Compositions and methods for treating cancer

Abstract

The present invention provides improved compositions for adoptive cell therapy against cancers expressing CD79A and / or CD20.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 991,314, filed on March 18, 2020, and U.S. Provisional Application No. 62 / 861,838, filed on June 14, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is BLBD_123_02WO_ST25. The text file is 152KB, created on June 5, 2020, and submitted electronically via EFS-Web at the same time as this specification is submitted. Background Art Technical Field

[0005] The present invention relates to improved compositions and methods for treating cancer. More specifically, 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 fusion polypeptides, optionally comprising one or more genome editings, and the use of these compositions for effectively treating cancer.

[0006] Existing technology

[0007] Cancer is a major health problem worldwide. Based on incidence rates from 2008 to 2010, 40.76% of men and women born today will be diagnosed with some form of cancer at some time in their lifetime. 20.37% of men will develop cancer between the ages of 50 and 70, compared to 15.30% of women. On January 1, 2010, approximately 13,027,914 men and women in the United States still had a history of cancer—6,078,974 men and 6,948,940 women. It is estimated that in 2013, 1,660,290 men and women (854,790 men and 805,500 women) in the United States will be diagnosed with cancer, and 580,350 will die from cancer of all sites. (Howlader et al. 2013).

[0008] Malignant transformation of B cells leads to cancer, including but not limited to lymphomas, such as multiple myeloma and non-Hodgkin's lymphoma. The vast majority of patients suffering from B cell malignancies, including non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), are an important contributing factor to cancer deaths. The response of B cell malignancies to various forms of treatment is mixed. Traditional methods of treating B cell malignancies, including chemotherapy and radiation therapy, have limited effectiveness due to toxic side effects.

[0009] There is an unmet need for new treatments for relapsed / refractory diffuse large B-cell lymphoma (DLBCL), as evidenced by several recent setbacks in clinical trials attempting to improve standard R-CHOP (rituximab [Rituxan] with cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy. The Phase III PHOENIX trial combining ibrutinib (Imbruvica) with R-CHOP failed its primary endpoint of improved event-free survival. Results from the CORAL and SCHOLAR-1 trials further demonstrate the high unmet need for patients with refractory DLBCL. In each of these studies, the long-term overall survival (OS) of patients who relapsed within 12 months after stem cell transplantation or had refractory disease was only 15% to 20%.

[0010] The CD19 CAR T-cell therapies Yescarta and Kymriah have been approved for the treatment of patients with relapsed / refractory DLBCL, and the CD19 CAR T-cell therapy JCAR017 is in the approval process. A major obstacle that continues to limit the efficacy of these CAR T-cell therapies is the recurrence of "antigen-negative" cancers. For example, although anti-CD19 CAR T-cell therapy initially resulted in modest response rates in relapsed and refractory acute DLBCL, the likelihood of relapse with CD19-negative blasts is substantial. The combination of modestly effective CAR T-cell therapies and the alarmingly high rate of antigen-negative relapse reaffirms the unmet medical need for providing highly effective CAR T immunotherapies to patients with DLBCL. Summary of the Invention

[0011] The present invention generally provides improved adoptive cell therapy and methods of using the same. More specifically, the present invention provides adoptive cell therapy for preventing, treating, or ameliorating at least one symptom of a cancer that expresses CD79A and / or CD20.

[0012] In various embodiments, a fusion polypeptide is provided, comprising an anti-CD79A chimeric antigen receptor (CAR), a polypeptide cleavage signal, and an anti-CD20 chimeric costimulatory receptor (CCR).

[0013] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof; a first transmembrane domain; a first intracellular co-stimulatory signaling domain; and a primary signaling domain.

[0014] In some 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), Fv, a single-chain Fv protein ("scFv"), a double scFv, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0015] In various embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof as a scFv.

[0016] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof, which comprises: a variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NO: 1-3 or 9-11 and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NO: 4-6 or 12-14.

[0017] 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.

[0018] In some 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.

[0019] In various embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof, comprising a variable light chain sequence as shown in any one of SEQ ID NOs: 7 or 15 and a variable heavy chain sequence as shown in any one of SEQ ID NOs: 8 or 16.

[0020] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof, which comprises the variable light chain sequence as shown in SEQ ID NO:7 and / or the variable heavy chain sequence as shown in SEQ ID NO:8.

[0021] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof, which comprises the variable light chain sequence shown in SEQ ID NO: 15 and / or the variable heavy chain sequence shown in SEQ ID NO: 16.

[0022] 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 a 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.

[0023] In some embodiments, the anti-CD79A CAR comprises the first transmembrane domain isolated from CD8α.

[0024] 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.

[0025] In certain embodiments, the anti-CD79A CAR comprises a first costimulatory signaling domain isolated from CD137.

[0026] In certain embodiments, 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.

[0027] In additional embodiments, the anti-CD79A CAR comprises a primary signaling domain isolated from CD3ζ.

[0028] In some embodiments, the fusion polypeptide comprises an anti-CD79A CAR comprising 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, a CD8α hinge domain, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain, a polypeptide cleavage signal, and an anti-CD20 CCR.

[0029] In certain embodiments, the polypeptide cleavage signal is a viral self-cleavage polypeptide.

[0030] In various embodiments, the polypeptide cleavage signal is a viral autolytic 2A polypeptide.

[0031] 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 virus type A (ERAV) 2A (E2A) peptide, cleaved beta-tetrasomal virus (TaV) 2A (T2A) peptide, porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, Theiler virus 2A peptide and encephalomyocarditis virus 2A peptide.

[0032] In certain embodiments, a fusion polypeptide is provided, comprising: an anti-CD79A CAR comprising an amino acid sequence as shown in any one of SEQ ID NOs: 17-20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR contemplated herein.

[0033] 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.

[0034] 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, (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single-domain antibody (sdAb, nanobody).

[0035] In various embodiments, the anti-CD20 CCR comprises an anti-CD20 antibody or antigen-binding fragment thereof that is a scFv.

[0036] 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.

[0037] 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 the variable heavy chain sequence set forth in SEQ ID NO:32.

[0038] 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 a 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.

[0039] In some embodiments, the anti-CD20 CCR comprises a second transmembrane domain isolated from CD8α.

[0040] In additional 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.

[0041] In various embodiments, the anti-CD20 CCR comprises a second costimulatory signaling domain isolated from CD28.

[0042] In certain embodiments, the anti-CD20 CCR comprises a CD8α hinge, a CD8α transmembrane domain, and a CD28 costimulatory signaling domain.

[0043] In a specific embodiment, a fusion polypeptide is provided, comprising: an anti-CD79A CAR comprising the amino acid sequence shown in any one of SEQ ID NOs: 17-20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR comprising the amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 35.

[0044] In additional embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37 or SEQ ID NO:39.

[0045] In certain embodiments, polynucleotides encoding the anti-CD79A CAR and anti-CD20 CAR contemplated herein are provided.

[0046] In some embodiments, polynucleotides encoding the fusion polypeptides contemplated herein are provided.

[0047] In various embodiments, a polynucleotide is provided comprising the sequence shown in SEQ ID NO: 38 or SEQ ID NO: 40.

[0048] In some embodiments, vectors comprising the polynucleotides contemplated herein are provided.

[0049] In certain embodiments, the vector is an expression vector.

[0050] In other embodiments, the vector is an episomal vector.

[0051] In certain embodiments, the vector is a viral vector.

[0052] In certain embodiments, the vector is a retroviral vector.

[0053] In various embodiments, the vector is a lentiviral vector.

[0054] In various embodiments, cells expressing the fusion polypeptides contemplated herein are provided.

[0055] In certain embodiments, cells comprising a polynucleotide encoding a fusion polypeptide contemplated herein or cells comprising a vector contemplated herein are provided.

[0056] In various embodiments, a cell is provided that comprises one or more polynucleotides encoding: an anti-CD79A CAR comprising 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; and an anti-CD20 CCR comprising an anti-CD20 antibody or an antigen-binding fragment thereof; a second transmembrane domain; and a second intracellular costimulatory signaling domain.

[0057] In a specific embodiment, the anti-CD79A antibody or antigen-binding fragment thereof and the anti-CD20 antibody or antigen-binding fragment thereof are 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), Fv, a single-chain Fv protein ("scFv"), a double scFv, (scFv)2, a miniantibody, a double antibody, a three-antibody, a four-antibody, a disulfide-stabilized Fv protein ("dsFv") and a single domain antibody (sdAb, nanobody).

[0058] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment thereof and the anti-CD20 antibody or antigen-binding fragment thereof are both scFvs.

[0059] In various embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises: a variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NO: 1-3 or 9-11 and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NO: 4-6 or 12-14.

[0060] In other 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.

[0061] 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.

[0062] In various embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in any one of SEQ ID NOs: 7 or 15 and a variable heavy chain sequence as shown in any one of SEQ ID NOs: 8 or 16.

[0063] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO:7 and / or the variable heavy chain sequence shown in SEQ ID NO:8.

[0064] In further embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO: 15 and / or the variable heavy chain sequence shown in SEQ ID NO: 16.

[0065] In certain embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NOs: 25-27 and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NOs: 28-30.

[0066] In various embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO:31 and the variable heavy chain sequence shown in SEQ ID NO:32.

[0067] In a specific embodiment, 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 a 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.

[0068] In further embodiments, the first transmembrane domain and the second transmembrane domain are both isolated from CD8α.

[0069] In various embodiments, the first costimulatory signaling domain and the second costimulatory signaling 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.

[0070] In certain embodiments, the first costimulatory signaling domain is isolated from CD137.

[0071] 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.

[0072] In certain embodiments, the primary signaling domain is isolated from CD3ζ.

[0073] In various embodiments, the second costimulatory signaling domain is isolated from CD28.

[0074] In certain embodiments, the anti-CD20 CCR comprises a CD8α hinge domain, a CD8α transmembrane domain, and a CD28 costimulatory signaling domain.

[0075] In a specific embodiment, 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.

[0076] In some embodiments, the cell comprises a first polynucleotide encoding an anti-CD79A CAR and a second polynucleotide encoding an anti-CD20 CCR.

[0077] In various embodiments, the isolated polynucleotides encode an anti-CD79A CAR and an anti-CD20 CCR.

[0078] In some embodiments, the isolated polynucleotide encodes an anti-CD79A CAR, an IRES sequence, and an anti-CD20 CCR.

[0079] In certain embodiments, the isolated polynucleotide encodes an anti-CD79A CAR, a polypeptide cleavage signal, and an anti-CD20 CCR.

[0080] In various embodiments, the polypeptide cleavage signal is a viral self-cleavage polypeptide.

[0081] In certain embodiments, the polypeptide cleavage signal is a viral autolytic 2A polypeptide.

[0082] 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 virus type A (ERAV) 2A (E2A) peptide, cleaved beta-tetrasomal virus (TaV) 2A (T2A) peptide, porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, Theiler virus 2A peptide and encephalomyocarditis virus 2A peptide.

[0083] In certain embodiments, the cells comprise an insertion or deletion of one or more nucleotides in a homing endonuclease (HE) variant cleavage target site or a megaTAL cleavage target site in the casitas B lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene.

[0084] In certain embodiments, the HE variant introduces one or more insertions or deletions into the HE target site in the CBLB gene shown in SEQ ID NO 55.

[0085] In some embodiments, the megaTAL introduces one or more insertions or deletions into the megaTAL target site in the CBLB gene shown in SEQ ID NO 56.

[0086] In certain embodiments, the insertion or deletion in the CBLB gene reduces CBLB expression, function and / or activity.

[0087] In additional embodiments, the cells comprise one or more modified CBLB alleles.

[0088] In additional embodiments, the cell comprises one or more modified CBLB alleles that do not express or produce CBLB, or express or produce non-functional CBLB.

[0089] In certain embodiments, the cells comprise an insertion or deletion of one or more nucleotides in a homing endonuclease (HE) variant cleavage target site or a megaTAL cleavage target site in the programmed cell death 1 (PDCD-1) gene.

[0090] In some embodiments, the HE variant introduces one or more insertions or deletions into the HE target site in the PDCD-1 gene shown in SEQ ID NO:51.

[0091] In additional embodiments, the megaTAL introduces one or more insertions or deletions into the megaTAL target site in the PDCD-1 gene shown in SEQ ID NO:52.

[0092] In certain embodiments, the insertion or deletion in the PDCD-1 gene reduces PDCD-1 expression, function and / or activity.

[0093] In some embodiments, the cells comprise one or more modified PDCD-1 alleles.

[0094] In certain embodiments, the cells comprise one or more modified PDCD-1 alleles that do not express or produce PDCD-1 or express or produce non-functional PDCD-1.

[0095] In further embodiments, the cell is a hematopoietic cell.

[0096] In certain embodiments, the cell is a hematopoietic stem or progenitor cell.

[0097] In other embodiments, the cells are CD34 + Hematopoietic stem or progenitor cells.

[0098] In certain embodiments, the cell is an immune effector cell.

[0099] In various embodiments, the cell is a T cell.

[0100] In certain embodiments, the cells are CD3 + , CD4+ and / or CD8 + cell.

[0101] In certain embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

[0102] In some embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0103] In various embodiments, cell populations comprising a plurality of cells contemplated herein are provided.

[0104] In additional embodiments, cell populations are provided that comprise one or more hematopoietic stem or progenitor cells contemplated herein and one or more immune effector cells.

[0105] In various embodiments, a method comprising one or more CD34 + A cell population of hematopoietic stem or progenitor cells and one or more T cells.

[0106] In certain embodiments, a composition comprises a genetically modified cell contemplated herein and a physiologically acceptable excipient.

[0107] In some embodiments, a composition comprises a cell population contemplated herein and a physiologically acceptable excipient.

[0108] In certain embodiments, methods are provided for killing cancer cells expressing CD79A or CD20 in a subject, the methods comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0109] In certain embodiments, methods are provided for killing cancer cells expressing CD79A and CD20 in a subject, the methods comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0110] In certain embodiments, methods are provided for killing cancer cells expressing CD79A and / or CD20 in a subject, the methods comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0111] In various embodiments, a method for reducing the number of cancer cells expressing CD79A and CD20 in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composition encompassed herein, sufficient to reduce the number of cancer cells expressing CD79A and CD20 as compared to the number of cancer cells expressing CD79A and CD20 prior to administration.

[0112] In various embodiments, a method for reducing the number of cancer cells expressing CD79A or CD20 in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a composition encompassed herein, the therapeutically effective amount sufficient to reduce the number of cancer cells expressing CD79A or CD20 as compared to the number of cancer cells expressing CD79A or CD20 prior to administration.

[0113] In various embodiments, a method for reducing the number of cancer cells expressing CD79A and / or CD20 in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a composition encompassed herein, the therapeutically effective amount sufficient to reduce the number of cancer cells expressing CD79A and / or CD20 as compared to the number of cancer cells expressing CD79A and / or CD20 prior to administration.

[0114] In some embodiments, provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0115] In certain embodiments, the cancer is a solid cancer.

[0116] In other embodiments, the solid cancer is osteosarcoma or Ewing's sarcoma.

[0117] In certain embodiments, the cancer is a liquid cancer.

[0118] In some embodiments, the cancer is a hematological malignancy.

[0119] In various embodiments, the cancer is non-Hodgkin lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0120] In certain embodiments, the non-Hodgkin lymphoma is Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL).

[0121] In various embodiments, the non-Hodgkin lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0122] 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, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0123] In various embodiments, provided is a method for treating a subject having DLBCL, the method comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0124] In certain embodiments, a method for ameliorating one or more symptoms associated with a cancer expressing CD79A and / or CD20 in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composition encompassed herein sufficient to ameliorate at least one symptom associated with a cancer cell expressing CD79A and / or CD20.

[0125] In certain embodiments, the at least one symptom that is improved is selected from the group consisting of weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal swelling or pain, bone or joint pain, bone fractures, unintentional weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination.

[0126] In certain embodiments, a method of producing a cell population expressing a fusion polypeptide contemplated herein is provided, the method comprising introducing a polynucleotide or vector contemplated herein into the cell population.

[0127] In various embodiments, a method of generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, the method comprising introducing one or more polynucleotides encoding the anti-CD79A CAR and anti-CD20 CCR contemplated herein into the cell population.

[0128] In some embodiments, a method of generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, the method comprising introducing into the cell population a polynucleotide encoding a fusion polypeptide contemplated herein.

[0129] In some embodiments, a method of generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, the method comprising introducing a polynucleotide encoding the fusion polypeptide sequence shown in SEQ ID NO: 37 or SEQ ID NO: 39 into the cell population.

[0130] In certain embodiments, a method of generating a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, the method comprising introducing into the cell population a first polynucleotide encoding the anti-CD79A CAR shown in any one of SEQ ID NOs: 17-20 and a second polynucleotide encoding the anti-CD20 CCR sequence shown in SEQ ID NO: 33 or SEQ ID NO: 35.

[0131] In certain embodiments, a method of producing a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, the method comprising introducing the polynucleotide sequence shown in SEQ ID NO: 38 or SEQ ID NO: 40 into the cell population.

[0132] In various embodiments, a method of producing a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR is provided, the method comprising introducing into the cell population a first polynucleotide sequence shown in any one of SEQ ID NOs: 21 to 24 and a second polynucleotide sequence shown in SEQ ID NO: 34 or SEQ ID NO: 36.

[0133] In certain embodiments, one or more cells in the cell population comprise one or more insertions or deletions in the PDCD-1 gene at the polynucleotide sequence shown in SEQ ID NO:51, which reduce or eliminate PDCD-1 expression and / or function.

[0134] In certain embodiments, a polynucleotide encoding a HE variant that binds to and cleaves the polynucleotide sequence shown in SEQ ID NO: 51 is introduced into the cell population.

[0135] In certain embodiments, one or more cells in the cell population comprise one or more insertions or deletions in the PDCD-1 gene at the polynucleotide sequence shown in SEQ ID NO: 52, which one or more insertions or deletions reduce or eliminate PDCD-1 expression and / or function.

[0136] In some embodiments, a polynucleotide encoding a megaTAL that binds to and cleaves the polynucleotide sequence shown in SEQ ID NO:52 is introduced into the cell population.

[0137] In certain embodiments, one or more cells in the cell population contain one or more insertions or deletions in the CBLB gene at the polynucleotide sequence shown in SEQ ID NO: 55, which reduce or eliminate CBLB expression and / or function.

[0138] In certain embodiments, a polynucleotide encoding a HE variant that binds to and cleaves the polynucleotide sequence shown in SEQ ID NO: 55 is introduced into the cell population.

[0139] In some embodiments, one or more cells in the cell population contain one or more insertions or deletions in the CBLB gene at the polynucleotide sequence shown in SEQ ID NO: 56, which reduce or eliminate CBLB expression and / or function.

[0140] In certain embodiments, a polynucleotide encoding a megaTAL that binds to and cleaves the polynucleotide sequence shown in SEQ ID NO:56 is introduced into the cell population.

[0141] In some embodiments, the cell population comprises hematopoietic stem or progenitor cells.

[0142] In certain embodiments, the cell population comprises CD34 + Hematopoietic stem or progenitor cells.

[0143] In various embodiments, the cell population comprises immune effector cells.

[0144] In certain embodiments, the cell population comprises T cells, NK cells and / or NKT cells.

[0145] In various embodiments, the cell population comprises T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1A-Figure 1C Anti-CD79A CAR and anti-CD20 CCR expression and activity are shown. A) CD79A CAR expression on untransduced PBMC (UTD) or PBMC transduced with a lentiviral vector encoding an anti-CD79A CAR, T2A, and anti-CD20 CCR fusion protein. B) This figure shows the expression of anti-CD79A CAR and anti-CD20 CCR in the absence of target cells or in combination with RD cells (CD79A - 、CD20 - ), RD.CD79A cells (CD79A + 、CD20 - ), RD.CD79A.CD20 cells (CD79A + 、CD20 + ) or Daudi cells (CD79A + 、CD20 + , high expression) co-cultured UTD T cells or T cells expressing anti-CD79A CAR and anti-CD20 CCR. C) This figure shows the IFNγ secretion of RD cells 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) co-cultured UTD T cells, anti-CD79A CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR.

[0147] Figure 2A and Figure 2B The activity of anti-CD79A CAR and anti-CD20 CCR against RD.CD20 cells is shown. A) This figure shows the activity of anti-CD79A CAR and anti-CD20 CCR against RD cells (CD79A - 、CD20 - ) or RD.CD20 cells (CD79A - 、CD20 + ) IFNγ secretion by co-cultured UTD T cells, anti-CD79CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR. C) This figure shows the IFNγ secretion by co-cultured UTD T cells, anti-CD79CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR. - 、CD20 - ) or RD.CD20 cells (CD79A - 、CD20 + ) IL-2 secretion by co-cultured UTD T cells, anti-CD79 CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR.

[0148] Figure 3A and Figure 3B Anti-CD79A CAR and anti-CD20 CAR expression and activity are shown. A) CD79A CAR expression on untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-CD28-CD3ζ fusion polypeptides (middle panel) or lentiviral vectors encoding anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-4-1BB-CD3ζ fusion polypeptides (right panel). B) The figure shows the expression of CD79A CAR on PBMCs in the absence of target cells or with RD cells (CD79A - 、CD20 - ), RD.CD79A cells (CD79A + 、CD20 - ), RD.CD20 cells (CD79A - 、CD20 + ) or Daudi cells (CD79A+ 、CD20 + , high expression) co-cultured 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.

[0149] Figures 4A-4C Anti-CD79A CAR and anti-CD20 CCR expression and activity are shown. A) CD79A CAR expression on untransduced PBMC (UTD) or PBMC transduced with a lentiviral vector encoding an anti-CD79A CAR, T2A, and anti-CD20 CCR fusion protein. B) This figure shows the expression of anti-CD79A CAR and anti-CD20 CCR in the absence of target cells or in combination with RD cells (CD79A - 、CD20 - ), RD.CD79A cells (CD79A + 、CD20 - ) or REC-1 cells (CD79A + 、CD20 + , high expression) co-cultured UTD T cells or T cells expressing anti-CD79A CAR and anti-CD20 CCR. B) This figure shows IFNγ secretion 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) co-cultured UTD T cells, anti-CD79A CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR.

