comprising: a) an anti-CD79A chimeric antigen receptor (CAR), an anti-CD79A antibody or an antigen-binding fragment thereof; CD8α hinge domain, CD8α transmembrane domain, the CD137 intracellular costimulatory signaling domain, and CD3ζ Primary Signaling Domain and an anti-CD79A CAR comprising: b) a 2A self-cleaving polypeptide; and c) an anti-CD20 chimeric costimulatory receptor (CCR), an anti-CD20 antibody or an antigen-binding fragment thereof; CD8α hinge domain, the CD8α transmembrane domain, and CD28 Costimulatory Signaling Domain and an anti-CD20 CCR comprising wherein the CCR does not include a primary signaling domain. Fusion polypeptides.

2. The fusion polypeptide described in claim 1, which is expressed in a cell and, when exposed to CD20 antigen, is capable of eliciting an immune response even in the absence of CD79A antigen.

3. (a) the anti-CD79A CAR comprises an anti-CD79A antibody, or an antigen-binding fragment thereof, selected from the group consisting of a Fab′ fragment, an F(ab′)2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single chain Fv protein ("scFv"), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody); and / or (b) the anti-CD20 CCR comprises an anti-CD20 antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab' fragment, an F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single chain Fv protein ("scFv"), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody); A fusion polypeptide according to claim 1 or claim 2.

4. The fusion polypeptide of any one of claims 1 to 3, wherein the anti-CD79A CAR and / or anti-CD20 CCR comprises an scFv.

5. (a) said CD79A-specific binding domain comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequences set forth in SEQ ID NOs: 1-3 or 9-11, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences set forth in SEQ ID NOs: 4-6 or 12-14; and / or (b) said anti-CD20 specific binding domain comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequences set forth in SEQ ID NOs:25-27 and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences set forth in SEQ ID NOs:28-30; The method according to any one of claims 1 to 4. Fusion polypeptides.

6. (a) the CD79A-specific binding domain comprises a variable light chain sequence as set forth in any one of SEQ ID NOs: 7 or 15, and a variable heavy chain sequence as set forth in any one of SEQ ID NOs: 8 or 16; and / or (b) the CD20-specific binding domain comprises a variable light chain sequence set forth in SEQ ID NO: 31 and a variable heavy chain sequence set forth in SEQ ID NO: 32; A fusion polypeptide according to any one of claims 1 to 5.

7. The polypeptide cleavage signal comprises: (a) a viral self-cleaving 2A polypeptide; or (b) a viral self-cleaving polypeptide selected from the group consisting of foot and mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, Thosea asigna virus (TaV) 2A (T2A) peptide, porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. The fusion polypeptide according to any one of claims 1 to 6,

8. A fusion polypeptide comprising an anti-CD79A CAR comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17 to 20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR.

9. A fusion polypeptide comprising an anti-CD79A CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 17 to 20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR comprising the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO:

35.

10. A fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO:37 or SEQ ID NO:

39.

11. A polynucleotide encoding the anti-CD79A CAR and anti-CD20 CCR of any one of claims 1 to 9.

12. A polynucleotide encoding the fusion polypeptide according to any one of claims 1 to 10.

13. A polynucleotide comprising the sequence set forth in SEQ ID NO:38 or SEQ ID NO:

40.

14. A vector comprising a polynucleotide encoding the fusion polypeptide according to any one of claims 1 to 10, or the polynucleotide according to any one of claims 11 to 13.

15. The vector described in claim 14, wherein the vector is an expression vector.

16. The vector described in claim 14 or claim 15, wherein the vector is an episomal vector.

17. The vector according to any one of claims 14 to 16, wherein the vector is a viral vector.

18. The vector described in any one of claims 14 to 17, wherein the vector is a retroviral vector.

19. The vector according to any one of claims 14 to 18, wherein the vector is a lentiviral vector.

20. A cell expressing a fusion polypeptide according to any one of claims 1 to 10.

21. A cell comprising a polynucleotide encoding the fusion polypeptide of any one of claims 1 to 10, a polynucleotide of any one of claims 11 to 13, or a vector of any one of claims 14 to 16.