[0150] Figure 5A and Figure 5B The activity of anti-CD79A CAR and anti-CD20 CCR against RD.CD20 cells is shown. A) This figure shows the activity of anti-CD79A CAR and anti-CD20 CCR against RD cells (CD79A - 、CD20 - ) or RD.CD20 cells (CD79A - 、CD20 +) IFNγ secretion by co-cultured UTD T cells, anti-CD79CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR. C) This figure shows the IFNγ secretion by co-cultured UTD T cells, anti-CD79CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR. - 、CD20 - ) or RD.CD20 cells (CD79A - 、CD20 + ) IL-2 secretion by co-cultured UTD T cells, anti-CD79 CAR T cells, or T cells expressing anti-CD79A CAR and anti-CD20 CCR.

[0151] Figure 6A and Figure 6B Anti-CD79A CAR and anti-CD20 CAR expression and activity are shown. A) CD79A CAR expression on untransduced PBMCs (UTD) or PBMCs transduced with lentiviral vectors encoding anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-CD28-CD3ζ fusion polypeptides (middle panel) or lentiviral vectors encoding anti-CD79A-CD8α-4-1BB-CD3ζ, T2A, anti-CD20-CD8α-4-1BB-CD3ζ fusion polypeptides (right panel). B) The figure shows the expression of CD79A CAR on PBMCs in the absence of target cells or with RD cells (CD79A - 、CD20 - ), RD.CD79A cells (CD79A + 、CD20 - ), RD.CD20 cells (CD79A - 、CD20 + ) or Daudi cells (CD79A + 、CD20 + , high expression) co-cultured 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.

[0152] Figure 7 The cytotoxicity of T cells expressing anti-CD79A CAR and anti-CD20 CCR against cell lines engineered to express CD79A or CD20 is shown. Untransduced PBMC (UTD) or PBMC transduced with a lentiviral vector encoding an anti-CD79A CAR, T2A, and anti-CD20 CCR fusion protein were cultured in the absence of RD cells (CD79A - 、CD20- ; Left), RD.79A cells (CD79A + 、CD20 - ; center panel) and RD.20 cells (CD79A - 、CD20 + ; right panel).

[0153] Figure 8 We show that T cells expressing anti-CD79A CAR and anti-CD20 CCR can target Daudi cells (CD79A) in vitro and in vivo. + 、CD20 + , high expression). Untransduced PBMC (UTD) or PBMC transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, and anti-CD20CCR fusion proteins were co-cultured in the presence of Daudi cells at a ratio of 1:1 for 24 hours, and the supernatants were collected and analyzed for IFNγ using Luminex (left panel, n=3). NSG mice were intravenously injected with 2×10 6 luciferase-expressing Daudi cells; 13 days later, five mice were injected with vehicle (culture medium), 10×10 6 UTD T cells or 10 × 10 6 T cells expressing anti-CD79A CAR and anti-CD20 CCR were injected into the mice; and the mice were monitored for 30 days (right panel).

[0154] Figure 9 The efficacy of T cells expressing anti-CD79A CAR and anti-CD20 CCR on NU-DUL-1 cells (DLBCL tumor model) in vitro and in vivo is shown. Untransduced PBMC (UTD) or PBMC transduced with a lentiviral vector encoding anti-CD79A CAR, T2A, and anti-CD20 CCR fusion protein were co-cultured in the presence of NU-DUL-1 cells at a ratio of 1:1 for 24 hours, and the supernatant was collected and analyzed for IFNγ using Luminex (left panel, n=3). NSG mice were injected intravenously with 2×10 6 luciferase-expressing NU-DUL-1 cells; after 15 days, five mice were injected with vehicle (culture medium), 10×10 6 UTD T cells or 5 × 10 6 T cells expressing anti-CD79A CAR and anti-CD20 CCR were injected into the mice; and the mice were monitored for 30 days (right panel).

[0155] Figure 10The efficacy of T cells expressing anti-CD79A CAR and anti-CD20 CCR in vitro and in vivo against Toledo (germinal center B cell (GCB) DLBCL tumor model) is shown. Untransduced PBMC (UTD) or PBMC transduced with a lentiviral vector encoding anti-CD79A CAR, T2A, and anti-CD20 CCR fusion protein were co-cultured in the presence of GCB DLBCL cells at a ratio of 1:1 for 24 hours, and the supernatant was collected and analyzed for IFNγ using Luminex (left panel, n=3). NSG mice were injected intravenously with 50×10 6 GCB DLBCL cells expressing luciferase; after 16 days (100 mm 3 Five mice were injected with vehicle (culture medium), 20×10 6 UTD T cells or 2.5 × 10 6 T cells expressing anti-CD79A CAR and anti-CD20 CCR were injected into the mice; and the mice were monitored for 30 days (right panel).

[0156] Figure 11 The efficacy of T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing is shown in vitro and in vivo against Toledo (Germinal Center B Cell (GCB) DLBCL tumor model). Untransduced PBMC (UTD) or PBMC transduced with lentiviral vectors encoding anti-CD79A CAR, T2A, and anti-CD20 CCR fusion proteins with and without genome editing at the CBLB locus were co-cultured at a 1:1 ratio for 24 hours in the presence of GCB DLBCL cells, and supernatants were collected and analyzed for IFNγ using Luminex (left panel, n=3). NSG mice were injected intravenously with 50×10 6 GCB DLBCL cells expressing luciferase; after 17 days (130 mm 3 Five mice were injected with vehicle (culture medium), 5×10 6 UTD T cells + / - CBLB editing or 1 × 10 6 T cells expressing anti-CD79A CAR and anti-CD20 CCR+ / -CBLB were expressed; and the mice were monitored for 21 days (right panel).

[0157] Figure 12 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 was shown. NSG mice were injected intravenously with 2×10 6luciferase-expressing Daudi.CD20KO cells; 14 days later, five mice were injected with vehicle (culture medium), 20×10 6 UTD T cells + / - CBLB editing or 10 × 10 6 The mice were then transfected with T cells expressing anti-CD79A CAR and anti-CD20 CCR+ / -CBLB editing; and the mice were monitored for 30 days.

[0158] Figure 13 Cytokine secretion in T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing and activated with anti-CD3 and anti-CD28 antibodies is shown. 6 UTD T cells + / - CBLB edited or 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 1 μg / mL to 0.063 μg / mL) and CD28 (5 μg / mL), and IL-2 (left panel) and IFNγ (right panel) were measured via Luminex (n=3).

[0159] Figure 14 Enhanced IL-2 secretion in T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing is shown. UTD T cells + / - CBLB editing and T cells expressing anti-CD79A CAR and anti-CD20 CCR + / - CBLB were co-cultured with Daudi tumor cells (Burkitt lymphoma; CD79 + 、CD20 + ) were co-cultured for 24 h, and the supernatants were collected and analyzed for IL-2 using Luminex.

[0160] Figure 15 T cells treated with CBLB megaTALs showed enhanced proliferation in a continuous restimulation assay compared to T cells treated with TCRα-dead megaTALs. 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 megaTALs or TCRα-dead megaTALs and subjected to a continuous restimulation assay (n=4).

[0161] Figure 16Enhanced cytokine responses of T cells expressing anti-CD79A CAR and anti-CD20 CCR in the presence of CBLB editing for the GCB DLBCL tumor model are shown. 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.

[0162] A brief description of sequence identifiers

[0163] SEQ ID NOs: 1-16 show the amino acid sequences of exemplary light chain CDR sequences, heavy chain CDR sequences, variable domain light chain, and variable domain heavy chain of anti-CD79A CARs encompassed herein.

[0164] SEQ ID NOs: 17-20 show the amino acid sequences of exemplary anti-CD79A CARs.

[0165] SEQ ID NOs: 21-24 show the nucleic acid sequences of exemplary anti-CD79A CARs.

[0166] SEQ ID NOs: 25-32 set forth the amino acid sequences of exemplary light chain CDR sequences, heavy chain CDR sequences, variable domain light chain, and variable domain heavy chain anti-CD20 CCRs encompassed herein.

[0167] SEQ ID NOs: 33 and 35 set forth the amino acid sequences of exemplary anti-CD20 CCRs.

[0168] SEQ ID NOs: 34 and 36 set forth the amino acid sequences of exemplary anti-CD20 CCRs.

[0169] SEQ ID NOs: 37 and 39 show the amino acid sequences of exemplary anti-CD79A CAR-T2A-anti-CD20 CCR fusion proteins.

[0170] SEQ ID NOs: 38 and 40 show the nucleic acid sequences of exemplary anti-CD79A CAR-T2A-anti-CD20 CCR fusion proteins.

[0171] SEQ ID NOs: 41 and 43 show the amino acid sequences of exemplary anti-CD79A CAR-T2A-anti-CD20 CD28zCAR fusion proteins.

[0172] SEQ ID NOs: 42 and 44 show the nucleic acid sequences of exemplary anti-CD79A CAR-T2A-anti-CD20 CD28zCAR fusion proteins.

[0173] SEQ ID NOs: 45 and 47 show the amino acid sequences of exemplary anti-CD79A CAR-T2A-anti-CD20 BBz CAR fusion proteins.

[0174] SEQ ID NOs: 46 and 48 show the nucleic acid sequences of exemplary anti-CD79A CAR-T2A-anti-CD20 BBz CAR fusion proteins.

[0175] SEQ ID NO: 49 is the amino acid sequence of an I-OnuI LHE variant that is reprogrammed to bind to and cleave a target site in the human PDCD-1 gene.

[0176] 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.

[0177] SEQ ID NO: 51 is the I-OnuI LHE variant target site in the human PDCD-1 gene.

[0178] SEQ ID NO: 52 is the megaTAL target site in the human PDCD-1 gene.

[0179] SEQ ID NO:53 is the amino acid sequence of an I-OnuI LHE variant that is reprogrammed to bind to and cleave a target site in the human CBLB gene.

[0180] 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.

[0181] SEQ ID NO: 55 is the I-OnuI LHE variant target site in the human CBLB gene.

[0182] SEQ ID NO: 56 is the megaTAL target site in the human CBLB gene.

[0183] SEQ ID NOs: 57-67 show the amino acid sequences of various linkers.

[0184] SEQ ID NO: 68-92 shows the amino acid sequence of the protease cleavage site and the self-cleaving polypeptide cleavage site. In the foregoing sequences, if X is present, it refers to any amino acid or the absence of a certain amino acid. DETAILED DESCRIPTION

[0185] A. Overview

[0186] Cancers are often a heterogeneous collection of cells that express varying levels of various antigens. Immunotherapies are typically initially selected to target antigens that are expressed on most cancer cells and largely absent on normal cells. Effective targeted immunotherapy will kill the majority of cancer cells expressing the target antigen, leading to partial or complete remission. However, because most cancers are inherently heterogeneous, residual cancer cells that do not express or express low levels of the target antigen remain and can potentially give rise to cancer cells that were not effectively targeted by the initial immunotherapy.

[0187] A major obstacle that still limits the efficacy of adoptive cell therapy is the recurrence of "antigen-negative" cancer. Surprisingly high antigen-negative recurrence rates represent the needs of adoptive cell therapy that have not been met so far. Without wishing to be bound by any particular theory, the inventors have solved the problem of killing the heterologous cancers expressing multiple target antigens by re-engineering immune effector cells (e.g., T cells, NK cells) to express chimeric antigen receptors (CAR) and chimeric costimulatory receptors (CCR) targeting multiple antigens, and synergistically activate multiple intracellular cell signaling pathways to strengthen inflammatory cytokine responses and kill tumor cells carrying one or two target antigens and prevent their recurrence. Surprisingly, the inventors have found that the immune effector cells expressing CAR and CCR for different antigens do not need two antigens to be present on the target cell, so as to trigger inflammatory cytokine responses and kill cells. This is surprising because CCR does not include a signaling domain, therefore, in theory, it should not be able to conduct signals on its own. Therefore, the compositions and methods encompassed herein represent important progress in T cell immunotherapy for heterologous cancers.

[0188] Another obstacle that limits the efficacy of adoptive cell therapy is that immune effector cells become hyporesponsive due to exhaustion mediated by the tumor microenvironment. For example, exhausted T cells have unique molecular markers that are significantly different from natural, 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; increased PD-1 expression is associated with reduced 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, which is involved in the negative regulation of effector T cell activity and persistence. CBLB knockout mouse T cells are overproliferative, produce elevated 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 wishing to be bound by any particular theory, it is expected that disruption of the PDCD-1 and / or CBLB genes in immune effector cells expressing a CAR to a first antigen and a CCR to a second antigen will result in more effective and durable adoptive cell therapy.

[0189] The present invention generally relates to compositions and methods for preventing or treating cancer expressing CD79A and / or CD20 or preventing, treating or improving at least one symptom associated with cancer expressing CD79A and / or CD20. In various embodiments, the present invention relates to adoptive cell therapy for the improvement of cancer expressing CD79A and / or CD20 using genetically modified immune effector cells, wherein the immune effector cells optionally include one or more reductions or elimination of PDCD1 and / or CBLB expression and / or functional genome editing. Genetic methods provide a potential means for enhancing immune recognition and eliminating cancer cells. In a specific embodiment, it is contemplated that the immune effector cells modified to express chimeric antigen receptors (CAR) and chimeric costimulatory receptors (CCR) are to redirect the cytotoxicity of cancer cells for expressing CAR target antigens or CCR target antigens, and to collaboratively enhance the immune effector cell responses to cancer. In a specific preferred embodiment, the immune effector cells modified to express CAR and CCR also include one or more reductions or elimination of PDCD-1 and / or CBLB expression and / or functional genome editing. In other particularly preferred embodiments, the immune effector cells modified to express CAR and CCR further comprise one or more genomic edits that reduce or eliminate the expression and function of CBLB.

[0190] Improved compositions and methods for adoptive cell therapy contemplated in certain embodiments herein provide genetically modified immune effector cells that can be readily expanded, exhibit long-term persistence in vivo, and demonstrate antigen-dependent cytotoxicity against cells expressing CD79A and / or CD20, as well as resistance to immunosuppressive signals in the tumor microenvironment.

[0191] CD79A is also known as B cell antigen receptor complex-associated protein α chain, membrane-bound immunoglobulin-associated protein (MB1, MB-1), surface IgM-associated protein, and Ig-α (IGA). Illustrative 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. Illustrative examples of polypeptide sequences encoding CD79a include, but are not limited to, P11912-1, P11912-2, ENSP00000400605ENSP00000468922, ENSP00000221972, NP_001774.1, and NP_067612.1.

[0192] CD79 consists of two proteins, CD79A and CD79B. CD79A is located on chromosome 19q13.2 and encodes a glycoprotein of 226 amino acids of approximately 47kDa. The exact molecular weight depends on the degree of glycosylation. CD79B is located on chromosome 17q23 and encodes a glycoprotein of 229 amino acids of approximately 37kDa. CD79A and CD79B share an exon-intron structure, both containing a single IgSF Ig domain (111 residues C-type for CD79A and 129 residues V-type for CD79B). Each also contains a highly conserved transmembrane domain and a cytoplasmic tail of 61 (CD79A) or 48 (CD79B) amino acids, which also exhibit remarkable amino acid evolutionary conservation. CD79A and CD79B are expressed by the earliest B cell progenitors. In the absence of μ heavy chains, CD79A / B heterodimers are also observed on the surface of early B cell progenitors, although the protein is not required for the progenitors to commit to the B cell lineage. Later in development, CD79A and CD79B are co-expressed as a mature BCR complex with all isotypes of Ig on the surface of B cells. The CD79 protein is specific for the B lineage and is expressed throughout B lymphocytes. CD79A and CD79B can be used to identify markers for B cell tumors, including DLBCL, most acute leukemias of the precursor B cell type, B cell lines, B cell lymphomas, and some myelomas.

[0193] CD20 is also known as membrane-spanning 4-domain A (MS4A), membrane-spanning 4-domain, subfamily A, member 1, leukocyte surface antigen Leu-16 (LEU-16), B-lymphocyte surface antigen B1 (B1), S7, and common variable immunodeficiency 5 (CVID5). Illustrative examples of polynucleotide sequences encoding CD79A include, but are not limited to, NM_021950.3, NM_152866.2, AK225630.1, X07203.1, AK292168.1, X12530.1, BC002807.2, NM_023945, NM_152867, ENST00000345732, and ENST00000389939. Illustrative examples of polypeptide sequences encoding CD79a include, but are not limited to, P11836, NX_P11836, ENSP00000432219, ENSP00000433519, ENSP00000432270, ENSP00000314620, ENSP00000437002, ENSP00000374589, ENSP00000433179, ENSP00000433277, NP_690605.1, and NP_068769.2.

[0194] CD20 is a member of the transmembrane 4A gene family. Members of this novel protein family are characterized by common structural features and similar intron / exon splicing boundaries and display distinct expression patterns between hematopoietic cells and non-lymphoid tissues. The CD20 gene encodes a B lymphocyte surface molecule that plays a role in B cell development and differentiation into plasma cells. CD20 is expressed in most B cell malignancies, including chronic lymphocytic leukemia, diffuse large B cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0195] In various embodiments, the immune effector cells modified to express CAR and CCR are highly potent; undergo robust in vivo expansion; and recognize and exhibit cytotoxic activity against cancer cells expressing CD79A and / or CD20.

[0196] In various preferred embodiments, the immune effector cells are modified to express a CAR and a CCR and further comprise one or more genome edits that reduce or eliminate the expression and / or function of PDCD-1 and / or CBLB.

[0197] In one embodiment, the immune effector cells are genetically modified to express anti-CD79 CAR and anti-CD20 CCR, and further comprise one or more genome edits that reduce or eliminate the expression and function of CBLB. T cells expressing CAR and CCR are referred to herein as CAR / CCR T cells or CAR / CCR modified T cells.

[0198] In one embodiment, a fusion polypeptide comprising an anti-CD79 CAR, a polypeptide cleavage signal, and an anti-CD20 CCR is contemplated.

[0199] In various embodiments, genetically modified immune effector cells are administered to subjects with cancer cells expressing 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 anti-CD79A CAR and anti-CD20 CCR are administered to subjects with DLBCL.

[0200] Techniques for 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 can generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology that are cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (updated July 2008 by John Wiley and Sons); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, GreenePub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, Volumes I and II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Editors: 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 ed., 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, ed., 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 ed., (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q. E. Colligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., 1991); Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0201] B. Definition

[0202] Before describing the present disclosure in greater detail, it may be helpful to understand the present disclosure by providing definitions of certain terms used herein.

[0203] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used to practice or test specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of this disclosure, the following terms are defined below.

[0204] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one or one or more) of the grammatical object of the article. As an example, "an element" means one element or one or more elements.

[0205] The use of alternatives such as "or" should be understood to mean one, two, or any combination of the alternatives.

[0206] The term "and / or" should be understood to mean one or both of the alternatives.

[0207] 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 quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is within the range of approximately ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0208] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to every numerical value encompassed within the range. 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.

[0209] As used herein, the term "substantially" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is about the same as a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0210] Throughout this specification, unless the context requires otherwise, the words "comprise / comprises / comprising" should be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or any other group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are essential or required, and that no other elements can be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or affect the activity or action described in the disclosure regarding the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are essential or required, and that no other elements are present that materially affect the activity or action of the listed elements.

[0211] Reference throughout this specification to "one embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "another embodiment" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the aforementioned phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that a positive recitation of a feature in one embodiment serves as a basis for excluding that feature from a particular embodiment.

[0212] 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 provide a CAR or CCR that is capable of specifically binding to a target antigen of interest. The binding domain can be derived from natural, synthetic, semisynthetic, or recombinant sources.

[0213] An "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 nucleic acid containing an antigenic determinant, such as those recognized by immune cells. An "isolated antibody or antigen-binding fragment thereof" is an antibody that has been identified and separated and / or recovered from a component of its natural environment.

[0214] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T cell response in an animal, including compositions injected or absorbed into an animal (e.g., compositions comprising a cancer-specific protein). Antigens react with products that elicit specific humoral or cellular immunity, including products induced by heterologous antigens such as the disclosed antigens. In certain embodiments, the target antigen is an epitope of a CD79A or CD20 polypeptide.

[0215] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds. An epitope can be formed by contiguous amino acids or by non-contiguous amino acids connected in parallel by the tertiary folding of the protein. Epitopes formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope typically comprises at least 3, and more typically at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0216] As will be understood by those skilled 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 comprising α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions (and for IgE and IgM, the fourth constant region) of the two heavy chains connected together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have a constant region consisting of three (a row) Ig domains in series, and a hinge domain for increasing flexibility; heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are referred to as "CH2 domains" and "CH3 domains," respectively. Each arm of the Y includes the variable region of a single heavy chain and the first constant region combined with the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0217] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs." CDRs can be defined or identified by conventional methods, such as by sequence according to 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, US Department of Health and Human Services, 1991, which are hereby incorporated by reference), or by structure according to Chothia et al. (Chothia, C. or Lesk, AM, J Mol. Biol., 196(4):901-917 (1987), Chothia, C. et al., Nature, 342:877-883 (1989)).

[0218] Illustrative examples of rules for predicting light chain CDRs include: CDR-L1 starts at approximately residue 24, is preceded by Cys, is about 10-17 residues, and is followed by Trp (typically Trp-Tyr-Gln, but also Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 starts approximately 16 residues after the end of CDR-L1, is typically preceded by Ile-Tyr, but also Val-Tyr, Ile-Lys, Ile-Phe, and is 7 residues; and CDR-L3 starts approximately 33 residues after the end of CDR-L2, is preceded by Cys, is 7-11 residues, and is followed by Phe-Gly-XXX-Gly (SEQ ID NO: 97) (XXX is any amino acid).

[0219] Illustrative examples of rules for predicting heavy chain CDRs include: CDR-H1 begins at approximately residue 26, is preceded by Cys-XXX-XXX-XXX (SEQ ID NO: 94), is 10-12 residues long, and is followed by a Trp (typically Trp-Val, but may also be Trp-Ile, Trp-Ala); CDR-H2 begins approximately 15 residues after the end of CDR-H1, is typically preceded by Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 95), or, in many variations, is 16-19 residues long, and is followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala; and CDR-H3 begins approximately 33 residues after the end of CDR-H2, is preceded by Cys-XXX-XXX (typically Cys-Ala-Arg), is 3 to 25 residues long, and is followed by Trp-Gly-XXX-Gly (SEQ ID NO: 96). ID NO:96).

[0220] In one embodiment, the light chain CDRs and heavy chain CDRs are determined according to the Kabat method.

[0221] In one embodiment, the light chain CDRs and 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 intermediate between the Kabat and Clothia methods, see, e.g., 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 publicly available, e.g., AbYsis (www.bioinf.org.uk / abysis / ).

[0222] Reference to "VL" or "VL" refers to the variable region of an immunoglobulin light chain.

[0223] Reference to "VH" or "VH" refers to the variable region of an immunoglobulin heavy chain.

[0224] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0225] A "chimeric antibody" has framework residues from one species, such as a human, and CDRs from another species, such as a mouse (which typically confer antigen binding). In certain preferred embodiments, the CAR comprises an antigen-specific binding domain that is a chimeric antibody or antigen-binding fragment thereof.

[0226] Human antibodies can be constructed by combining Fv clone variable domain sequences selected from human-derived phage display libraries with known human constant domain sequences as described above. Alternatively, human monoclonal antibodies can be prepared by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described, for example, by 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 human antibody library in the absence of endogenous immunoglobulin production. See, for example, 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, where the human antibodies have similar affinity and specificity to the starting non-human antibodies. (See PCT WO 93 / 06213 published on April 1, 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technology provides completely human antibodies that do not have FR or CDR residues from non-human sources.

[0227] Humanized antibody is the immunoglobulin that comprises people's framework region and one or more CDRs from non-human (such as mouse, rat or synthetic) immunoglobulin.The non-human immunoglobulin that CDR is provided is called " donor ", and the human immunoglobulin that framework is provided is called " acceptor ".In one embodiment, all CDRs are from the donor immunoglobulin in humanized immunoglobulin.Do not need to have constant region, but if exist, they must be substantially identical with human immunoglobulin constant region, i.e. at least about 85-90%, such as about 95% or more identical.Therefore, except possible CDR, all parts of humanized immunoglobulin are substantially identical with the corresponding part of natural human immunoglobulin sequence.Humanization or other monoclonal antibodies can have other conservative amino acid substitutions, and it has basically no influence on antigen binding or other immunoglobulin functions.Humanized antibody can be constructed by genetic engineering (referring to, for example, U.S. Patent number 5,585,089).

[0228] Antibodies include antigen-binding fragments thereof, such as camelid Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein ("scFv"), bis-scFv, (scFv)2, miniantibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv") and single domain antibodies (sdAb, nanobodies), as well as the portion of the full-length antibody responsible for antigen binding. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., 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., WH Freeman & Co., New York, 1997.

[0229] A "heavy chain antibody" is one containing two V H domains and no light chain (Riechmann L. et al., J. Immunol. Methods 231: 25-38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent 6,005,079). "Camelid antibodies" are antibodies containing two V H An antibody isolated from a camel, alpaca, or llama that has a VHH domain and no light chain. "Humanized VHH" or "humanized camelid antibody" refers to a non-human VHH or camelid antibody that has undergone humanization to reduce the potential immunogenicity of the antibody in a human recipient.

[0230] "IgNAR" for "immunoglobulin new antigen receptor" refers to a class of antibodies from the shark immune repertoire that are composed of homodimers of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains. IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and are highly stable and possess potent binding characteristics. The inherent stability can be attributed to (i) the underlying Ig scaffold, which presents a large number of charged and hydrophilic surface-exposed residues compared to conventional antibody VH and VL domains found in murine antibodies; and (ii) structural features in the stabilizing complementarity determining region (CDR) loops, including a pattern of inter-loop disulfide bonds and intra-loop hydrogen bonds.

[0231] 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, whose name reflects its 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.

[0232] "Fv" is the smallest antibody fragment containing a complete antigen binding site. In one embodiment, the two-chain Fv species consists of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly and non-covalently bound. In single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and the heavy chain can associate in a "dimer" structure similar to that in the two-chain Fv species. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. The six HVRs together confer antibody antigen binding specificity. However, even a single variable domain (or half of an Fv comprising only three antigen-specific HVRs) has the ability to recognize and bind antigens, although its affinity is lower than that of the entire binding site.

[0233] Fab fragments contain heavy and light chain variable domains, and also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The difference between Fab' fragments and Fab fragments is that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge domain. Fab'-SH is referred to as Fab' in this article, in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. Bispecific Fab dimer (Fab2) has two Fab' fragments, each of which binds to different antigens. Trispecific Fab trimer (Fab3) has three Fab' fragments, each of which binds to different antigens.

[0234] The term "diabody" refers to an antibody fragment with two antigen-binding sites, the fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VH) in the same polypeptide chain (VH-VL). By using a linker that is too short to pair between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described 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).

[0235] "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).

[0236] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain and in either orientation (e.g., VL-VH or VH-VL). Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the structure required for antigen binding. For a review of scFv, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, New York, 1994), pp. 269-315.

[0237] "Joint " is the amino acid sequence of the adjacent domain of connecting polypeptide or fusion polypeptide.Joint sequence includes " variable region linking sequence ", it is amino acid sequence, and the amino acid sequence connects the VH and VL domains of antibody or its antigen-binding fragment and provides the spacer function compatible with the interaction of two sub-binding domains, so that the resulting polypeptide retains specific binding affinity to the target molecule identical with the antibody including identical light chain variable region and heavy chain variable region.Illustrative examples of connector include glycine polymer (G) n;Glycine-serine polymer (G1-5S1-5) n, wherein n is at least one, two, three, four or five integers;Glycine-alanine polymer;Alanine-serine polymer;And other flexible joints known in the art.Glycine and glycine-serine polymers are relatively unstructured, and therefore can serve as neutral tethers between the domains of fusion protein such as CAR as described herein. Glycine enters a significantly larger phi-psi space than even alanine and is less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Linkers are 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) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 59) (Pomerantz et al. 1995, supra); (GGGGS)n where 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); KESGSVSSEQLAQFRSLD (SEQ ID NO: 62) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO:63); LRQRDGERP (SEQ ID NO:64); LRQKDGGGSERP (SEQ ID NO:65); LRQKD(GGGS)2ERP (SEQ ID NO:66).Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling 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)).

[0238] "Spacer domain" refers to a region that moves the antigen binding domain away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The spacer domain can be derived from natural, synthetic, semisynthetic, or recombinant sources. The spacer domain can be part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge domain or an altered immunoglobulin hinge domain.

[0239] "Hinge domain" is a type of spacer domain that plays a role in positioning the antigen binding domain away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding and activation. The hinge domain is placed between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semisynthetic or recombinant sources. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge domain or an altered immunoglobulin hinge domain.

[0240] By "altered hinge domain" is meant (a) a naturally occurring hinge domain that has up to 30% amino acid variation (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 in length) that has up to 30% amino acid variation (e.g., up to 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions), or (c) a portion of a naturally occurring hinge domain that comprises a core hinge domain (which can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length). The hinge domain can be altered by replacing one or more cysteine ​​and / or proline residues in a naturally occurring immunoglobulin hinge domain with one or more other amino acid residues (e.g., one or more serine residues).

[0241] A "transmembrane domain" refers to the portion of a polypeptide that fuses the extracellular domain to the intracellular domain and anchors the polypeptide to the plasma membrane of the cell. A TM domain can be derived from natural, synthetic, semisynthetic, or recombinant sources.

[0242] "Intracellular signaling domain" refers to a polypeptide that is involved in transducing information about the effective binding of a target antigen to a receptor expressed on an immune effector cell into the interior of the immune effector cell to induce effector cell functions (e.g., activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors), or other cellular responses triggered by the binding of an antigen to a receptor expressed on an immune effector cell.

[0243] The term "effector function" refers to a specialized function of an immune effector cell. The effector function of a T cell can be, for example, cytolytic activity or help or activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Although the entire intracellular signaling domain can generally be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, as long as it transduces an effector function signal. The term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0244] It is well known that the signal generated by the TCR alone is insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Therefore, T cell activation can be considered to be mediated by two different classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex), and co-stimulatory signaling domains that act in an antigen-independent manner to provide secondary or co-stimulatory signals.

[0245] "Primary signaling domain" refers to a signaling domain that regulates primary activation of the TCR complex in a stimulatory or inhibitory manner. A primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0246] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the efficient activation and function of T lymphocytes after binding to an antigen.

[0247] The terms "selectively binds" or "selectively bound" or "selectively binding" or "selectively targeting" describe the preferential binding of a molecule to a target molecule (on-target binding) in the presence of multiple off-target molecules. In certain embodiments, the frequency of selective binding of a HE or megaTAL targeting the PDCD1 gene or CBLB gene to the target DNA binding site is about 5, 10, 15, 20, 25, 50, 100, or 1000 times higher than the frequency of the off-target DNA target binding site.

[0248] "On target" refers to the target site sequence.

[0249] "Off-target" refers to sequences that are similar but not identical to the target site sequence.

[0250] 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 will bind and / or cleave under conditions sufficient to achieve binding and / or cleavage. When referring to a polynucleotide sequence or SEQ ID NO. that relates to only one strand of a target site or target sequence, it is understood that the target site or target sequence to which the nuclease variant binds and / or cleaves is double-stranded and comprises the reference sequence and its complement. In a preferred embodiment, the target site is a sequence in the human PDCD1 gene. In a preferred embodiment, the target site is a sequence in the human CBLB gene.

[0251] "Recombination" refers to a procedure for exchanging genetic information between two polynucleotides, including but not limited to donor capture by non-homologous end joining (NHEJ) and homologous recombination. For the purposes of this disclosure, "homologous recombination (HR)" refers to a special form of such exchange that occurs during the repair of double-strand breaks in cells, such as by homology-directed repair (HDR) mechanisms. This procedure requires nucleotide sequence homology, uses a "donor" molecule as a template to repair a "target" molecule (i.e., a molecule that undergoes a double-strand break), and is variously referred to as "non-crossover gene conversion" or "short tract gene conversion" because it causes genetic information to be transferred from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve mismatch correction of the heteroduplex DNA formed between the target and the donor of the break; and / or "synthesis-dependent strand annealing," wherein the donor is used to resynthesize genetic information that will become part of the target; and / or related procedures. Such specific HR typically results in an alteration of the target molecule sequence such that a portion or all of the donor polynucleotide sequence is incorporated into the target polynucleotide.

[0252] "NHEJ" or "non-homologous end joining" refers to the elimination of double-strand breaks in the absence of a donor repair template or homologous sequence. NHEJ can cause insertions and deletions at the break site. NHEJ is mediated by several subpathways, each of which has different mutational outcomes. The classical NHEJ pathway (cNHEJ) requires the KU / DNA-PKcs / Lig4 / XRCC4 complex to reconnect the two ends together through minimal processing and generally results in precise repair of the break. Alternative NHEJ pathways (altNHEJ) are also active in eliminating dsDNA breaks, but these pathways are significantly more susceptible to mutagenesis and generally cannot precisely repair breaks marked by insertions and deletions. Although not wishing to be bound by any particular theory, it is expected that the use of end-processing enzymes, such as exonucleases, such as Trex2, to modify dsDNA breaks may increase the likelihood of imprecise repair.

[0253] "Cleavage" refers to the breakage 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 cleavage and double-strand cleavage are possible. Double-strand cleavage can occur as a result of two different single-strand cleavage events. DNA cleavage can result in blunt ends or staggered ends. In certain embodiments, the polypeptides and nuclease variants encompassed herein, such as homing endonuclease variants, megaTALs, etc., are used for targeted double-stranded DNA cleavage. The endonuclease cleavage recognition site can be on either DNA strand.

[0254] "Exogenous" molecules are molecules that are not normally present in cells, but are introduced into cells by one or more genetic, biochemical or other methods. Exemplary exogenous molecules include, but are not limited to, small organic molecules, proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the aforementioned molecules, or any complex comprising one or more of the aforementioned molecules. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, biopolymer nanoparticles, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0255] An "endogenous" molecule is a molecule that is normally present in a particular cell at a particular stage of development under specific environmental conditions. Additional endogenous molecules may include proteins.

[0256] "Gene" refers to a DNA region that encodes a gene product, as well as all DNA regions that regulate the production of a gene product, whether or not these regulatory sequences are adjacent to the coding sequence and / or transcribed sequence. A gene includes, but is not limited to, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcriptional response elements, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, origins of replication, matrix attachment sites, and locus control regions.

[0257] "Gene expression" refers to the conversion of the information contained in 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, ribozymes, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNA modified by methods such as capping, polyadenylation, methylation, and editing, as well as proteins modified by methods such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0258] 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 that restores, corrects, disrupts, and / or modifies the expression of a gene or gene product. Genome editing encompassed in certain embodiments comprises introducing one or more nuclease variants (including but not limited to homing endonuclease variants or megaTALs) into a cell to generate DNA damage at or near a target site in the genome of the cell, optionally in the presence of a donor repair template.

[0259] Additional definitions are set forth throughout this disclosure.

[0260] C. Chimeric Antigen Receptor

[0261] In various embodiments, immune effector cells are modified to express a chimeric antigen receptor (CAR) targeting CD79A and a chimeric co-stimulatory receptor targeting CD20 to redirect the cytotoxicity of immune effector cells to cancer cells expressing CD79A and / or CD20. In various embodiments, immune effector cells are modified to express anti-CD79 CAR and anti-CD20 CCR, and the cells also have one or more genome edits that reduce the function and expression of CBLB and / or PDCD-1.

[0262] Chimeric antigen receptor (CAR) is a molecule that combines the specificity of the desired antigen based on antibody with the intracellular domain of T cell receptor activation to produce a chimeric protein that exhibits antigen-specific cellular immune activity. As used herein, the term "chimeric" description is composed of different proteins or DNA parts from different sources. In a specific embodiment, CAR includes an extracellular domain (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain, a costimulatory signaling domain, and a primary signaling domain that are bound to CD79A. The main characteristic of CAR is that it can redirect immune effector cell specificity using the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors, thereby triggering proliferation, cytokine production, phagocytosis, or the generation of molecules that can mediate the cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0263] In various embodiments, the CAR comprises an extracellular binding domain comprising a CD79A-specific binding domain; a transmembrane domain; a co-stimulatory signaling domain and / or a primary signaling domain.

[0264] In certain embodiments, the CAR comprises an extracellular binding domain comprising an anti-CD79A antibody or an antigen-binding fragment thereof; one or more hinge domains or spacer domains; a transmembrane domain; and a co-stimulatory signaling domain and / or a primary signaling domain.

[0265] In a specific embodiment, the CAR comprises an extracellular binding domain comprising an anti-CD79A antibody or an antigen-binding fragment thereof that specifically binds to a human CD79A polypeptide expressed on a target cell (e.g., a cancer cell).

[0266] In certain embodiments, the CD79A-specific binding domain comprises a light chain CDR sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the light chain CDR sequence shown in SEQ ID NO: 1-3 or 9-11. In certain embodiments, the CD79A-specific binding domain comprises a light chain CDR sequence shown in SEQ ID NO: 1-3 or 9-11.

[0267] In a specific embodiment, the CD79A-specific binding domain comprises a heavy chain CDR sequence that has 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 sequence shown in SEQ ID NO: 4-6 or 12-14. In one embodiment, the CD79A-specific binding domain comprises a heavy chain CDR sequence shown in SEQ ID NO: 4-6 or 12-14.

[0268] In certain embodiments, the antigen-specific binding domain is a scFv that binds to a human CD79A polypeptide.

[0269] In a specific embodiment, the antigen-specific binding domain is a humanized Camelid VHH that binds to the human CD79A polypeptide.

[0270] In some embodiments, an 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 sequences shown 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 sequences shown in SEQ ID NOs 4-6 or 12-14.

[0271] In various embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NO: 1-3 or 9-11 and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NO: 4-6 or 12-14.

[0272] In preferred embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in any one of SEQ ID NOs: 7 or 15 and / or a variable heavy chain sequence as shown in any one of SEQ ID NOs: 8 or 16.

[0273] In certain embodiments, anti-CD79A CAR includes linker residues between various domains, for example, linker residues added for the appropriate spacing and conformation of the molecule. In a specific embodiment, the linker is a variable region connecting sequence. Anti-CD79A CAR can include one, two, three, four or five or more linkers. In a specific embodiment, 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 intermediate length of amino acids. In some embodiments, the length of 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. Exemplary linkers include but are not limited to those encoded by SEQ ID NO: 57-67.

[0274] In certain embodiments, the binding domain of an 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 comprises CH2 and CH3 of IgG1, IgG4, or IgD.

[0275] In some embodiments, the binding domain of anti-CD79A CAR is typically followed by one or more "hinge domains", which play a role in positioning the antigen binding domain away from the effector cell surface so that appropriate cell / cell contact, antigen binding and activation can be achieved. Illustrative hinge domains suitable for CARs described herein include hinge domains derived from the extracellular region of type 1 membrane proteins such as CD8 α and CD4, which may be wild-type hinge domains from these molecules or may be changed. In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge. In a preferred embodiment, the hinge domain comprises a CD8 α hinge domain.

[0276] In certain embodiments, anti-CD79A CAR includes a transmembrane (TM) domain derived from (i.e., at least including) a transmembrane region of a T cell receptor α chain or β chain, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, and PD1. In a specific embodiment, the TM domain is synthetic and primarily includes hydrophobic residues, such as leucine and valine.

[0277] In one embodiment, the anti-CD79A CAR comprises a TM domain derived from PD1, CD152, CD28 or CD8α. In another embodiment, the anti-CD79A CAR comprises a TM domain derived from PD1, CD152, CD28 or CD8α and a short oligopeptide or polypeptide linker preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, which connects the TM domain of the CAR to the intracellular signaling domain. In a preferred embodiment, the TM domain is derived from CD8α.

[0278] In a specific embodiment, the anti-CD79A CAR comprises one or more intracellular signaling domains. In a 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 connected in series to the carboxyl terminus of the transmembrane domain in any order.

[0279] 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, CD 134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activating protein 10 (DAP10), adaptor for activated 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 T-cell receptor-associated protein kinase 70 zeta chain (ZAP70).

[0280] In a preferred embodiment, the anti-CD79A CAR comprises a CD28, CD137 or CD134 costimulatory signaling domain.

[0281] In a specific embodiment, the anti-CD79A CAR comprises an ITAM-containing primary signaling domain isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0282] In a preferred embodiment, the anti-CD79A CAR comprises the CD3ζ primary signaling domain.

[0283] In certain embodiments, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof that specifically binds to a CD79A polypeptide expressed on cancer cells.

[0284] In one embodiment, the anti-CD79A CAR comprises an anti-CD79A antibody or antigen-binding fragment that binds to a CD79A polypeptide; a transmembrane domain derived from a polypeptide selected from the group consisting of: an alpha or beta chain of a T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD and one or more intracellular costimulatory signaling domains 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, 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.

[0285] In one embodiment, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide; a hinge domain selected from the group consisting of: IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, PD1 hinge, CD152 hinge, and CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of: an α chain or a β chain of a T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD and one or more intracellular costimulatory signaling domains 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, 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.

[0286] In one embodiment, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide; a hinge domain selected from the group consisting of: IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, PD1 hinge, CD152 hinge, and CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of: an α chain or a β chain of a 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; a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, which connects the TM domain to the intracellular signaling domain of the CAR; and one or more intracellular costimulatory signaling domains 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, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNRFS25, and ZAP70; and the primary signaling domains from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0287] In a specific embodiment, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide; a hinge domain comprising an IgG1 hinge / CH2 / CH3 polypeptide and a CD8α hinge domain; a CD8α transmembrane domain comprising a polypeptide linker having about 3 to about 10 amino acids; a CD137 intracellular co-stimulatory signaling domain; and a CD3ζ primary signaling domain.

[0288] In a specific embodiment, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide; a CD8α hinge domain; a CD8α transmembrane domain comprising a polypeptide linker having about 3 to about 10 amino acids; a CD134 intracellular co-stimulatory signaling domain; and a CD3ζ primary signaling domain.

[0289] In a specific embodiment, the anti-CD79A CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide; a CD8α hinge domain; a CD8α transmembrane domain comprising a polypeptide linker having about 3 to about 10 amino acids; a CD28 intracellular co-stimulatory signaling domain; and a CD3ζ primary signaling domain.

[0290] In a specific embodiment, the anti-CD79A CAR comprises one or more anti-CD79AVHHs that bind to a CD79A polypeptide; a CD8α hinge domain; a CD8α transmembrane domain comprising a polypeptide linker having about 3 to about 10 amino acids; a CD28 intracellular co-stimulatory signaling domain; and a CD3ζ primary signaling domain.

[0291] D. Chimeric co-stimulatory receptors

[0292] In various embodiments, the immune effector cells are modified to express anti-CD79A and anti-CD20 CCRs to redirect the cytotoxicity of the immune effector cells to cancer cells expressing CD79A and / or CD20 and synergistically increase the effectiveness of the immune effector cell therapy. In various embodiments, the immune effector cells are modified to express anti-CD79A and anti-CD20 CCRs to redirect the cytotoxicity of the immune effector cells to cancer cells expressing CD79A or CD20 and further comprise one or more gene edits that disrupt or eliminate PDCD-1 and / or CBLB function and / or activity to synergistically increase the effectiveness of the immune effector cell therapy.

[0293] Chimeric costimulatory receptor (CCR) is a molecule that combines the specificity of the desired antigen based on antibody with T cell receptor-costimulatory domain but lacks primary signal transduction domain. In a specific embodiment, CCR includes an extracellular domain (also referred to as binding domain or antigen-specific binding domain) combined with CD20, a transmembrane domain and a costimulatory signal transduction domain, and lacks primary signal transduction domain. The main feature of CCR is that it redirects immune effector cell specificity in an MHC-independent manner and enhances the ability of immune effector cell responses in the presence of CAR.

[0294] In various embodiments, a CCR comprises an extracellular binding domain comprising a CD20-specific binding domain; a transmembrane domain; and a costimulatory signaling domain, but does not comprise a primary signaling domain.