22. A cell comprising a first polynucleotide encoding an anti-CD79A CAR and a second polynucleotide encoding an anti-CD20 CCR according to any one of claims 1 to 9.

23. The cell of claim 21, wherein an isolated polynucleotide encodes the anti-CD79A CAR and the anti-CD20 CCR.

24. The cell described in claim 23, wherein the isolated polynucleotide encodes the anti-CD79A CAR, an IRES sequence and the anti-CD20 CCR.

25. 25. The cell of any one of claims 20 to 24, wherein the cell comprises an insertion or deletion of one or more nucleotides in a homing endonuclease (HE) variant cleavage target site or a megaTAL cleavage target site within the Casitas B-lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene and / or the programmed cell death 1 (PDCD-1) gene, and wherein the insertion or deletion in the CBLB and / or PDCD-1 genes reduces the expression, function, and / or activity of CBLB and / or PDCD-1, respectively.

26. (a) the HE variant introduces one or more insertions or deletions into the HE target site of the CBLB gene set forth in SEQ ID NO:55; (b) the megaTAL introduces one or more insertions or deletions into the megaTAL target site of the CBLB gene set forth in SEQ ID NO:56; (c) the HE variant introduces one or more insertions or deletions into the HE target site of the PDCD-1 gene set forth in SEQ ID NO:51; and / or (d) the megaTAL introduces one or more insertions or deletions into the megaTAL target site of the PDCD-1 gene set forth in SEQ ID NO:52; The cell of claim 25.

27. The cell, a) one or more altered CBLB alleles; b) one or more altered PDCD-1 alleles; c) one or more altered CBLB alleles that do not express or produce CBLB or that express or produce non-functional CBLB; and / or d) one or more altered PDCD-1 alleles that do not express or produce PDCD-1 or that express or produce non-functional PDCD-1 The cell according to any one of claims 20 to 25, comprising:

28. The cell comprising: (a) Hematopoietic cells; (b) immune effector cells; (c) T cells; (d) CD3+, CD4+, and / or CD8+ cells; (e) a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell; or (f) natural killer (NK) cells or natural killer T (NKT) cells The cell according to any one of claims 20 to 27, 29. A population of cells comprising a plurality of cells according to any one of claims 20 to 28.

30. A method for the treatment of a disease comprising administering to a subject a therapeutically effective amount of a hematopoietic stem or progenitor cell of claim 28, comprising administering to said subject a therapeutically effective amount of said hematopoietic stem or progenitor cell of claim 28, and / or administering to said subject a therapeutically effective amount of said hematopoietic stem or progenitor cell of claim 28. b) one or more CD34+ hematopoietic stem or progenitor cells according to claim 28 and one or more T cells according to claim 28 A population of cells containing

31. A composition comprising the cells of any one of claims 20 to 28 and a physiologically acceptable excipient.

32. For use in killing cancer cells expressing CD79A and / or CD20 in a subject; or For use in reducing the number of cancer cells expressing CD79A and / or CD20 in a subject, compared to the number of said cancer cells expressing CD79A and / or CD20 prior to said use.

32. The composition of claim 31.

33. 32. The composition of claim 31 for use in treating cancer in a subject.

34. 34. The composition of claim 32 or claim 33, wherein the cancer is a hematological malignancy.

35. 35. The composition of claim 33 or claim 34, wherein the cancer is osteosarcoma or Ewing's sarcoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myelogenous leukemia (AML), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, isolated plasmacytoma of bone, extramedullary plasmacytoma or chronic myelogenous leukemia (CML).

36. 36. The composition of claim 35, wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL).

37. 37. The composition of claim 36, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL).

38. A method for generating a population of cells according to claim 29 or claim 30.

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