[0295] In certain embodiments, a 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 does not comprise a primary signaling domain.

[0296] In certain embodiments, the CCR comprises an extracellular binding domain comprising 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).

[0297] In certain embodiments, the CD20-specific binding domain comprises a light chain CDR sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the light chain CDR sequence shown in SEQ ID NOs: 25-27. In certain embodiments, the CD20-specific binding domain comprises a light chain CDR sequence shown in SEQ ID NOs: 25-27.

[0298] In a specific embodiment, the CD20-specific binding domain comprises a heavy chain CDR sequence that has 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 sequence shown in SEQ ID NOs: 28-30. In one embodiment, the CD20-specific binding domain comprises the heavy chain CDR sequence shown in SEQ ID NOs: 28-30.

[0299] In certain embodiments, the antigen-specific binding domain is a scFv that binds to a human CD20 polypeptide.

[0300] In a specific embodiment, the antigen-specific binding domain is a humanized Camelid VHH that binds to the human CD20 polypeptide.

[0301] 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 sequences shown 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 sequences shown in SEQ ID NOs: 28-30.

[0302] In various embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequence set forth in SEQ ID NOs: 25-27 and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence set forth in SEQ ID NOs: 28-30.

[0303] In preferred embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO:31 and / or the variable heavy chain sequence shown in SEQ ID NO:32.

[0304] In certain embodiments, the anti-CD20 CCR comprises linker residues between the various domains, e.g., linker residues added for proper spacing and conformation of the molecule. In certain embodiments, the linker is a variable region linker sequence. The anti-CD20 CCR may comprise one, two, three, four, or five or more linkers. In certain embodiments, 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 in length, or any intermediate length of amino acids.

[0305] In certain embodiments, the binding domain of an 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 comprises CH2 and CH3 of IgG1, IgG4, or IgD.

[0306] In some embodiments, the binding domain of an anti-CD20 CCR is typically followed by one or more "hinge domains," which play a role in positioning the antigen binding domain away from the surface of effector cells so that appropriate cell / cell contact, antigen binding, and activation can be achieved. Illustrative hinge domains suitable for use with the CCRs described herein include hinge domains derived from the extracellular regions of type 1 membrane proteins such as CD8α and CD4, which can be wild-type hinge domains from these molecules or can be altered. In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge. In a preferred embodiment, the hinge domain comprises a CD8α hinge domain.

[0307] In certain embodiments, the anti-CD20 CCR comprises a transmembrane (TM) domain derived from (i.e., comprising at least) the transmembrane region of a T cell receptor α or β chain, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD 1. In a specific embodiment, the TM domain is synthetic and comprises primarily hydrophobic residues, such as leucine and valine.

[0308] 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 and a short oligopeptide or polypeptide linker preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that connects the TM domain to the intracellular signaling domain of the CCR. In a preferred embodiment, the TM domain is derived from CD8α.

[0309] In certain embodiments, the anti-CD20 CCR comprises one or more intracellular signaling domains. In preferred specific embodiments, the anti-CD20 CCR comprises one or more costimulatory signaling domains but lacks a primary signaling domain. The costimulatory signaling domains can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0310] 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, CD 134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activating protein 10 (DAP10), adaptor for activated 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 T-cell receptor-associated protein kinase 70 zeta chain (ZAP70).

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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.

[0315] In one embodiment, the anti-CD20 CCR comprises an anti-CD20 scFv that binds to a CDCD20 polypeptide; a transmembrane domain derived from a polypeptide selected from the group consisting of the alpha or beta chain of a 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; and one or more intracellular costimulatory signaling domains 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, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNRFS25 and ZAP70.

[0316] In one embodiment, the anti-CD20 CCR comprises an anti-CD20 scFv that binds to a CD20 polypeptide; a hinge domain selected from the group consisting of an IgG1 hinge / CH2 / CH3, an IgG4 hinge / CH2 / CH3, a PD1 hinge, a CD152 hinge, and a CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of an α or β chain of a 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; a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, which connects the TM domain to the intracellular signaling domain of the CAR; and one or more intracellular costimulatory signaling domains 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, CD134(OX 40), CD137(4-1BB), CD278(ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNRFS25 and ZAP70.

[0317] In a specific embodiment, the anti-CD20 CCR comprises an anti-CD20 scFv that binds to a CD20 polypeptide; a hinge domain comprising an IgG1 hinge / CH2 / CH3 polypeptide and a CD8α hinge domain; a CD8α transmembrane domain comprising a polypeptide linker having about 3 to about 10 amino acids; and a CD28 intracellular costimulatory signaling domain.

[0318] In a specific embodiment, an anti-CD20 CCR comprises an anti-CD20 scFv that binds a CD20 polypeptide; a CD8α hinge domain; a CD8α transmembrane domain comprising a polypeptide linker having about 3 to about 10 amino acids; and a CD28 intracellular costimulatory signaling domain.

[0319] E. Nuclease variants

[0320] In various embodiments, the immune effector cells modified to express anti-CD79A CAR and anti-CD20 CCR also use nuclease variants, such as homing endonuclease (meganuclease) variants or megaTAL to genetically modify to reduce or eliminate the expression and / or function of PDCD-1 and / or CBLB. The nuclease variants covered in a particular embodiment are suitable for the target site in genome editing people PDCD-1 gene or people CBLB gene and include one or more DNA binding domains and one or more DNA cleavage domains (such as one or more endonucleases and / or exonucleases domains), and optionally one or more joints covered herein. The terms "reprogrammed nuclease", "engineered nuclease" or "nuclease variants" are used interchangeably and refer to the nuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the nuclease has been designed and / or modified from parent or naturally occurring nuclease to combine with and crack double-stranded DNA target sequence.

[0321] In certain embodiments, the nuclease variant binds to and cleaves a target sequence in exon 1 of the PDCD-1 gene, preferably SEQ ID NO: 51 in exon 1 of the PDCD-1 gene, and more preferably the sequence "ATCC" in SEQ ID NO: 51 in exon 1 of the PDCD-1 gene.

[0322] In a preferred embodiment, the nuclease variant binds to and cleaves a target sequence in exon 6 of the CBLB gene, preferably SEQ ID NO: 55 in exon 6 of the CBLB gene, and more preferably the sequence "ATCC" in SEQ ID NO: 55 in exon 6 of the CBLB gene.

[0323] Nuclease variants can be designed and / or modified from naturally occurring nucleases or previous nuclease variants. The nuclease variants encompassed in the specific embodiments can also comprise one or more other functional domains, such as an end processing enzyme domain that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity.

[0324] Illustrative examples of nuclease variants that bind to and cleave target sequences in the PDCD-1 or CBLB gene include, but are not limited to, homing endonuclease (meganuclease) variants and megaTALs.

[0325] 1. Homing endonuclease (meganuclease) variants

[0326] In various embodiments, the homing endonuclease or meganuclease is reprogrammed to introduce a double-strand break (DSB) in a target site in the PDCD-1 gene or the CBLB gene. In a preferred embodiment, the homing endonuclease or meganuclease is reprogrammed to introduce a double-strand break (DSB) in a target site in the CBLB gene.

[0327] In a specific embodiment, the homing endonuclease variant introduces a double-strand break in exon 1 of the PDCD-1 gene, preferably at SEQ ID NO: 51 in exon 1 of the PDCD-1 gene, and more preferably at the sequence "ATCC" in SEQ ID NO: 51 in exon 1 of the PDCD-1 gene.

[0328] In a preferred embodiment, the homing endonuclease variant introduces a double-strand break in exon 6 of the CBLB gene, preferably at SEQ ID NO:55 in exon 6 of the CBLB gene, and more preferably at the sequence "ATCC" in SEQ ID NO:55 in exon 6 of the CBLB gene.

[0329] "Homing endonuclease" and "meganuclease" are used interchangeably and refer to naturally occurring homing endonucleases that recognize cleavage sites with 12-45 base pairs and are generally divided into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK.

[0330] A "reference homing endonuclease" or "reference meganuclease" refers to a wild-type homing endonuclease or a 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.

[0331] An "engineered homing endonuclease," "reprogrammed homing endonuclease," "homing endonuclease variant," "engineered meganuclease," "reprogrammed meganuclease," or "meganuclease variant" refers to a homing endonuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the homing endonuclease has been designed and / or modified from a parent or naturally occurring homing endonuclease to bind to and cleave a DNA target sequence. A homing endonuclease variant can be designed and / or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant. The homing endonuclease variants contemplated in particular embodiments may also comprise one or more additional functional domains, such as an end processing enzyme domain that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity in an end processing enzyme.

[0332] Homing endonuclease (HE) variants do not exist in nature and can be obtained by recombinant DNA technology or by random mutagenesis. HE variants can be obtained by altering one or more amino acids in a naturally occurring HE or HE variant, such as by mutation, substitution, addition, or deletion of one or more amino acids. In certain embodiments, HE variants comprise one or more amino acid changes in the DNA recognition interface.

[0333] In a specific embodiment, the homing endonuclease is an I-OnuI HE variant that binds to and cleaves the human PDCD-1 gene shown in SEQ ID NO:51, wherein the variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:49, or a biologically active fragment thereof.

[0334] In a preferred embodiment, the nested endonuclease is an I-OnuI HE variant that binds to and cleaves the human CBLB gene shown in SEQ ID NO:55, said variant comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:53 or a biologically active fragment thereof.

[0335] 2. MEGATAL

[0336] In various embodiments, megaTALs comprising homing endonuclease variants are reprogrammed to introduce double-strand breaks (DSBs) at target sites in the PDCD-1 gene or the CBLB gene. In preferred embodiments, megaTALs comprising homing endonuclease variants are reprogrammed to introduce double-strand breaks (DSBs) at target sites in the CBLB gene.

[0337] In a specific embodiment, the megaTAL introduces a DSB in exon 1 of the PDCD-1 gene, preferably at SEQ ID NO: 52 in exon 1 of the PDCD-1 gene, and more preferably at the sequence "ATCC" in SEQ ID NO: 52 in exon 1 of the PDCD-1 gene.

[0338] 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, and more preferably at the sequence "ATCC" in SEQ ID NO: 56 in exon 6 of the CBLB gene.

[0339] "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, such as a terminal processing enzyme domain that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, or template-independent DNA polymerase activity.

[0340] A "TALE DNA binding domain" is the DNA binding portion of a transcription activator-like effector (TALE or TAL effector) that mimics plant transcriptional activators to manipulate the plant transcriptome (see, eg, Kay et al., 2007. Science 318:648-651). TALE DNA binding domains contemplated in particular embodiments are engineered de novo or from naturally occurring TALEs, such as AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae, and brg11 and hpx17 from Ralstonia solanacearum. Illustrative examples of TALE proteins used to derive and design DNA binding domains are disclosed in U.S. Patent No. 9,017,967 and references cited therein, all of which are incorporated herein by reference in their entirety.

[0341] In a specific embodiment, the megaTAL binds to and cleaves the target site in the human PDCD-1 gene at SEQ ID NO:52 and comprises the amino acid sequence shown in SEQ ID NO:50.

[0342] In a preferred embodiment, the megaTAL binds to and cleaves the target site in the human CBLB gene at SEQ ID NO:56 and comprises the amino acid sequence shown in SEQ ID NO:54.

[0343] F. Peptide

[0344] Various 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 as set forth in any one of SEQ ID NOs: 1-20, 25-33, 35, 37, 39, 41, 43, 45, and 47.

[0345] Unless otherwise indicated, "polypeptide," "peptide," and "protein" are used interchangeably and are defined in their conventional sense, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length; for example, it may comprise a full-length polypeptide or a polypeptide fragment, and may include one or more post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other naturally occurring and non-naturally occurring modifications known in the art.

[0346] As used herein, "isolated polypeptide" and the like refer to a peptide or polypeptide molecule that has been synthesized, separated, and / or purified in vitro from the cellular environment and from association with other components of the cell, i.e., the peptide or polypeptide molecule is not significantly associated with substances in the body. In certain embodiments, the isolated polypeptide is a synthetic polypeptide, a semisynthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0347] Polypeptides include "polypeptide variants". A polypeptide variant may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the above-described polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of a CAR and / or CCR by introducing one or more substitutions, deletions, additions and / or insertions into a binding domain, hinge, TM domain, co-stimulatory signaling domain, or primary signaling domain (if present). 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, typically wherein 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.

[0348] Polypeptides include "polypeptide fragments". A polypeptide fragment refers to a polypeptide, which may be monomeric or multimeric, having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. Illustrative examples of biologically active polypeptide fragments include antibody fragments. As used herein, the terms "biologically active fragment" or "minimal biologically active fragment" refer 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 activity of a naturally occurring polypeptide. In certain embodiments, a polypeptide fragment may comprise an amino acid chain having a length of at least 5 to about 500 amino acids. , 300, 350, 400, or 450 amino acids in length.

[0349] The polypeptide can also be fused in frame or conjugated to a linker or other sequence to facilitate synthesis, purification or identification of the polypeptide (eg, poly-His), or to enhance binding of the polypeptide to a solid support.

[0350] As described above, in particular embodiments, the polypeptide can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well 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).

[0351] In certain embodiments, polypeptide variants comprises one or more conservative replacements." conservative replacement " is that amino acid is replaced by another amino acid with similar characteristics, makes the technical staff of peptide chemistry field can expect the secondary structure of polypeptide and the replacement that hydrophilicity is constant basically.Modification can be carried out at the structure of the polynucleotide and polypeptide contained in the specific embodiment and still obtained the variant or the derivative polypeptide with desired characteristics being encoded functional molecule.When wishing to change the amino acid sequence of polypeptide to produce the variant polypeptide of equivalence or even improvement, those skilled in the art for example can change one or more codons of coding DNA sequence, for example according to Table 1.

[0352] Table 1 - Amino acid codons

[0353]

[0354]

[0355] Guidance on determining which amino acid residues can be substituted, inserted or deleted without eliminating biological activity can be found using computer programs well known in the art such as DNASTAR, DNA Strider, Geneious, MacVector or Vector NTI software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., replacements of similarly charged or uncharged amino acids. Conservative amino acid changes involve replacement of one of a group of amino acids associated with a side chain. Naturally occurring amino acids are generally divided into four groups: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), non-polar 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 collectively classified as aromatic amino acids. In peptides or proteins, suitable conservative amino acid substitutions 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 will recognize that, generally speaking, single amino acid substitutions in non-essential regions of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0356] As described above, amino acid substitutions can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0357] Polypeptide variants also include glycosylated forms, aggregated conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). As is known in the art, covalent variants can be prepared by linking functional groups to groups found in amino acid chains or N-terminal or C-terminal residues. Variants also include allelic variants, species variants, and mutant proteins. Truncations or deletions in regions that do not affect the functional activity of the protein are also variants.

[0358] In certain embodiments, it is desired that the CAR and CCR be expressed in the same cell. The polynucleotide sequences encoding the CAR and CCR can be separated by an IRES sequence as discussed elsewhere herein.

[0359] In preferred embodiments, fusion polypeptides are contemplated herein.

[0360] In a particularly preferred embodiment, the CAR and CCR are expressed as a fusion polypeptide comprising one or more self-cleaving polypeptide sequences that separate the CAR and CCR.

[0361] Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine or ten or more polypeptide segments. Fusion polypeptides are typically C-terminally connected to the N-terminus, but they can also be C-terminally connected to the C-terminus, N-terminally connected to the N-terminus, or N-terminally connected to the C-terminus. The polypeptides of the 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.

[0362] In a preferred embodiment, the fusion protein comprises an anti-CD79A CAR, a polypeptide cleavage signal, and an anti-CD20 CCR. In another preferred embodiment, the fusion protein comprises an anti-CD20 CCR, a polypeptide cleavage signal, and an anti-CD79A CAR.

[0363] In certain embodiments, the fusion protein comprises an anti-CD79A CAR comprising: a variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NOs: 1-3 or 9-11, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown 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 shown in SEQ ID NOs: 25-27, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NOs: 28-30.

[0364] In a specific embodiment, the fusion polypeptide comprises an anti-CD79A CAR comprising the variable light chain sequence as set forth in SEQ ID NO:7 and / or the variable heavy chain sequence as set forth in SEQ ID NO:8; a polypeptide cleavage signal; and an anti-CD20 CCR comprising the variable light chain sequence as set forth in SEQ ID NO:31 and the variable heavy chain sequence as set forth in SEQ ID NO:32.

[0365] In a specific embodiment, the fusion protein comprises an anti-CD79A CAR comprising: a variable light chain sequence comprising the CDRL1-CDRL3 sequences shown in SEQ ID NOs: 1-3 or 9-11, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences shown in SEQ ID NOs: 4-6 or 12-14, a CD8α hinge and transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain; a polypeptide cleavage signal; and an anti-CD20 CCR comprising: a variable light chain sequence comprising the CDRL1-CDRL3 sequences shown in SEQ ID NOs: 25-27, and a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences shown in SEQ ID NOs: 28-30, a CD8α hinge and transmembrane domain, and a 4-1BB co-stimulatory domain.

[0366] In a specific embodiment, the fusion polypeptide comprises an anti-CD79A CAR comprising the variable light chain sequence as set forth in SEQ ID NO:7 and / or the variable heavy chain sequence as set forth in SEQ ID NO:8, a CD8α hinge and transmembrane domains, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain; a polypeptide cleavage signal; and an anti-CD20 CCR comprising the variable light chain sequence as set forth in SEQ ID NO:31 and the variable heavy chain sequence as set forth in SEQ ID NO:32, a CD8α hinge domain, a CD8α transmembrane domain, and a 4-1BB co-stimulatory domain.

[0367] Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (eg, 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).

[0368] Suitable protease cleavage sites and self-cleaving peptides are known to the skilled person (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary 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 PI (P35) protease, byovirus NIa protease, protease encoded by byovirus RNA-2, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, rice tungro sphericalvirus (RTSV) 3C-like protease, parsnip yellow fleck virus (PYVF) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. In one embodiment, TEV (Tobacco Etch Virus) protease cleavage sites are preferred due to their higher cleavage stringency, such as EXXYXQ(G / S) (SEQ ID NO:68), such as ENLYFQG (SEQ ID NO:69) and ENLYFQS (SEQ ID NO:70), where X represents any amino acid (TEV cleavage occurs between Q and G or Q and S).

[0369] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0370] Illustrative examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041). In certain embodiments, the viral 2A peptide is an aphthous 2A peptide, a potato virus 2A peptide, or a cardiovirus 2A peptide.

[0371] In one embodiment, the viral 2A peptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis virus A (ERAV) 2A peptide, cleometrial beta-tetrasomal virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0372] Illustrative examples of 2A sites are provided in Table 2.

[0373] Table 2

[0374]

[0375]

[0376] 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: 17-20; 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.

[0377] In certain preferred embodiments, the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO:37 or SEQ ID NO:39.

[0378] G. Polynucleotides

[0379] In a preferred embodiment, there is provided a polynucleotide encoding one or more CAR polypeptides, CCR polypeptides or a fusion polypeptide comprising 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 are recombinant, synthesized or isolated. Polynucleotides include but are not limited to: pre-mRNA, messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA or recombinant DNA. A polynucleotide is a length 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, as well as all intermediate lengths of nucleotides, i.e., polymeric forms of ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. It will be readily understood that in this context, "intermediate lengths" means any length between the recited values, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. In certain embodiments, the 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.

[0380] Illustrative examples of polynucleotides include, but are not limited to, SEQ ID NOs: 21-24, 34, 36, 38, 40, 42, 44, 46, and 48 and polynucleotides encoding SEQ ID NOs: 1-20, 25-33, 35, 37, 39, 41, 43, 45, and 47.

[0381] As used herein, "isolated polynucleotide" refers to a polynucleotide that has been purified from its naturally occurring sequences on both sides, for example, a DNA fragment that has been removed from sequences that are usually adjacent to it. In certain embodiments, "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and have been manufactured by human hands. In certain embodiments, the isolated polynucleotide is a synthetic polynucleotide, a semisynthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0382] In various embodiments, the polynucleotides include mRNAs encoding the polypeptides contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0383] In certain embodiments, the polynucleotide may undergo codon optimization. As used herein, the term "codon optimized" refers to replacing codons in a polynucleotide encoding a polypeptide so as 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) variations in codon preference between two or more organisms or genes or synthetically constructed preference tables; (ii) variations in the degree of codon preference within an organism, gene, or gene set; (iii) systematic variations in codons, including environmental variations; (iv) variations in codons derived from decoding tRNAs; (v) variations in codons derived from GC% in one position of a triplet; (vi) variations in the degree of similarity to a reference sequence, such as a naturally occurring sequence; (vii) variations in the codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence upon which the codon substitution set was designed; (x) systematic variations in the codon set for each amino acid; and / or (xi) isolated removal of pseudo-translation start sites.

[0384] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or that hybridize to a reference sequence under stringent conditions as defined below. These terms include polynucleotides in which one or more nucleotides are added, deleted, or modified, or substituted with different nucleotides, as compared to a reference polynucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0385] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1500, 2000, 21000, 22000, 23000, 24000, 25000, 26000, 27000, 28000 100, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides that encode a polypeptide variant 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 the naturally occurring polypeptide. A polynucleotide fragment is a polynucleotide that encodes a 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.

[0386] As used herein, the description "sequence identity" or, for example, "a sequence that is 50% identical to..." refers to the degree to which the sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within the comparison window. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions at which identical nucleic acid bases (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) appear in the two sequences to obtain 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 obtain the percentage of sequence identity. comprising 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, typically wherein the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0387] 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" is at least 12, but often 15 to 18 and usually at least 25 monomer units in length, including nucleotides and amino acid residues. Because two polynucleotides can each contain (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence); and (2) a sequence that is different between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6, typically about 50 to about 100, more typically about 100 to about 150 contiguous positions, in which one sequence is compared after optimally aligning it with a reference sequence having the same number of contiguous positions. In order to carry out optimal comparison to two sequences, the comparison window can include additions or deletions of about 20% or less (i.e., gaps) compared to the reference sequence (not including added or deleted sequences). The optimal sequence alignment for comparing the comparison window can be performed by computer-implemented algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by an inspection and optimal comparison (i.e., producing the highest homology percentage within the comparison window) generated by any of the various selected methods. Reference can also be made to the BLAST program family disclosed in, for example, 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, Unit 19.3 of Chapter 15.

[0388] Terms describing the orientation of polynucleotides include: 5' (usually the end of the polynucleotide with a free phosphate group) and 3' (usually the end of the polynucleotide with a free hydroxyl group (OH)). Polynucleotide sequences can be annotated in a 5' to 3' orientation or a 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated as the "sense" strand, "positive" strand, or "coding" strand because its sequence is consistent with the sequence of the pre-messenger (premRNA) [although it is uracil (U) in RNA and 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" strand, "antisense" strand, "minus" strand, or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in a 3' to 5' orientation or a 3' to 5' sequence written in a 5' to 3' orientation.

[0389] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., nucleotide sequences) that are related by the base pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG 3' is 3'TCA GT AC 5'. The latter sequence is usually written as a reverse complementary sequence, with the 5' end on the left and the 3' end on the right, i.e., 5'CATGACT 3'. A sequence identical to its reverse complementary sequence is called a palindrome. Complementarity can be "partial", in which only the bases of some nucleic acids match according to the base pairing rules. Alternatively, there can be "complete" or "overall" complementarity between nucleic acids.

[0390] In addition, it will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides or their variant fragments, as described herein. In these polynucleotides, some have minimal homology to the nucleotide sequence of any natural gene. Nevertheless, in particular embodiments, polynucleotides that vary due to codon usage differences are encompassed, for example, polynucleotides optimized for human and / or primate codon selection. In addition, alleles of the genes comprising the polynucleotide sequences provided herein can also be used. Alleles are endogenous sequences that change due to one or more sudden changes in nucleotides, such as deletions, additions, and / or substitutions.

[0391] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a gene sequence in a vector that can express RNA and subsequently express a polypeptide. In one embodiment, the nucleic acid cassette contains a target gene, such as a target polynucleotide. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, such as a promoter, an enhancer, a polyadenylic acid sequence, and a target gene, such as a target polynucleotide. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassette is oriented sequentially by position within the vector so that the nucleic acid in the cassette can be transcribed into RNA and translated into a protein or polypeptide when necessary, undergoing the appropriate post-translational modification required for activity in the transformed cell, and translocated to the appropriate compartment by targeting the appropriate intracellular compartment or secreting into the extracellular compartment to obtain biological activity. Preferably, the 3' and 5' ends of the cassette are suitable for rapid insertion into the vector, for example, it has a restriction endonuclease site at each end. In a preferred embodiment, the nucleic acid cassette encodes anti-CD79A CAR and anti-CD20 CCR. The cassette can be removed as a single unit and inserted into a plasmid or viral vector.

[0392] Polynucleotides include target polynucleotides. As used herein, the term "target polynucleotide" refers to a polynucleotide encoding a polypeptide, polypeptide variant, or fusion polypeptide. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 target polynucleotides. In certain embodiments, the target polynucleotide encodes a polypeptide that provides a therapeutic effect in treating or preventing a disease or condition. Target polynucleotides and polypeptides encoded therefrom include polynucleotides encoding wild-type polypeptides and functional variants and fragments thereof. In specific embodiments, the functional variant has at least 80%, at least 90%, at least 95%, or at least 99% identity to the corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, the functional variant or fragment has 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.

[0393] As disclosed elsewhere herein or as known in the art, regardless of the length of the coding sequence itself, the polynucleotides encompassed herein can 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 sites, FRT sites, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, such that the overall length of the polynucleotides can vary significantly. Thus, it is contemplated that polynucleotide fragments of virtually any length can be employed in a particular embodiment, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0394] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of well-established techniques known and available in the art. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0395] Illustrative 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.

[0396] Additional illustrative 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.

[0397] Illustrative examples of viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0398] Illustrative examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; the pLenti4 / V5-DEST vector (Promega) for lentivirus-mediated gene transfer and expression in mammalian cells; TM 、pLenti6 / V5-DEST TM and pLenti6.2 / V5-GW / lacZ (Invitrogen—). In certain embodiments, the coding sequence of the polypeptides disclosed herein can be ligated into such expression vectors to express the polypeptides in mammalian cells.

[0399] In a specific embodiment, carrier is an episomal vector or maintains an extrachromosomal carrier. As used herein, term "episomal" refers to a carrier that can replicate and is not integrated into the host's chromosomal DNA and can not gradually lose from the host cell of division, which also means that the carrier replicates extrachromosomally or additionally.

[0400] "Control elements" or "regulatory sequences" present in an expression vector are those untranslated regions of the vector—origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylation sequence, 5' and 3' untranslated regions—that interact with host cell proteins to effect transcription and translation. These elements can vary in their strength and specificity. Depending on the vector system and host utilized, a variety of suitable transcription and translation elements can be used, including ubiquitous promoters and inducible promoters.

[0401] In certain embodiments, vectors include, but are not limited to, expression vectors and viral vectors, and will include exogenous, endogenous, or heterologous control sequences, such as promoters and / or enhancers. "Endogenous" control sequences are sequences naturally associated with a given gene in the genome. "Exogenous" control sequences are sequences that have been placed in juxtaposition with a gene by genetic manipulation (i.e., molecular biology techniques) such that transcription of the gene is directed by the associated enhancer / promoter. "Heterologous" control sequences are exogenous sequences that are from a different species than the cell being genetically manipulated.

[0402] As used herein, the term "promoter" refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In a specific embodiment, a promoter that functions in mammalian cells includes an AT-rich region approximately 25 to 30 bases upstream of the start transcription site and / or another sequence found 70 to 80 bases upstream of the start of transcription, i.e., a CNCAAT region in which N can be any nucleotide.

[0403] The term "enhancer" refers to a segment of DNA that contains sequences that can provide enhanced transcription and, in some cases, can function independently of its orientation relative to another control sequence. An enhancer can function synergistically or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences that can provide both promoter and enhancer functions.

[0404] The term "operably linked" refers to a juxtaposition in which 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 (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0405] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that continuously or continually permits transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a wide variety of cell and tissue types, or a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in a restricted variety of cell and tissue types, respectively.

[0406] Exemplary ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, 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, H5, P7.5 promoter and P11 promoter from vaccinia virus, elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate 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 β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer negative control region deleted and d1587rev primer binding site replaced (MND) U3 promoter (Haas et al., Journal of Virology. 2003; 77(17): 9439-9450).

[0407] In one embodiment, the vector includes the MNDU3 promoter.

[0408] In one embodiment, the vector comprises an EF1a promoter comprising the first intron of the human EF1a gene.

[0409] In one embodiment, the vector includes the EF1a promoter lacking the first intron of the human EF1a gene.

[0410] As used herein, "conditional expression" can refer to any type of conditional expression, including but not limited to: inducible expression; repressible expression; expression in cells or tissues having a specific physiological, biological or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, by controlling expression by subjecting a cell, tissue, organism, etc., to a treatment or condition that results in increased or decreased expression of the polynucleotide or a polynucleotide encoded by the polynucleotide of interest.

[0411] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoid 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-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0412] Conditional expression can also be achieved by using site-specific DNA recombinase. According to some embodiments, carrier comprises at least one (usually two) site, for carrying out the restructuring mediated by site-specific recombinase. As termed herein, term " recombinase " or " site-specific recombinase " include the excision property or integrative protein, enzyme, cofactor or related protein of the recombination reaction that participates in the one or more recombination sites (for example, two, three, four, five, six, seven, ten, twelve, fifteen, twenty, thirty, forty etc.), it can be wild-type protein (referring to Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutant, derivative (for example, containing the fusion protein of recombinant protein sequence or its fragment), fragment and variant thereof. Illustrative examples of recombinases suitable for use in particular 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.

[0413] The vector may comprise one or more recombination sites for any of a variety of site-specific recombinases. It should be understood that the target site of a site-specific recombinase is a site other than any site required for integration of the vector (e.g., a retroviral vector or a 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.

[0414] For example, one recombination site of Cre recombinase is loxP, which is a 34 base pair sequence comprising two 13 base pair inverted repeats flanking an 8 base pair core sequence (serving as recombinase binding sites) (see Figure 1 of 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).

[0415] Suitable recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).

[0416] Other examples of recognition sequences are the attB, attP, attL and attR sequences, which are recognized by the recombinase lambda integrase, such as phi-c31. SSR only mediates recombination between the heteroplasmic sites attB (length 34 bp) and attP (length 39 bp) (Groth et al., 2000). attB and attP, named after the attachment sites of phage integrase on the bacterial and phage genomes, respectively, contain potential The product sites attL and attR are important for further The mediated recombination is effectively inert (Belteki et al., 2003), making the reaction irreversible. For catalytic insertion, it has been found that DNA carrying attB inserts more easily into genomic attP sites than into attP sites within genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy is to position an attP-bearing "docking site" into a defined locus by homologous recombination, which is then paired with an input sequence carrying attB for insertion.

[0417] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct entry of internal ribosomes into a cistron (protein coding region) into a start codon such as ATG, thereby resulting in cap-independent translation of the gene. 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 a specific embodiment, the vector includes one or more polynucleotides of interest encoding one or more polypeptides. In a specific embodiment, in order to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequence can be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0418] As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome 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 a specific embodiment, the vector comprises a polynucleotide having a consensus Kozak sequence and encoding a desired polypeptide, such as a CAR.

[0419] Elements that guide efficient termination and polyadenylation of heterologous nucleic acid transcripts will increase expression of heterologous genes. Transcription termination signals are generally found downstream of the polyadenylation signal. In specific embodiments, the vector comprises a polyadenylation sequence at the 3' end of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that guides the termination and polyadenylation of nascent RNA transcripts caused by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a polyadenylic acid tail to the 3' end of the coding sequence, thereby promoting increased translation efficiency. Cleavage and polyadenylation are guided by the polyadenylic acid sequence in the RNA. The core polyadenylic acid sequence of mammalian pre-mRNA has two recognition elements flanking the cleavage-polyadenylation site. Typically, the nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of the more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to add up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 poly(A) sequence, the bovine growth hormone poly(A) sequence (BGHpA), the rabbit β-globin poly(A) sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous poly(A) sequence known in the art.

[0420] In some embodiments, the polynucleotide or cell containing the polynucleotide utilizes a suicide gene, including an inducible suicide gene for reducing the risk of direct toxicity and / or uncontrolled proliferation. In specific aspects, the suicide gene does not confers immunity to the host containing the polynucleotide or cell. An example of a suicide gene that can be used is caspase-9 or caspase-8 or cytosine deaminase. Specific chemical inducers of dimerization (CID) can be used to activate caspase-9.

[0421] In a specific embodiment, one or more polynucleotides encoding anti-CD79A CAR and anti-CD20 CCR are introduced into cells (e.g., immune effector cells) by non-viral or viral vectors. In a specific embodiment, the polycistronic polynucleotides encoding anti-CD79A CAR and anti-CD20 CCR are introduced into cells by non-viral or viral vectors. In a specific embodiment, the polycistronic polynucleotides encoding fusion protein are introduced into cells by non-viral or viral vectors, and the fusion protein encodes anti-CD79A CAR, 2A self-cleavage polypeptide and anti-CD20 CCR.

[0422] The term "vector" is used herein to refer to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The transferred nucleic acid is generally connected to a carrier nucleic acid molecule, for example, inserted into a carrier nucleic acid molecule. The vector may include a sequence that guides autonomous replication in the cell, or may include a sequence sufficient to allow integration into the host cell DNA. In a specific embodiment, a non-viral vector is used to deliver one or more polynucleotides encompassed herein into T cells. In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding a polycistronic message encoding anti-CD79A CAR and anti-CD20 CCR.

[0423] 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.

[0424] Illustrative 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.

[0425] Illustrative methods of non-viral delivery of polynucleotides encompassed in particular embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, gene guns, virions, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0426] Illustrative examples of polynucleotide delivery systems suitable for use in the specific embodiments contemplated herein include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofectamine transfection reagents are commercially available (e.g., Transfectam TM and Lipofectin TM Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides have been 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, bacterial-derived, inanimate nanocell-based delivery is also contemplated in certain embodiments.

[0427] In various embodiments, the polynucleotide is an mRNA that is introduced into a cell to transiently express a desired polypeptide. As used herein, "transient" refers to expression of an unintegrated transgene over a period of hours, days, or weeks, wherein the period of expression is less than the period of expression of the polynucleotide if integrated into the genome or contained within a stable plasmid replicon in the cell.

[0428] In a specific embodiment, the mRNA encoding the polypeptide is an in vitro transcribed mRNA. As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. Typically, the in vitro transcribed RNA is produced by an in vitro transcription vector. The in vitro transcription vector comprises a template for producing in vitro transcribed RNA.

[0429] In certain embodiments, the mRNA may further comprise a 5' cap or a modified 5' cap and / or a polyadenylation sequence. As used herein, a 5' cap (also referred to as an RNA cap, an RNA 7-methylguanosine cap, or an RNA mRNA) may further comprise a 5' cap or a modified 5' cap and / or a polyadenylation sequence. 7G The 5' cap is a modified guanine nucleotide added to the "front" or 5' end of a eukaryotic messenger RNA shortly after transcription begins. The 5' cap comprises a terminal group that is linked to the first transcribed nucleotide and is recognized by the ribosome and protected from RNases. The capping portion can be modified to regulate the functionality of the mRNA, such as its stability or translation efficiency. In a specific embodiment, the mRNA comprises a poly(A) sequence of between about 50 and about 5000 adenines. In one embodiment, the mRNA comprises a poly(A) sequence of between about 100 and 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 or more bases. The poly(A) sequence can be chemically or enzymatically modified to regulate mRNA functionality, such as positioning, stability, or translation efficiency.

[0430] In certain embodiments, the viral vectors comprising polynucleotides encompassed herein can be delivered by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical administration, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells transplanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsies, etc.) or universal donor hematopoietic stem cells, which are then reimplanted into the patient.

[0431] In one embodiment, a viral vector comprising a polynucleotide encoding anti-CD79A CAR and anti-CD20 CCR is directly administered to an organism for in vivo transduction of cells. In one embodiment, a viral vector comprising a polynucleotide encoding anti-CD79A CAR, 2A self-cleavage polypeptide and anti-CD20 CCR is directly administered to an organism for in vivo transduction of cells. Alternatively, naked DNA can be administered. It is administered by any approach commonly used for introducing molecules into final contact with blood or tissue cells, including but not limited to injection, infusion, topical administration and electroporation. Suitable methods for administering this nucleic acid are available to those skilled in the art and are well known, and although more than one approach can be used to administer a particular composition, a particular approach can generally provide a more direct and more effective reaction than another approach.

[0432] Illustrative examples of viral vector systems suitable for use in specific embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0433] In various embodiments, one or more polynucleotides encoding a polycistronic message or fusion protein encoding an anti-CD79A CAR and an anti-CD20 CCR are introduced into immune effector cells, optionally comprising one or more genome edits that reduce or eliminate the expression and / or function of PDCD-1 or CBLB, e.g., T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) comprising one or more polynucleotides encoding an anti-CD79A CAR, a 2A autolytic polypeptide, and an anti-CD20 CCR.

[0434] AAV is a small (about 26 nm), replication-defective, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can incorporate its genome into the genome of the host cell. Recombinant AAV (rAAV) typically consists of at least a transgene and its regulatory sequences and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequence is about 145 bp in length. In a specific embodiment, rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0435] In some embodiments, chimeric rAAV is used, ITR sequences are isolated from one AAV serotype, and capsid sequences are isolated from a different AAV serotype. For example, a rAAV having ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2 / AAV6. In specific embodiments, the rAAV vector can include 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 includes ITR sequences derived from AAV2 and capsid sequences derived from AAV6. In a preferred embodiment, the rAAV includes ITR sequences derived from AAV2 and capsid sequences derived from AAV2.

[0436] In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest.

[0437] The construction, preparation, and purification of rAAV vectors have been 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, each of which is incorporated herein by reference in its entirety.

[0438] In various embodiments, one or more polynucleotides encoding a polycistronic message or fusion protein encoding an anti-CD79A CAR and an anti-CD20 CCR are introduced into immune effector cells, optionally comprising one or more genome edits that reduce or eliminate the expression and / or function of PDCD-1 or CBLB, by transducing the cells with a retrovirus, e.g., a lentivirus, comprising one or more polynucleotides encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR.

[0439] 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 subsequently covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in specific 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), foamy virus, Friedreich's murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0440] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; comprising 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, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0441] In various embodiments, the lentiviral vectors encompassed herein include 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 include a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.

[0442] In certain embodiments, the lentiviral vectors encompassed herein can be integrating or non-integrating or integration-defective lentiviruses. As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus that lacks an integrase capable of integrating the viral genome into the host cell's genome. Integration-incompetent viral vectors have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety.

[0443] Exemplary mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116 N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E 157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K1 86T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0444] In one embodiment, the HIV-1 integrase-deficient pol gene comprises a D64V, D116I, D116A, E152G, or E152A mutation; a D64V, D116I, and E152G mutation; or a D64V, D116A, and E152A mutation.

[0445] In one embodiment, the HIV-1 integrase-deficient pol gene comprises a D64V mutation.

[0446] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA that is a direct repeat in its native sequence context and contains the U3, R, and U5 regions.

[0447] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence comprises the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus (e.g., HIV-1 and HIV-2). Suitable FLAP elements are described in U.S. Patent 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 element. In yet another embodiment, the lentiviral vector comprises a cPPT or CTS element. In yet another embodiment, the lentiviral vector does not comprise a cPPT or CTS element.

[0448] As used herein, the term "packaging signal" or "packaging sequence" refers to a psi[Ψ] sequence located within the retroviral genome that is required for insertion of viral RNA into the viral capsid or particle, see, e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0449] The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al. 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE).

[0450] In certain embodiments, expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally transcription termination signals into the vector. A variety of post-transcriptional regulatory elements can increase expression of heterologous nucleic acids on proteins, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); etc. (Liu et al., 1995, Genes Dev., 9:1766).

[0451] Due to the modification of LTR, lentiviral vectors preferably contain several safety enhancements. "Self-inactivation" (SIN) vectors refer to replication-defective vectors, for example, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription from exceeding the first round of viral replication. Additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, viral 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.

[0452] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus that has a viral envelope protein that has been replaced with an envelope protein from another virus that has preferred properties. For example, HIV can be pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope protein, which allows HIV to infect a wider range of cells because the HIV envelope protein (encoded by the env gene) normally targets the virus to CD4 + Presenting cells.

[0453] In certain embodiments, lentiviral vectors are generated according to known methods. See, for example, 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.

[0454] According to certain specific embodiments encompassed herein, most or all of the viral vector backbone sequences are derived from lentiviruses, such as HIV-1. However, it will be appreciated that retroviral and / or lentiviral sequences from many different sources may be used or combined, and that a variety of substitutions and alterations of certain lentiviral sequences in the lentiviral sequences may be accommodated without compromising the ability of the transfer vector to perform the functions described herein. In addition, a variety of 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 which may be suitable for producing viral vectors or transfer plasmids encompassed herein.

[0455] In various embodiments, one or more polynucleotides encoding a polycistronic message or fusion protein encoding an anti-CD79A CAR and an anti-CD20 CCR are introduced into immune effector cells, optionally comprising one or more genome edits that reduce or eliminate the expression and / or function of PDCD-1 or CBLB, by transducing the cells with an adenovirus comprising one or more polynucleotides encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR.

[0456] Adenovirus-based vectors are capable of extremely high transduction efficiencies in many cell types and do not require cell division. High titers and high expression levels have been achieved using these vectors. These vectors can be produced in large quantities in relatively simple systems. Most adenovirus vectors are engineered so that the transgene replaces the Ad E1a, E1b, and / or E3 genes; the replication-defective vectors are then propagated in human 293 cells that provide the missing gene function in trans. Ad vectors can transduce a variety of tissue types in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0457] The production and propagation of replication-deficient current adenoviral vectors can utilize a unique helper cell line named 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses the E1 protein (Graham et al., 1977). Since the E3 region can be allocated from the adenoviral genome (Jones and Shenk, 1978), current adenoviral vectors carry foreign DNA in the E1, D3 region, or both regions with the help of 293 cells (Graham and Prevec, 1991). Adenoviral 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). Studies on the administration of recombinant adenoviruses to different tissues include intratracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic intracerebral inoculation (Le Gal La Salle et al., 1993). An example of the use of Ad vectors in clinical trials involves polynucleotide therapy for anti-tumor immunity with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0458] In various embodiments, one or more polynucleotides encoding a polycistronic message or fusion protein encoding an anti-CD79A CAR and an anti-CD20 CCR are introduced into immune effector cells that optionally contain one or more genome edits that reduce or eliminate the expression and / or function of PDCD-1 or CBLB by using a herpes simplex virus (e.g., HSV-1, HSV-2) comprising one or more polynucleotides encoding an anti-CD79A CAR and an anti-CD20 CCR, and the fusion protein encoding an anti-CD79A CAR, a 2A self-cleaving polypeptide, and an anti-CD20 CCR.

[0459] Mature HSV virions are composed of an enveloped icosahedral capsid, wherein the viral genome is composed of a 152kb linear double-stranded DNA molecule. In one embodiment, the viral vector based on HSV lacks one or more essential or non-essential HSV genes. In one embodiment, the viral vector based on HSV is replication-defective. Most replication-defective HSV vectors contain deletions to remove one or more early, early or late HSV genes to prevent replication. For example, the HSV vector may lack the immediate early genes selected from the group consisting of: ICP4, ICP22, ICP27, ICP47 and combinations thereof. The advantage of the HSV vector is its ability to enter a latent period that can lead to long-term DNA expression, and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25kb. HSV-based vectors are described, for example, in U.S. Patent Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0460] H. Genetically modified cells

[0461] In various embodiments, there is provided a cell genetically modified to express anti-CD79A CAR and anti-CD20 CCR encompassed herein for the treatment of cancer. In various preferred embodiments, the immune effector cells genetically modified to express anti-CD79A CAR and anti-CD20 CCR and comprising one or more reductions or elimination of PDCD-1 and / or CBLB function and / or expression of genome editing are used for cancer treatment. In another preferred embodiment, T cells or NK cells genetically modified to express anti-CD79A CAR and anti-CD20 CCR and comprising one or more reductions or elimination of CBLB function and / or expression of genome editing are used for cancer treatment.

[0462] In a specific embodiment, the fusion protein of anti-CD79A CAR and anti-CD20 CCR or coding anti-CD79A CAR, 2A self-cleavage polypeptide and anti-CD20 CCR is introduced into immune effector cells and expressed in immune effector cells to redirect their target antigens of interest, such as CD79A or CD20 specificity. In a preferred embodiment, the fusion protein of anti-CD79A CAR and anti-CD20 CCR or coding anti-CD79A CAR, 2A self-cleavage polypeptide and anti-CD20 CCR is introduced into immune effector cells and expressed in immune effector cells, and the immune effector cells include one or more genome editing in CBLB to redirect their specificity to CD79A and CD20." immune effector cell " is any cell of the immune system, which has one or more effector functions (for example, cytotoxic cell killing activity, cytokine secretion, ADCC and / or CDC induction). The exemplary immune effector cells encompassed herein are T lymphocytes, including but not limited to cytotoxic T cells (CTL; ​​CD8 + T cells), TILs, and helper T cells (HTL; CD4 + In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T (NKT) cells.

[0463] Immune effector cells can be autologous / one's own ("self") or non-autologous ("non-self," e.g., allogeneic, isogenic, or xenogeneic). As used herein, "autologous" refers to cells from the same subject. As used herein, "allogeneic" refers to cells from the same species that are genetically different from the comparison cells. As used herein, "isogenic" refers to cells from different subjects that are genetically identical to the comparison cells. As used herein, "xenogeneic" refers to cells from a different species than the comparison cells. In preferred embodiments, the cells are autologous.

[0464] Exemplary immune effector cells for use with anti-CD79A CAR and anti-CD20 CCR encompassed 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 can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 + T cells)CD4 +cells, cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other T cell subsets. Other exemplary T cell populations 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 ).

[0465] As will be understood by technicians, other cells can also be used as immune effector cells with anti-CD79A CAR and anti-CD20 CCR as encompassed herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include the progenitor cells of effector cells, wherein such progenitor cells can be induced in vivo or in vitro to differentiate into immune effector cells. Therefore, in specific embodiments, immune effector cells include the progenitor cells of immune effector cells, such as hematopoietic stem cells (HSC) contained in CD34+ cell groups of peripheral blood derived from cord blood, bone marrow or mobilized, the HSC is differentiated into mature immune effector cells after application in a subject, or the HSC can be induced in vitro to differentiate into mature immune effector cells.

[0466] As used herein, the term "CD34+ cells" refers to cells expressing CD34 protein on their cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin) that typically acts as a cell-cell adhesion factor and participates in the entry of T cells into lymph nodes. The CD34+ cell population contains hematopoietic stem cells (HSCs), which differentiate and contribute to all hematopoietic lineages when administered to a patient, including cells of the T cell, NK cell, NKT cell, neutrophil, and monocyte / macrophage lineages.

[0467] In a specific embodiment, a method for preparing immune effector cells expressing anti-CD79A CAR and anti-CD20 CCR as contemplated herein is provided. In one embodiment, the method includes transfection or transduction of immune effector cells isolated from an individual such that the immune effector cells express polycistronic messages encoding anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding anti-CD79A CAR, 2A self-cleaving polypeptide, and anti-CD20 CCR as contemplated herein.

[0468] 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 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. The method further comprises introducing a polynucleotide encoding a HE variant or megaTAL into the cell that binds to and cleaves a target site in the PDCD-1 gene or the CBLB gene, preferably the CBLB gene. In a specific embodiment, the transduced and edited cells are then cultured for expansion prior to administration to the subject.

[0469] In certain embodiments, immune effector cells are separated from individuals and genetically modified and / or edited without further manipulation in vitro.Then, this cell can be directly applied to individuals again.In other embodiments, immune effector cells are first activated and stimulated to proliferate in vitro, then genetically modified to express anti-CD79A CAR and anti-CD20 CCR, and then edited using targeting PDCD-1 or CBLB, preferably HE variants of CBLB or megaTAL.In this regard, immune effector cells can be cultivated before and / or after genetic modification and / or genome editing (that is, transduction or transfection to express anti-CD79A CAR and anti-CD20 CCR encompassed herein).

[0470] In certain embodiments, prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, a source of cells is obtained from a subject. In certain embodiments, the modified immune effector cells comprise T cells.

[0471] In certain embodiments, PBMCs can be genetically modified directly using the methods encompassed 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, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets before or after genetic modification and / or expansion.

[0472] Immune effector cells, such as T cells, can be genetically modified after being separated using known methods, or immune effector cells can be activated and amplified in vitro (or differentiated in the case of progenitor cells) before being genetically modified and / or genome editing. In a specific embodiment, immune effector cells, such as T cells are activated and stimulated for amplification, and then genetically modified (for example, with a viral vector transduction comprising a nucleic acid encoding a polycistronic message or fusion protein) using a chimeric antigen receptor as encompassed herein, and then activated and amplified in vitro, the polycistronic message encoding anti-CD79A CAR and anti-CD20 CCR, the fusion protein encoding anti-CD79A CAR, 2A self-cleavage polypeptide and anti-CD20 CCR. In various embodiments, T cells can be activated and expanded before or after genetic modification using the methods described in, for example, U.S. Patents 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.

[0473] In one embodiment, CD34 + Cells are transduced with the nucleic acid constructs contemplated herein. In certain embodiments, the transduced CD34 + The cells differentiate into mature immune effector cells in vivo after administration to a subject (typically the subject from whom the cells were initially isolated). In another embodiment, the CD34 + Cells can be stimulated in vitro according to the methods described previously 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 (Asheuer et al., 2004; Imren et al., 2004).

[0474] In certain embodiments, the immune effector cell group of the modification for treating cancer includes CAR and CCR as contemplated herein.For example, the immune effector cell group of modification is prepared by peripheral blood mononuclear cells (PBMC) obtained from patients (autologous donors) diagnosed with B cell malignancies as described herein.PBMC formation can be a heterogeneous population of T lymphocytes of CD4+, CD8+ or CD4+ and CD8+.

[0475] PBMC may also include other cytotoxic lymphocytes, such as NK cells or NKT cells. The expression vector of the coding sequence of the CAR and CCR covered in the specific embodiment is introduced into the colony of human donor T cells, NK cells or NKT cells. In a specific embodiment, the T cells successfully transduced by carrying the expression vector can be sorted using flow cytometry to separate CD3 positive T cells, and then in addition to using anti-CD3 antibodies and / or anti-CD28 antibodies and IL-2 or any other method known in the art as described elsewhere herein to activate cells, further propagate to increase the number of these expression CAR and CCR T cells. Standard procedures are used for the freezing preservation of T cells, for storage and / or preparation in human subjects. In one embodiment, in the absence of non-human animal derived products such as fetal bovine serum (fetal calf serum / fetalbovine serum), T cells are transduced in vitro, cultured and / or expanded. Since the heterogeneous PBMC population is genetically modified, 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 a specific embodiment, the heterogeneous PBMC population is genetically modified and genome-edited, and 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 comprising one or more genome edits that reduce or eliminate PDCD-1 and / or CBLB function and expression.

[0476] In other embodiments, a mixture of, for example, one, two, three, four, five or more different expression vectors can be used to genetically modify a donor population of immune effector cells, wherein each vector encodes a different chimeric antigen receptor protein as contemplated herein. The resulting modified immune effector cells form a mixed population of modified cells.

[0477] IT cell production method

[0478] In various embodiments, the genetically modified T cells are expanded by contacting them with an agent that stimulates signaling associated with the CD3 TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells.

[0479] In certain embodiments, PBMCs or isolated T cells are contacted with stimulatory and co-stimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies attached to beads or other surfaces, in culture medium with appropriate cytokines such as IL-2, IL-7, and / or IL-15.

[0480] In certain embodiments, PBMCs or isolated T cells are contacted with stimulatory and co-stimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies attached 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.

[0481] In one embodiment, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell production methods contemplated herein. PBMCs can be CD4 + 、CD8 + or CD4 + and CD8 + In one embodiment, the present invention provides a heterogeneous group of T lymphocytes and can include other mononuclear cells, such as monocytes, B cells, NK cells and NKT cells. An expression vector comprising a polynucleotide encoding a polycistronic message or a fusion protein is introduced into a human donor T cell, NK cell or NKT cell population, wherein the polycistronic message encodes anti-CD79A CAR and anti-CD20 CCR, and the fusion protein encodes the anti-CD79A CAR, 2A self-cleaving polypeptide and anti-CD20 CCR covered in a specific embodiment. In a specific embodiment, the T cells successfully transduced with the expression vector can be sorted using flow cytometry to separate CD3 positive T cells, and then further proliferated to increase the number of modified T cells in addition to cell activation using anti-CD3 antibodies and / or anti-CD28 antibodies and IL-2, IL-7 and / or IL-15.

[0482] In order to achieve a sufficient therapeutic dose of a T cell composition, the T cells are typically subjected to one or more rounds of stimulation, activation, and / or expansion. T cells can generally be activated and expanded using methods described in, for example, U.S. Patents 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, each of which is incorporated herein by reference in its entirety.

[0483] In preferred embodiments, T cells manufactured by the methods encompassed herein provide improved adoptive immunotherapy compositions. Without wishing to be bound by any particular theory, it is believed that the T cell compositions manufactured by the methods in the specific embodiments encompassed herein have excellent properties, including increased survival, expansion in the absence of relative differentiation, and in vivo persistence. In one embodiment, the method of manufacturing T cells comprises contacting the cells with one or more agents that modulate the PI3K cell signaling pathway.

[0484] In a specific embodiment, T cells are produced by stimulating T cells to become activated and proliferate in the presence of one or more stimulatory signals and a PI3K inhibitor.

[0485] T cells can then be modified to express polycistronic messages encoding anti-CD79A CAR and anti-CD20 CCR or fusion proteins encoding anti-CD79A CAR, 2A self-cleaving polypeptides and anti-CD20 CCR. In one embodiment, T cells are modified by transducing T cells with viral vectors, and the viral vectors include polycistronic messages encoding anti-CD79A CAR and anti-CD20 CCR or fusion proteins encoding anti-CD79A CAR, 2A self-cleaving polypeptides and anti-CD20 CCR encompassed herein. In certain embodiments, in the presence of an inhibitor of the PI3K cell signaling pathway, T cells are modified before stimulation and activation. In another embodiment, in the presence of an inhibitor of the PI3K cell signaling pathway, T cells are modified after stimulation and activation. In a specific embodiment, in the presence of an inhibitor of the PI3K cell signaling pathway, T cells are modified within 12 hours, 24 hours, 36 hours or 48 hours of stimulation and activation. In a specific embodiment, T cells are modified in the presence of a PI3K inhibitor.

[0486] In certain embodiments, after immune effector cells are transduced with a viral vector comprising polynucleotides 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, a polynucleotide encoding a HE variant or megaTAL that binds to and cleaves a target site in the PDCD-1 gene or the CBLB gene, preferably in the CBLB gene, is introduced into the cell.

[0487] After transduction and / or editing of the T cells, the cells are cultured to proliferate. The T cells can 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 longer, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion. In a specific embodiment, the T cells are cultured to proliferate in the presence of a PI3K inhibitor.

[0488] In various embodiments, T cell compositions are manufactured in the presence of a PI3K inhibitor. Without wishing to be bound by any particular theory, it is contemplated that 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 preferentially increases young T cells, thereby resulting in superior therapeutic T cell compositions.

[0489] As used herein, the term "PI3K inhibitor" refers to a nucleic acid, peptide, compound or small organic molecule that binds to and inhibits at least one activity of PI3K. PI3K proteins can be divided into three categories: Class 1 PI3K, Class 2 PI3K and Class 3 PI3K. Class 1 PI3K exists as a heterodimer that consists 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 will show selectivity for one or more isoforms of the Class 1 PI3K inhibitor (i.e., selectivity for one or more of p110α, p110β, p110δ and p110γ and p110α, p110β, p110δ and p110γ). In another aspect, the PI3K inhibitor will not show isoform selectivity and will be considered a "pan-PI3K inhibitor." In one embodiment, the PI3K inhibitor will compete with ATP for binding to the PI3K catalytic domain.

[0490] In certain embodiments, a PI3K inhibitor can, for example, target PI3K as well as another protein in the PI3K-AKT-mTOR pathway. In certain embodiments, a PI3K inhibitor that targets both mTOR and PI3K can be referred to as an mTOR inhibitor or a PI3K inhibitor. A PI3K inhibitor that targets only PI3K can be referred to as a selective PI3K inhibitor. In one embodiment, a selective PI3K inhibitor can be understood to refer to an agent that exhibits a 50% inhibitory concentration relative to PI3K that is at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more lower than the IC50 of the inhibitor relative to mTOR and / or other proteins in the pathway.

[0491] In a specific embodiment, exemplary PI3K inhibitors inhibit PI3K with an IC50 (concentration that inhibits 50% of activity) of 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.

[0492] Illustrative examples of PI3K inhibitors suitable for use in the T cell manufacturing methods contemplated in specific embodiments include, but are not limited to, BKM120 (a class 1 PI3K inhibitor, Novartis), XL147 (a class 1 PI3K inhibitor, Exelixis), (a pan-PI3K inhibitor, GlaxoSmithKline), and PX-866 (a class 1 PI3K inhibitor; p110α, p110β, and p110γ isoforms, Oncothyreon).

[0493] Other illustrative examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145.

[0494] Further illustrative examples of pan-PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

[0495] In a preferred embodiment, the PI3K inhibitor is ZSTK474.

[0496] In a specific embodiment, a method for increasing the proliferation of T cells expressing an engineered T cell receptor is provided. Such methods can include, for example, harvesting a T cell source 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 cell genome with an HE variant or megaTAL, and expanding the T cells in culture, wherein the T cells are manufactured in the presence of one or more inhibitors of the PI3K pathway.

[0497] The manufacturing methods encompassed herein may also include cryopreservation of modified T cells for storage and / or preparation in human subjects. In one embodiment, the method for storing genetically modified immune effector cells includes cryopreservation of immune effector cells so that the cells remain viable after thawing. T cells are cryopreserved so that the cells remain viable after thawing. When necessary, the cryopreserved converted immune effector cells can be thawed, grown, and expanded more such cells. As used herein, "cryopreservation" refers to preserving cells by cooling to subzero temperatures, such as (usually) 77K or -196°C (the boiling point of liquid nitrogen). Cryoprotectants are often used at subzero temperatures to prevent cells from being damaged by freezing or warming to room temperature at low temperatures. Cryoprotectants and optimal cooling rates can prevent 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, polyvinyl pyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 1°C / min to 3°C / min. After at least two hours, the T cells have reached a temperature of -80°C and can be placed directly in liquid nitrogen (-196°C) for permanent storage, such as in a long-term cryogenic storage container.

[0498] J. Compositions and Formulations

[0499] The compositions encompassed herein may include one or more CAR polypeptides, CCR polypeptides, polynucleotides, vectors comprising same, genetically modified immune effector cells, etc. as encompassed herein. Compositions include, but are not limited to, pharmaceutical compositions. In a preferred embodiment, the composition comprises one or more cells modified to express anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding anti-CD79A CAR, 2A self-cleaving polypeptide, and anti-CD20 CCR. In a preferred embodiment, the composition comprises one or more cells modified to express anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding anti-CD79A CAR, 2A self-cleaving polypeptide, and anti-CD20 CCR, wherein the cells are further subjected to genome editing to reduce or eliminate the expression and function of CBLB.

[0500] "Pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution that is administered to cells or animals alone or in combination with one or more other therapeutic modalities. It should also be understood that the composition can also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies or other various pharmaceutically active agents, if necessary. There is actually no restriction on other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent or excipient and one or more genome-edited cells that are also modified to express anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding anti-CD79A CAR, 2A self-cleaving polypeptide and anti-CD20 CCR.

[0501] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0502] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock. Exemplary pharmaceutically 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 such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; astragalus; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, 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 glycerol, 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; ethanol; phosphate buffered saline; and any other compatible substances employed in pharmaceutical formulations.

[0503] In certain embodiments, compositions include the immune effector cells of expression anti-CD79A CAR and anti-CD20 CCR as encompassed herein.In preferred embodiments, compositions include a certain amount of immune effector cells of the genome editing of expression anti-CD79A CAR and anti-CD20 CCR, wherein genome editing reduces or eliminates PDCD-1 and / or CBLB, preferably CBLB expression and function.As used herein, term " amount " refers to the therapeutic cells of the gene modification for achieving beneficial or desired prevention or treatment results (including clinical results), such as " effective amount " or " effective amount " of T cells.

[0504] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic amount is less than a therapeutically effective amount because a prophylactic dose is used in subjects prior to or at an early stage of disease.

[0505] A "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutic beneficial effects outweigh any toxic or deleterious effects of the virus or transduced therapeutic cells. The term "therapeutically effective amount" includes an amount that effectively "treats" a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the composition to be administered can be determined by a physician encompassing individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).

[0506] Generally speaking, the pharmaceutical composition comprising the T cells described herein can be taken as 10 2 to 10 10 cells / kg body weight, preferably 10 5 to 10 6 The dosage of cells / kg body weight (including all integer values ​​within those ranges) is preferably administered. The number of cells will depend on the desired end use of the composition, as will the type of cells contained therein. For the uses provided herein, the volume of cells is typically one liter or less, and can be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is typically greater than 10 6 cells / mL and usually greater than 10 7 cells / mL, usually 10 8 cells / mL or greater. Clinically relevant numbers of immune cells can be allocated to cumulatively equal to or greater than 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10, 10 11 or 10 12 In some aspects, particularly since all infused cells will be redirected to a specific target antigen, 10 6 / kg(10 6 -10 11 The composition can be administered multiple times at doses within these ranges. For patients receiving treatment, the cells can be allogeneic, isogenic, xenogeneic, or autologous. If desired, treatment can also include administering mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance the induction of an immune response.

[0507] Typically, the compositions comprising activated and amplified cells as described herein can be used to treat and prevent diseases occurring in immunocompromised individuals. In a specific embodiment, immune effector cells modified to express anti-CD79A CAR and anti-CD20 CCR or encoding anti-CD79A CAR, 2A self-cleavage polypeptide and anti-CD20 CCR fusion proteins encompassed herein are included and include one or more compositions that reduce or eliminate PDCD-1 and / or CBLB, preferably CBLB genome editing for treatment. The modified immune effector cells can be administered alone or as a pharmaceutical composition with a carrier, diluent, excipient and / or in combination with other components such as IL-2 or other cytokines or cell populations. In a specific embodiment, the pharmaceutical composition includes a certain amount of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0508] The immune effector cells modified to express anti-CD79A CAR and anti-CD20 CCR or the fusion protein encoding anti-CD79A CAR, 2A self-cleavage polypeptide and anti-CD20 CCR, such as T cell pharmaceutical compositions can include buffers, such as neutral buffered saline, phosphate buffered saline, etc.;Carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol;Protein;Polypeptide or amino acid, such as glycine;Antioxidant;Chelating agent, such as EDTA or glutathione;Adjuvant (for example, aluminum hydroxide);And preservative. Composition is preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

[0509] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline solutions (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils that can serve as solvents or suspension media (such as synthetic mono- or diglycerides), polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; 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 formulations can be placed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0510] In one embodiment, the T cell compositions encompassed herein are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In a specific embodiment, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0511] Serum-free culture medium has several advantages over serum-containing culture medium, including that the composition is simplified and better defined, the degree of contaminants is reduced, possible sources of infectious agents are eliminated, and costs are reduced. In various embodiments, serum-free culture medium is animal-free and can optionally be protein-free. Optionally, the culture medium can contain biopharmaceutically acceptable recombinant proteins. "Animal-free" culture medium refers to a culture medium in which the components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in the animal-free culture medium, and nutrients are obtained from synthetic, plant or microbial sources. In contrast, "protein-free" culture medium is defined as being substantially protein-free.

[0512] Illustrative examples of serum-free culture media used in certain embodiments include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0513] In a preferred embodiment, the compositions comprising the immune effector cells contemplated herein are formulated in a solution comprising PlasmaLyte A.

[0514] In another preferred embodiment, the composition comprising the immune effector cells encompassed herein is formulated in a solution comprising a cryopreservation medium. For example, a cryopreservation medium with a cryopreservation agent can be used to maintain high cell viability results after thawing. Illustrative examples of cryopreservation medium used in specific embodiments include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0515] In a more preferred embodiment, the composition comprising the immune effector cells contemplated herein is formulated in a solution comprising 50:50 PlasmaLyte A:CryoStor CS10.

[0516] In a specific embodiment, the composition comprises an effective amount of genome-edited immune effector cells, which are modified to express anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding anti-CD79A CAR, 2A self-cleaving polypeptide and anti-CD20 CCR alone or in combination with one or more therapeutic agents. Therefore, the immune effector cell composition expressing CAR can be administered alone or in combination with other known cancer treatments, such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The composition can also be administered in combination with antibiotics. This area can accept this therapeutic agent as a standard treatment for specific disease states such as specific cancers as described herein. Exemplary therapeutic agents encompassed in specific embodiments include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutics, radiotherapeutics, therapeutic antibodies or other active agents and adjuvants.

[0517] In certain embodiments, compositions comprising genome-edited 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 can be administered with any number of chemotherapeutic agents.

[0518] A variety of other therapeutic agents can be used in conjunction with the compositions described herein. In one embodiment, a composition comprising genome-edited immune effector cells, anti-CD79A CAR and anti-CD20 CCR or a fusion protein encoding anti-CD79A CAR, 2A self-cleaving polypeptide and anti-CD20 CCR is administered together with an anti-inflammatory agent.

[0519] In one embodiment, a composition comprising a genome-edited immune effector cell, 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 is administered with a therapeutic antibody. Illustrative examples of therapeutic antibodies suitable for combination with the 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, durvalumab, elotuzumab (HuLuc63), and selegiline (Sergidizumab). , gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0520] K. Target cells and antigens

[0521] In certain embodiments, genetically modified immune effector cells are provided that are redirected to target cells, such as cancer cells, and express anti-CD79A CAR and anti-CD20 CCR, and one or more comprise PDCD-1 and / or CBLB, preferably genome editing in CBLB. As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without control and may invade nearby tissues.

[0522] As used herein, the term "malignant" refers to a group of cancer cells that show one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via the lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. Tumors formed by cells that have spread are called "metastatic tumors" or "metastasis". Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0523] As used herein, the term "benign" or "non-malignant" refers to a tumor that can grow larger but does not spread to other parts of the body. Benign tumors are self-limited and usually do not invade or metastasize.

[0524] "Cancer cell" refers to a single cell of a cancerous growth or tissue. Cancer cells include solid cancers and liquid cancers. "Tumor" or "tumor cell" generally refers to a swelling or lesion formed by an abnormal growth of cells, which can be benign, pre-malignant, or malignant. Most cancers form tumors, but liquid cancers such as leukemias do not necessarily form tumors. For those 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.

[0525] The term "relapse" refers to the return of cancer diagnosis or signs and symptoms after a period of improvement or remission.

[0526] "Remission" is also called "clinical remission" and includes both partial remission and complete remission. In a partial remission, some but not all signs and symptoms of cancer have disappeared. In a complete remission, all signs and symptoms of cancer have disappeared, although the cancer may still be in the body.

[0527] "Refractory" means that the cancer is resistant or unresponsive to treatment with a particular therapeutic agent. A cancer can be refractory at the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent), or it can become refractory because it develops resistance to the therapeutic agent during the first treatment period or during subsequent treatment periods.

[0528] In one embodiment, the target cell expresses an antigen, eg, a target antigen that is substantially absent from the surface of other normal (desired) cells.

[0529] In one embodiment, the target cell is an osteosarcoma cell or an Ewing's sarcoma cell.

[0530] In one embodiment, the target cell is a hematopoietic cell, a lymphocyte, or a myeloid cell.

[0531] In certain embodiments, the target cell is part of blood, lymphoid tissue, or myeloid tissue.

[0532] In a specific embodiment, the target cell is a cancer cell or cancer stem cell that expresses CD79A and / or CD20. In a specific embodiment, the target cell is a cancer cell or cancer stem cell that expresses CD79A and CD20. In a specific embodiment, the target cell is a cancer cell or cancer stem cell that expresses CD79A or CD20.

[0533] In a specific embodiment, the target cell is a liquid cancer cell or a blood cancer cell that expresses CD79A and / or CD20. In a specific embodiment, the target cell is a liquid cancer cell or a blood cancer cell that expresses CD79A and CD20. In a specific embodiment, the target cell is a liquid cancer cell or a blood cancer cell that expresses CD79A or CD20.

[0534] Illustrative examples of liquid or blood cancers that can be prevented, treated, or ameliorated using the compositions contemplated in particular embodiments include, but are not limited to, leukemias, lymphomas, and multiple myeloma.

[0535] Illustrative examples of cells that can be targeted by immune effector cells expressing the anti-CD79A CAR and anti-CD20 CCR contemplated in particular embodiments include, but are not limited to, those in the following leukemias: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblasts, promyelocytic cells, myelomonocytic cells, monocytes, erythroleukemias, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera.

[0536] Illustrative examples of cells that can be targeted by the compositions comprising immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR and the methods encompassed in specific embodiments include, but are not limited to, those in the following lymphomas: Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, and non-Hodgkin lymphomas, 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-lymphocytic lymphoma, and mantle cell lymphoma; and T-cell non-Hodgkin lymphomas: mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, and precursor T-lymphoblastic lymphoma.

[0537] Illustrative examples of cells that can be targeted by the composition of immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR and by the methods encompassed in specific embodiments include, but are not limited to, those of the following multiple myeloma: overt multiple myeloma, smoldering multiple myeloma (MGUS), plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0538] In a preferred embodiment, the target cell expressing CD79A and / or CD20 is a DLBCL cancer cell. In a preferred embodiment, the target cell expressing CD79A and CD20 is a DLBCL cancer cell. In a preferred embodiment, the target cell expressing CD79A or CD20 is a DLBCL cancer cell.

[0539] L. Treatment methods

[0540] Genetically modified immune effector cells expressing the anti-CD79A CAR and anti-CD20 CCR contemplated herein provide improved methods for adoptive immunotherapy for preventing, treating, and ameliorating cancers that express CD79A and / or CD20, or for preventing, treating, or ameliorating at least one symptom associated with cancers that express CD79A and / or CD20.

[0541] In various embodiments, the genetically modified immune effector cells encompassed herein provide improved methods for adoptive immunotherapy 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.

[0542] In certain embodiments, the specificity of primary immune effector cells is redirected to cells expressing CD79A and / or CD20, such as cancer cells, by genetically modifying primary immune effector cells with a CAR encompassed herein. In various embodiments, viral vectors are used to genetically modify immune effector cells with specific polynucleotides encoding anti-CD79A CAR and anti-CD20 CCR.

[0543] In one embodiment, a cell therapy is provided in which T cells are genetically modified to express anti-CD79A CAR and anti-CD20 CCR targeting cancer cells expressing CD79A and / or CD20, and the T cells are infused into a recipient in need. The infused cells are able to kill the cells that cause the disease in the recipient. Unlike antibody therapy, T cell therapy is able to replicate in vivo, resulting in long-term persistence that can lead to sustained cancer treatment.

[0544] In one embodiment, T cells expressing anti-CD79A CAR and anti-CD20 CCR can undergo robust in vivo T cell expansion and can last for an extended period of time. In another embodiment, T cells expressing anti-CD79A CAR and anti-CD20 CCR evolve into specific memory T cells or stem cell memory T cells, which can be reactivated to suppress any additional tumor formation or growth.

[0545] In certain embodiments, compositions comprising immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR contemplated herein are used to treat a disorder associated with cancer cells or cancer stem cells expressing CD79A and / or CD20.

[0546] Illustrative examples of conditions that are treated, prevented, or ameliorated using immune effector cells expressing an anti-CD79A CAR and an anti-CD20 CCR are contemplated in certain embodiments.

[0547] In a specific embodiment, a composition comprising T cells expressing an anti-CD79A CAR and an anti-CD20 CCR contemplated herein is used to treat osteosarcoma or Ewing's sarcoma.

[0548] In a specific embodiment, a composition comprising T cells expressing an anti-CD79A CAR and an anti-CD20 CCR contemplated herein is used to treat a liquid or hematological cancer.

[0549] In certain embodiments, the liquid or blood cancer is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.

[0550] In certain embodiments, the liquid cancer 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 myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt 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, Sézary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0551] 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 myeloid leukemia (CML).

[0552] In a preferred embodiment, the liquid or blood cancer is DLBCL.

[0553] In preferred embodiments, the liquid or blood cancer is relapsed / refractory DLBCL.

[0554] In certain embodiments, there is provided a method comprising administering to a patient in need thereof a therapeutically effective amount of an immune effector cell expressing an anti-CD79A CAR and an anti-CD20 CCR, or a composition comprising the same, alone or in combination with one or more therapeutic agents. In certain embodiments, the cell is used to treat a patient at risk of a disorder associated with cancer cells expressing CD79A and / or CD20. Thus, in certain embodiments, the present invention encompasses a method for treating or preventing or ameliorating at least one symptom of cancer, comprising administering to a subject in need thereof a therapeutically effective amount of modified T cells expressing anti-CD79A CAR and anti-CD20 CCR.

[0555] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of a disease, condition, or disorder that can be treated with gene therapy vectors, cell-based therapeutics, and methods encompassed elsewhere herein. In preferred embodiments, subjects include any animal that exhibits symptoms of a disease, condition, or disorder associated with cancer that can be treated with gene therapy vectors, cell-based therapeutics, and methods encompassed elsewhere herein. Suitable subjects (e.g., patients) include experimental animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (e.g., cats or dogs). Non-human primates and preferably human patients are included. Typical subjects include human patients who have a cancer that expresses CD79A and / or CD20, have been diagnosed with a cancer that expresses CD79A and / or CD20, or are at risk of a cancer that expresses CD79A and / or CD20, such as DLBCL.

[0556] As used herein, the term "patient" refers to a subject who has been diagnosed with a particular disease, disorder, or symptom that can be treated with the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein.

[0557] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally involve alleviation of the disease or condition, or delay in progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms.

[0558] As used herein, "prevent" and similar words such as "prevented" or "preventing" refer to methods used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition. Prevention also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0559] As used herein, the phrase "alleviating at least one symptom of" refers to reducing one or more symptoms of the disease or condition being treated in the subject. In certain embodiments, the disease or condition being treated is cancer, wherein the one or more symptoms alleviated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal swelling or pain (due to enlarged abdominal organs), bone or joint pain, bone fractures, unintentional weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).

[0560] "Enhance" or "promote" or "increase" or "amplify" generally refers to the ability of the compositions encompassed herein, such as genetically modified T cells expressing anti-CD79A CAR and anti-CD20 CCR, to produce, elicit or cause a greater physiological response (i.e., downstream effect) compared to the response caused by a vehicle or control molecule / composition. Measurable physiological responses can include an increase in T cell expansion, activation, persistence and / or cancer cell killing ability, as well as other aspects apparent from the understanding of the art and the description herein. An "increased" or "enhanced" amount is generally a "statistically significant" amount and can include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) than the response produced by vehicle or a control composition.

[0561] "Decrease" or "attenuate" or "become less" or "reduce" or "mitigate" generally refers to the ability of the compositions encompassed herein to produce, elicit or cause less of a response (i.e., a physiological response) than the response elicited by vehicle or a control molecule / composition. A "decreased" or "reduced" amount is generally a "statistically significant" amount and can include a decrease of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) compared to the response produced by vehicle, a control composition, or a response in a particular cell lineage (reference response).

[0562] "Maintain" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a similar physiological response (i.e., downstream effect) in a cell compared to the response elicited by vehicle, a control molecule / composition, or a response in a particular cell lineage. A comparable response is one that is not significantly different or measurably different from a reference response.

[0563] 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 genetically modified immune effector cells as contemplated herein. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials.

[0564] In one embodiment, the amount of immune effector cells (e.g., T cells expressing anti-CD79A CAR and anti-CD20 CCR) in the composition administered to a subject is at least 0.1×10 5 cells, at least 0.5×10 5 cells, at least 1×10 5 cells, at least 5×10 5 cells, at least 1×10 6 cells, at least 0.5×10 7 cells, at least 1×10 7 cells, at least 0.5×10 8 cells, at least 1×10 8 cells, at least 0.5×10 9 cells, at least 1×10 9 cells, at least 2×10 9 cells, at least 3×10 9 cells, at least 4×10 9cells, at least 5×10 9 cells or at least 1×10 10 cells.

[0565] In certain embodiments, about 1×10 7 T cells to about 1×10 9 T cells, about 2×10 7 T cells to about 0.9×10 9 T cells, about 3×10 7 T cells to about 0.8×10 9 T cells, about 4×10 7 T cells to about 0.7×10 9 T cells, about 5×10 7 T cells to about 0.6×10 9 T cells, or about 5×10 7 T cells to about 0.5×10 9 T cells.

[0566] In one embodiment, the amount of immune effector cells (e.g., T cells expressing anti-CD79A CAR and anti-CD20 CCR) in the composition administered to a subject is at least 0.1×10 4 cells / kg body weight, at least 0.5×10 4 cells / kg body weight, at least 1×10 4 cells / kg body weight, at least 5×10 4 cells / kg body weight, at least 1×10 5 cells / kg body weight, at least 0.5×10 6 cells / kg body weight, at least 1×10 6 cells / kg body weight, at least 0.5×10 7 cells / kg body weight, at least 1×10 7 cells / kg body weight, at least 0.5×10 8 cells / kg body weight, at least 1×10 8 cells / kg body weight, at least 2×10 8 cells / kg body weight, at least 3×10 8 cells / kg body weight, at least 4×10 8 cells / kg body weight, at least 5×10 8 cells / kg body weight or at least 1×10 9 cells / kg body weight.

[0567] In certain embodiments, about 1×10 6 T cells / kg body weight is about 1×108 T cells / kg body weight, about 2×10 6 T cells / kg body weight is about 0.9×10 8 T cells / kg body weight, about 3×10 6 T cells / kg body weight is about 0.8×10 8 T cells / kg body weight, about 4×10 6 T cells / kg body weight is about 0.7×10 8 T cells / kg body weight, about 5×10 6 T cells / kg body weight is about 0.6×10 8 T cells / kg body weight, or about 5×10 6 T cells / kg body weight is about 0.5×10 8 T cells / kg body weight.

[0568] Those of ordinary skill in the art will recognize that multiple administrations of the compositions contemplated herein may be required to achieve the desired therapy. For example, the compositions can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more over a span 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.

[0569] In certain embodiments, it may be desirable to administer activated immune effector cells to a subject, and subsequently redraw blood (or perform apheresis), activate immune effector cells therefrom and re-infuse these activated and amplified immune effector cells to the patient. This process can be performed repeatedly every few weeks. In certain embodiments, 10cc to 400cc of blood can be drawn to activate immune effector cells. In certain embodiments, 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc or 400cc or more of the blood is drawn to activate immune effector cells. Without being bound by theory, using this multiple blood draw / multiple re-infusion scheme can be used to select certain immune effector cell populations.

[0570] The administration of the compositions encompassed herein can be carried out in any convenient manner, including by aerosol inhalation, injection, intake, infusion, implantation or transplantation. In a preferred embodiment, parenteral administration compositions. As used herein, phrases "parenteral administration (parenteral administration)" and "parenteral administration (administered parenterally)" refer to modes of administration except enteral and topical administration, usually by injection, and include but are not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions encompassed herein are administered to the subject by direct injection into tumor, lymph node or site of infection.

[0571] In one embodiment, an effective amount of a composition is administered to a subject in need thereof to increase a cellular immune response to a B cell-related disorder of 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 mediated primarily by helper T cells that can activate B cells, thereby leading to antibody production, can also be induced. A variety of techniques can be used to analyze the type of immune response induced by the composition, and the composition is fully described in the art; for example, Current Protocols in Immunology, eds.: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0572] In one embodiment, a method for treating a subject diagnosed with a cancer expressing CD79A and / or CD20 is provided, the method comprising removing immune effector cells from the subject, modifying the immune effector cells with vector genes comprising nucleic acids encoding anti-CD79A CAR and anti-CD20 CCRs encompassed herein, thereby producing modified immune effector cell populations, and administering the modified immune effector cell populations to the same subject. In a preferred embodiment, the immune effector cells include T cells.

[0573] In certain embodiments, a method for stimulating an immune effector cell-mediated immunomodulatory response to a target cell population in a subject is provided, the method comprising the step of 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.

[0574] The method of administering the cell composition encompassed in the specific embodiment includes any method that effectively results in ex vivo reintroduction of genetically modified immune effector cells that directly express anti-CD79A CAR and anti-CD20 CCR in the subject, or when reintroducing the progenitor cells of the genetically modified immune effector cells, the immune effector cells differentiate into mature immune effector cells expressing anti-CD79A CAR and anti-CD20 CCR when introduced into the subject. One method includes ex vivo transduction of peripheral blood T cells with the nucleic acid constructs encompassed herein and returning the transduced cells to the subject.

[0575] 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.

[0576] Although the foregoing embodiments have been described in considerable detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will readily appreciate, based on the teachings contained herein, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided for illustration only and are not intended to be limiting. Those skilled in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results.

[0577] Example

[0578] Example 1

[0579] Construction of an anti-CD79A CAR-anti-CD20 CCR bicistronic construct

[0580] A lentiviral vector containing a bicistronic construct comprising a humanized anti-CD79A CAR, a T2A self-cleaving peptide, and an anti-CD20 CCR was designed, constructed, and validated. A construct comprising the MNDU3 promoter operably linked to the 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, and an anti-CD20 CCR comprising an anti-CD20 scFv, a CD8α hinge and transmembrane domain, and a CD28 costimulatory domain was cloned into the 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.

[0581] Example 2

[0582] T cells expressing both anti-CD79A CAR and anti-CD20 CCR exhibit antigen-dependent cytokine release

[0583] Peripheral blood mononuclear cells (PBMC) are harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells are transduced with lentiviral vectors, which include polynucleotides encoding anti-CD79A 41BB CD3ζCAR (SEQ ID NO:18) and anti-CD20 CCR (SEQ ID NO:33). Polynucleotides are expressed as fusion proteins (SEQ ID NO:37), wherein anti-CD79A CAR and anti-CD20 CCR are separated by T2A virus self-cleavage polypeptides. Untransduced (UTD) cells are used as controls. After transduction, cells are expanded 10 days in T cell growth medium containing IL-2.

[0584] After expansion, transduced and non-transduced T cell cultures were evaluated for anti-CD79A CAR expression by flow cytometry using recombinant Fc-CD79A fusion protein conjugated to PE fluorescent dye. Compared with non-transduced control T cells, high expression was observed in T cells transduced with lentiviral vectors encoding anti-CD79A CAR. Figure 1A .

[0585] Anti-CD79A CAR-anti-CD20 CCR T cell function was also assessed. T cells that were not transduced or lentiviral vectors transduced with anti-CD79A CAR (SEQ ID NO: 18) or anti-CD79A 41BB CD3 ζ CAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 37) were cultured alone or in a 1: 1 ratio with tumor cells lacking target antigens (RD; Rhabdomyosarcoma cell line); and co-cultured with RD.79A cells (modified to express CD79A RD), RD.CD79A.CD20 cells (modified to express CD79A and CD20 RD) and Daudi cells (Burkitt's lymphoma, which have high endogenous expression of both CD79a and CD20). After 24 hours of co-culture, supernatants were collected, and IFNγ and IL-2 cytokines were analyzed using Luminex assays. Anti-CD79A CAR–anti-CD20 CCR T cells produced IFNγ cytokines only in response to CD79A-expressing cell lines and not against RD cells. Figure 1B IL-2 production was increased in anti-CD79A CAR-anti-CD20 CCR T cells compared with anti-CD79A CAR T cells, as the anti-CD20 CCR induced the PI3K pathway and promoted IL-2 expression through the activity of its CD28 costimulatory domain. Figure 1C Graphs show mean + SEM of replicates from a single PBMC donor.

[0586] Example 3

[0587] T cells expressing both anti-CD79A CAR and anti-CD20 CCR respond to target cells expressing CD20

[0588] PBMCs were harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors, which included coding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 37) or polynucleotides encoding only anti-CD79A CAR (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 amplification, 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 (modified to express CD20 RD cells) at a ratio of 1: 1. After co-cultivation for 24 hours, supernatants were collected, and IFNγ and IL-2 cytokines were analyzed using Luminex.

[0589] Surprisingly, anti-CD79A CAR-anti-CD20 CCRT cells produced IFNγ and IL-2 cytokines after coculture with RD.CD20 cells. Figure 2A and Figure 2B .lack of activity for RD parental lines shows specific activity for CD20 targets.These data confirm the independent activity of CAR antigen to CCR antigen expressing cells (lack of CAR antigen).This discovery is unexpected, because CD28 signal transduction usually amplifies T cell receptor signal transduction, and therefore in the absence of CAR activity by CD3ζ signal transduction, it is expected that CAR-CCR T cells will not be active for cells expressing only CCR antigens.These data show that anti-CD79A CAR-anti-CD20 CCR T cells may be able to participate in the cytotoxic activity for single CAR or CCR positive target cells and dual CAR and CCR positive target cells.Graphs show the mean+SEM of single PBMC donor replicas.

[0590] Example 4

[0591] Expressing anti-CD79A CAR and anti-CD20 C AR T cells display antigen-dependent cytokine release

[0592] PBMCs were harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with anti-lentiviral vectors comprising coding CD79A 41BB CD3 ζ CAR, T2A self-cleavage polypeptides and anti-CD20 CD28 CD3 ζ CAR (SEQ ID NO: 41) polypeptides, or with anti-CD79A 41BB CD3 ζ CAR, T2A self-cleavage polypeptides and anti-CD20 41BBC CD3 ζ CAR (SEQ ID NO: 45) polypeptides. Untransduced (UTD) cells were used as controls. After transduction, cells were expanded 10 days in T cell growth medium containing IL-2.

[0593] After expansion, transduced and non-transduced T cell cultures were assessed for anti-CD79A CAR expression by flow cytometry using recombinant Fc-CD79A fusion protein conjugated to PE fluorescent dye. Expression was observed in T cells transduced with lentiviral vectors encoding the anti-CD79A CAR compared to non-transduced control T cells. Figure 3A .

[0594] Anti-CD79A CAR-anti-CD20 CAR T cell functionality was also assessed. Untransduced T cells and T cells transduced with lentiviral vectors encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CD28 CD3ζCAR fusion polypeptide (SEQ ID NO: 41) or anti-CD79A41BB CD3ζCAR, T2A, anti-CD20 41BB CD3ζCAR fusion polypeptide (SEQ ID NO: 45) were cultured alone or in a 1: 1 ratio with tumor cells lacking the target antigen (RD; rhabdomyosarcoma cell line) and co-cultured with RD.79A, RD.CD20 cells and Daudi cells. After 24 hours of co-culture, supernatants were collected and IFNγ was analyzed using Luminex assay. Both dual CD79A-CD20 CAR T cells produced IFNγ cytokines in response to RD cells expressing only either target antigen, although the responses were slightly attenuated compared to the responses of CD79A CAR–CD20 CCR T cells to either antigen. Both dual CD79A-CD20 CAR T cells also displayed robust responses against Daudi cells, but again, the responses were slightly attenuated compared to the responses of CD79A CAR–CD20 CCR T cells against Daudi cells. Figure 3B Graphs show mean + SEM of replicates from a single PBMC donor.

[0595] Example 5

[0596] T cells expressing both anti-CD79A CAR and anti-CD20 CCR exhibit antigen-dependent cytokine release

[0597] PBMCs were harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors, which included polynucleotides encoding anti-CD79A 41BB CD3ζCAR (SEQ ID NO:19) and anti-CD20 CCR (SEQ ID NO:33). Polynucleotides were expressed as fusion proteins (SEQ ID NO:39), in which anti-CD79A CAR and anti-CD20 CCR were separated by T2A virus self-cleavage polypeptides. Untransduced (UTD) cells were used as controls. After transduction, cells were expanded for 12 days in T cell growth medium containing IL-2.

[0598] After expansion, transduced and non-transduced T cell cultures were evaluated for anti-CD79A CAR expression by flow cytometry using recombinant Fc-CD79A fusion protein conjugated to PE fluorescent dye. Compared with non-transduced control T cells, high expression was observed in T cells transduced with lentiviral vectors encoding anti-CD79A CAR. Figure 4A .

[0599] Anti-CD79A CAR-anti-CD20 CCR T cell function was also assessed.Untransduced T cells and T cells transduced with anti-CD79A CAR (SEQ ID NO:19) or anti-CD79A 41BB CD3 ζ CAR, T2A, anti-CD20 CCR fusion polypeptide (SEQ ID NO:39) lentiviral vector were cultured alone or with a 1:1 ratio and lack of target antigen (RD; Rhabdomyosarcoma cell line) tumor cells;And co-cultured with RD.79A cells (modified to express CD79A RD), RD.CD79A.CD20 cells (modified to express CD79A and CD20 RD) and REC1 cells (mantle cell lymphoma, which has high endogenous expression of both CD79a and CD20). After co-cultivation for 24 hours, supernatant was collected, and IFNγ and IL-2 cytokines were analyzed using Luminex assays. Anti-CD79A CAR–anti-CD20 CCR T cells produced IFNγ cytokines only in response to CD79A-expressing cell lines and not against RD cells. Figure 4B IL-2 production was increased in anti-CD79A CAR-anti-CD20 CCR T cells compared with anti-CD79A CAR T cells, as the anti-CD20 CCR induced the PI3K pathway and promoted IL-2 expression through the activity of its CD28 costimulatory domain. Figure 4C Graphs show mean + SEM of replicates from a single PBMC donor.

[0600] Example 6

[0601] T cells expressing both anti-CD79A CAR and anti-CD20 CCR respond to target cells expressing CD20

[0602] PBMCs were harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors, which included coding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 39) or polynucleotides encoding only anti-CD79A CAR (SEQ ID NO: 19). Untransduced (UTD) cells were used as controls. After transduction, cells were expanded for 10 days in T cell growth medium containing IL-2. After amplification, 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 (modified to express CD20 RD cells) at a ratio of 1: 1. After co-cultivation for 24 hours, supernatants were collected, and IFNγ and IL-2 cytokines were analyzed using Luminex.

[0603] Surprisingly, anti-CD79A CAR-anti-CD20 CCRT cells produced IFNγ and IL-2 cytokines after coculture with RD.CD20 cells. Figure 5A and Figure 5B .lack of activity for RD parental lines shows specific activity for CD20 targets.These data confirm the independent activity of CAR antigen to CCR antigen expressing cells (lack of CAR antigen).This discovery is unexpected, because CD28 signal transduction usually amplifies T cell receptor signal transduction, and therefore in the absence of CAR activity by CD3ζ signal transduction, it is expected that CAR-CCR T cells will not be active for cells expressing only CCR antigens.These data show that anti-CD79A CAR-anti-CD20 CCR T cells may be able to participate in the cytotoxic activity for single CAR or CCR positive target cells and dual CAR and CCR positive target cells.Graphs show the mean+SEM of single PBMC donor replicas.

[0604] Example 7

[0605] Expressing anti-CD79A CAR and anti-CD20 C AR T cells display antigen-dependent cytokine release

[0606] PBMCs were harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with anti-lentiviral vectors comprising coding CD79A 41BB CD3 ζ CAR, T2A self-cleavage polypeptides and anti-CD20 CD28 CD3 ζ CAR (SEQ ID NO: 43) polypeptides, or with anti-CD79A 41BB CD3 ζ CAR, T2A self-cleavage polypeptides and anti-CD20 41BBC CD3 ζ CAR (SEQ ID NO: 47) polypeptides. Untransduced (UTD) cells were used as controls. After transduction, cells were expanded 10 days in T cell growth medium containing IL-2.

[0607] After expansion, transduced and non-transduced T cell cultures were assessed for anti-CD79A CAR expression by flow cytometry using recombinant Fc-CD79A fusion protein conjugated to PE fluorescent dye. Expression was observed in T cells transduced with lentiviral vectors encoding the anti-CD79A CAR compared to non-transduced control T cells. Figure 6A .

[0608] Anti-CD79A CAR-anti-CD20 CAR T cell functionality was also assessed. Untransduced T cells and T cells transduced with lentiviral vectors encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CD28 CD3ζCAR fusion polypeptide (SEQ ID NO: 43) or anti-CD79A41BB CD3ζCAR, T2A, anti-CD20 41BB CD3ζCAR fusion polypeptide (SEQ ID NO: 47) were cultured alone or in a 1: 1 ratio with tumor cells lacking the target antigen (RD; rhabdomyosarcoma cell line) and co-cultured with RD.79A, RD.CD20 cells and Daudi cells. After 24 hours of co-culture, supernatants were collected and IFNγ was analyzed using Luminex assay. Both dual CD79A-CD20 CAR T cells produced IFNγ cytokines in response to RD cells expressing only either target antigen, although the responses were slightly attenuated compared to the responses of CD79A CAR–CD20 CCR T cells to either antigen. Both dual CD79A-CD20 CAR T cells also displayed robust responses against Daudi cells, but again, the responses were slightly attenuated compared to the responses of CD79A CAR–CD20 CCR T cells against Daudi cells. Figure 6B Graphs show mean + SEM of replicates from a single PBMC donor.

[0609] Example 8

[0610] Expression of anti-CD79A CAR and anti-CD20 CCR T cells effectively kill

[0611] Cell lines expressing CD79A or CD20

[0612] PBMCs were harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector comprising polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (eg, SEQ ID NO: 37 or 39). After transduction, cells were expanded for 10 days in T cell growth medium containing IL-2.

[0613] Following expansion, the in vitro cytotoxic function of transduced and non-transduced T cell cultures was assessed by xCELLigence Real-Time Cell Analysis (RTCA) using non-invasive electrical impedance monitoring.

[0614] 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 CD79A (RD.CD79A) or CD20 (RD.CD20) at a ratio of 10: 1, 5: 1, and 2.5: 1. After 6 hours of co-culture, the cell index was measured by non-invasive electrical impedance on CELLigence RTCA MP. The cytotoxicity percentage was calculated by normalizing the T cell condition cell index to the tumor control cell index alone. The lack of activity against RD blasts indicates specific activity against CD79A CAR targets and CD20 CCR targets. Figure 7 (Left panel). Transduced T cells to RD.CD79A cells ( Figure 7 , middle panel) and RD.CD20 cells ( Figure 7 , right panel) all exhibited cytotoxicity. The figure shows the mean + SD cytotoxicity of replicates from a single PBMC donor at effector:target ratios of 10:1, 5:1, and 2.5:1.

[0615] The ability of T cells expressing anti-CD79A CAR and anti-CD20 CCR to kill single positive targets of CD79a and CD20 demonstrates a novel dual targeting capability of these cells. Without wishing to be bound by any particular theory, it is believed that CCR-mediated cytotoxicity can occur through CD20 engagement independent of the CAR target and is a unique and innovative property of these cells.

[0616] Example 9

[0617] Expression of anti-CD79A C ARand anti-CD20 CCR T cells in

[0618] D AUDI (Burkitt lymphoma) tumor model is effective

[0619] PBMCs are harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells are transduced with a lentiviral vector comprising polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 37). After transduction, cells are expanded for 10 days in T cell growth medium containing IL-2 and then cryopreserved or their function is evaluated.

[0620] In vitro, UTD T cells or T cells expressing anti-CD79A 41BB CD3ζCAR, T2A, and anti-CD20 CCR fusion protein were inoculated with Daudi tumor cells (CD79A + 、CD20 + ) were co-cultured for 24 hours, and then the supernatants were collected and analyzed for IFNγ using Luminex. Figure 8 (Left panel) Shown are the mean + SEM of replicate IFNγ cytokine measurements from three PBMC donors.

[0621] In vivo. NSG mice were injected with 2×10 6 After 13 days, mice (5) were injected with vehicle (culture medium), 10×10 6 UTD T cells or 10 × 10 6 After 20 days, mice treated with only the CD79A CAR / CD20 CCR-treated mice cleared tumor cells and maintained this clearance until the end of the study on day 30. Figure 8 (Right panel) Shown are the mean + SEM of N = 5 mice for vehicle, except for time points after day 6, where N = 4, N = 5 for UTD, and N = 5 for CD79A CAR / CD20 CCR. *Asterisks indicate animals sacrificed due to tumor size and animal health.

[0622] Example 10

[0623] Expression of anti-CD79A C AR and anti-CD20 CCR T cells in

[0624] NU-DUL-1ABC is effective in DLBCL tumor models

[0625] PBMCs are harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells are transduced with a lentiviral vector comprising polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 37). After transduction, cells are expanded for 10 days in T cell growth medium containing IL-2 and then cryopreserved or their function is evaluated.

[0626] In vitro, UTD T cells or T cells expressing anti-CD79A 41BB CD3ζCAR, T2A, and anti-CD20 CCR fusion protein were mixed with NU-DUL-1ABC cells (CD79A + 、CD20 + ) were co-cultured for 24 hours, and then the supernatants were collected and analyzed for IFNγ using Luminex. Figure 9 (Left panel) Shown are the mean + SEM of replicate IFNγ cytokine measurements from three PBMC donors.

[0627] In vivo. NSG mice were injected with 10×10 6 After 15 days, mice (5) were injected with vehicle (culture medium), 10×10 6 UTD T cells or 5 × 10 6 In this model, only mice treated with CD79A CAR / CD20 CCR-treated mice experienced tumor regression. Figure 9 (Right panel) Shown are the mean + SEM of N = 5 mice for vehicle, except for time points after day 23, where N = 4, N = 5 for UTD, and N = 5 for CD79A CAR / CD20 CCR. *Asterisks indicate animals sacrificed due to tumor size and animal health.

[0628] Example 11

[0629] Expression of anti-CD79A C AR and anti-CD20 CCR T cells in

[0630] T OLEDO Effective in germinal center B cell (GCB) DLBCL tumor models

[0631] PBMCs are harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells are transduced with a lentiviral vector comprising polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 37). After transduction, cells are expanded for 10 days in T cell growth medium containing IL-2 and then cryopreserved or their function is evaluated.

[0632] In vitro, UTD T cells or T cells expressing anti-CD79A 41BB CD3ζCAR, T2A, and anti-CD20 CCR fusion protein were mixed with Toledo GCB DLBCL cells (CD79A 低 、CD20 高 ) were co-cultured for 24 hours, and then the supernatants were collected and analyzed for IFNγ using Luminex. Figure 10 (Left panel) Shown are the mean + SEM of replicate IFNγ cytokine measurements from three PBMC donors.

[0633] In vivo. NSG mice were injected with 50×10 6 luciferase-expressing Toledo GCB DLBCL tumor cells. After 16 days (tumor size approximately 100 mm 3 ), mice (5) were injected with vehicle (culture medium), 20×10 6 UTD T cells or 2.5 × 10 6 The researchers tested T cells expressing both anti-CD79A CAR and anti-CD20 CCR. Mice treated with only the CD79A CAR / CD20 CCR cleared tumor cells and maintained this clearance until the end of the study on day 29, with complete tumor clearance in 3 / 5 mice. Figure 10 (Right panel) Shown are the mean + SEM of N = 5 mice per condition. *Asterisks indicate animals sacrificed due to tumor size and animal health.

[0634] Example 12

[0635] Expression of anti-CD79A C AR and anti-CD20 CCR CBLB-edited T cells in T OLEDO GCB demonstrates increased efficacy in DLBCL tumor models

[0636] PBMC is harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies.Activated cells are transduced with lentiviral vectors, which include polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO:37). Three days after activation, transduced cells are electroporated with the mRNA encoding the megaTAL for cracking CBLB genes (SEQ ID NO:54), and expanded in T cell growth medium containing IL-2 for 10 days, then cryopreserved or evaluated for function.

[0637] In vitro, UTD T cells (+ / - CBLB editing) or T cells expressing anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20CCR fusion protein (+ / - CBLB editing) were co-cultured with Toledo GCB DLBCL cells (CD79A 低 , CD20 高 ) were co-cultured for 24 hours, and then the supernatants were collected and analyzed for IFNγ using Lumineex. Figure 11 (Left panel) Shown are the mean + SEM of replicate IFNγ cytokine measurements from three PBMC donors.

[0638] In vivo. NSG mice were injected with 50×10 6 luciferase-expressing Toledo GCB DLBCL tumor cells. After 17 days (tumor size approximately 130 mm 3 ), mice (5) were injected with vehicle (culture medium), 5×10 6 UTD T cells (+ / - CBLB editing) or 1×10 6 Mice treated with CBLB-edited T cells expressing anti-CD79A CAR and anti-CD20 CCR cleared tumor cells and maintained clearance until the end of the study on day 21, with complete tumor clearance in 1 / 5 mice. Figure 11 (Right panel) shows N=5 mice for vehicle, except for time points after day 17, where N=3, for CD79A CAR / CD20 CCR, N=5, except for time points after day 21, where N=4, and for CD79A CAR / CD20 CCR (+CBLB editing), N=5. *Asterisks indicate animals sacrificed due to tumor size and animal health.

[0639] Example 13

[0640] Expression of anti-CD79A C ARand anti-CD20 CCR CBLB-edited T cells in D AUDI Demonstrates increased efficacy in CD20 knockout tumor models

[0641] PBMC is harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies.Activated cells are transduced with lentiviral vectors, which include polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO:37). Three days after activation, transduced cells are electroporated with the mRNA encoding the megaTAL for cracking CBLB genes (SEQ ID NO:54), and expanded in T cell growth medium containing IL-2 for 10 days, then cryopreserved or evaluated for function.

[0642] In vivo. NSG mice were injected with 2×10 6 Daudi.CD20KO cells expressing luciferase (CD79A + 、CD20 - ). After 14 days, mice (5) were injected with vehicle (culture medium), 20×10 6 UTD T cells (+ / - CBLB editing) or 10 × 10 6 T cells expressing anti-CD79A CAR and anti-CD20 CCR (+ / - CBLB editing). Mice treated with T cells expressing CD79A CAR / CD20 CCR showed anti-tumor activity. CBLB-edited T cells expressing CD79A CAR / CD20 CCR showed enhanced anti-tumor activity compared to all other conditions. Figure 12 Shown are the mean + SEM of N = 5 mice for all conditions. *Asterisks indicate animals sacrificed due to tumor size and animal health.

[0643] Example 14

[0644] Cytokine secretion in CBLB-edited T cells

[0645] PBMC is harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies.Activated cells are transduced with lentiviral vectors, which include polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO:37). Three days after activation, transduced cells are electroporated with the mRNA encoding the megaTAL for cracking CBLB genes (SEQ ID NO:54), and expanded in T cell growth medium containing IL-2 for 10 days, then cryopreserved or evaluated for function.

[0646] UTD T cells (+ / -CBLB editing) or T cells expressing anti-CD79A CAR and anti-CD20 CCR (+ / -CBLB editing) were cultured at 1×10 6 The cells were plated at a concentration of 10 cells / mL in T cell growth medium lacking exogenous IL-2 in a 96-well high binding plate pre-coated with CD3 (1 μg / mL to 0.063 μg / mL) and CD28 (5 μg / mL) monoclonal antibodies. After 24 hours, the supernatant was harvested and cytokine assays were measured via Luminex. CBLB-edited UTD T cells and cells expressing CD79ACAR / CD20 CCR showed IL-2 ( Figure 13 , left panel) and IFNγ ( Figure 13 , right panel) produced an increase. UDD T cell conditions were plated in duplicate, and transduced T cell conditions were plated in quadruplicate. Figure 13 Means + SEM of N = 3 donors are shown.

[0647] Example 15

[0648] Expression of anti-CD79A C AR and anti-CD20 CCR CBLB-edited T cells in D AUDI Increased IL-2 secretion demonstrated in tumor models

[0649] PBMC is harvested from healthy donors and activated using anti-CD3 and anti-CD28 antibodies.Activated cells are transduced with lentiviral vectors, which include polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO:37). Three days after activation, transduced cells are electroporated with the mRNA encoding the megaTAL for cracking CBLB genes (SEQ ID NO:54), and expanded in T cell growth medium containing IL-2 for 10 days, then cryopreserved or evaluated for function.

[0650] UTD T cells (+ / -CBLB editing) or T cells expressing anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion protein (+ / -CBLB editing) were co-cultured with Daudi cells at a ratio of 1:1 for 24 hours, and the supernatant was then collected and analyzed for IL-2 using Luminex. Compared to all other conditions tested, CBLB-edited T cells expressing CD79A CAR / CD20 CCR produced increased amounts of IL-2. Figure 14 Shown are the mean + SEM of IL-2 cytokine production by replicates from a single PBMC donor.

[0651] Example 16

[0652] Proliferation of CBLB-edited T cells

[0653] PBMCs were harvested from healthy human donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with a lentiviral vector comprising a polynucleotide encoding anti-CD79A 41BB CD3ζCAR, T2A, and anti-CD20 CCR fusion protein (SEQ ID NO: 37). Three days after activation, the transduced cells were transduced with mRNA encoding a megaTAL for the cleavage of the CBLB gene (SEQ ID NO: 54) or encoding an inactivated TCRα megaTAL (TCRα DEAD ) mRNA and expanded in T cell growth medium containing IL-2 for 10 days before cryopreservation or functional assessment.

[0654] GFP-expressing Daudi cells were seeded at a density of 50,000 cells / well in 96-well plates in T cell growth medium containing low levels of IL-2. Daudi cells were then mixed with 50,000 UTD T cells (TCRα DEAD ) or expressing anti-CD79A41BB CD3ζCAR, T2A, anti-CD20 CCR fusion protein (TCRα DEAD ) T cells were co-cultured. After four days, half of the T cell growth medium was removed and replaced with fresh medium containing low levels of IL-2. Three days later, 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. Four days later (day 11), the medium was removed and replaced with fresh medium containing low levels of IL-2. Three days later (day 14), the T cells were resuspended and counted. A total of 50,000 T cells (UTD or transduced (+ / - editing)) were then transferred to a fresh 96-well plate containing 50,000 Daudi-GFP tumor cells. Three days later (day 17), the medium was removed and replaced with fresh medium containing low levels of IL-2. Three days later (day 20), the T cells were resuspended and counted. After the third round of tumor cell stimulation, CBLB-edited T cells expressing the CD79 CAR / CD20 CCR showed increased proliferation compared with cells treated with a TCRα-dead control. Figure 15 Shown are the means + SD of 4 replicates per condition.

[0655] Example 17

[0656] Expression of anti-CD79A CAR and anti-CD20 CCR CBLB-edited T cells in T OLEDO Increased IFNγ was demonstrated in the GCB DLBCL tumor model

[0657] PBMCs were harvested from three healthy donors and three DLBCL diseased donors and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were transduced with lentiviral vectors, which included polynucleotides encoding anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion proteins (SEQ ID NO: 37). Three days after activation, transduced cells were electroporated with the mRNA encoding the megaTAL for the cracking CBLB gene (SEQ ID NO: 54), and expanded in T cell growth medium containing IL-2 for 10 days, then cryopreserved or evaluated for function.

[0658] UTD T cells or T cells expressing anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion protein (+ / -CBLB editing) were co-cultured with Toledo GCB DLBCL cells (CD79A low, CD20 high) at a 1:1 ratio for 24 hours, and the supernatants were then collected and analyzed for IFNγ using Luminex. In all healthy and DLBCL donors, CBLB-edited T cells expressing anti-CD79A 41BB CD3ζCAR, T2A, anti-CD20 CCR fusion protein produced more IFNγ cytokines in response to tumor cells compared to T cells expressing UTD or unedited CD79A CAR / CD20 CCR. Figure 16 (Left panel) Shown are the mean + SEM of duplicate IFNγ cytokine measurements in all donors.

[0659] 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 encompass all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure. Sequence Listing <110> bluebird bio, Inc. Friedman, Kevin <120> Compositions and methods for treating cancer <130> BLUE-123.PC <150> US 62 / 861,838 <151> 2019-06-14 <150> US 62 / 991,314 <151> 2020-03-18 <160> 97 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-01 CDRL1 <400> 1 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-01 CDRL2 <400> 2 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-01 CDRL3 <400> 3 Phe Gln Gly Ser His Val Pro Phe Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-01 CDRH1 <400> 4 Gly Tyr Thr Phe Ser Thr Ser Trp Met Asn 1 5 10 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-01 CDRH2 <400> 5 Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 Gly <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-01 CDRH3 <400> 6 Phe Tyr Tyr Gly Asn Thr Phe Ala Met Asp Tyr 1 5 10 <210> 7 <211> 112 <212> PRT <213> Artificial Seqauence <220> <223> Laboratory-made - anti-CD79A-01 light chain <400> 7 Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 8 <211> 120 <212> PRT <213> Artificial Seqauence <220> <223> Laboratory - made - anti - CD79A - 01 heavy chain <400> 8 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Thr Ser 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys 85 90 95 Glu Arg Phe Tyr Tyr Gly Asn Thr Phe Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-10 CDRL1 <400> 9 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-10 CDRL2 <400> 10 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-10 CDRL3 <400> 11 Ser Gln Ser Thr His Val Pro Pro Tyr Thr 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-19 CDRH1 <400> 12 Gly Tyr Ala Phe Ser Phe Ser Trp Met Asn 1 5 10 <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-10 CDRH2 <400> 13 Arg Ile Tyr Pro Gly Asn Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 Gly <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - anti-CD79A-10 CDRH3 <400> 14 Trp Val Tyr Ser Gly Asn Asn Tyr Ala Val Asp Tyr 1 5 10 <210> 15 <211> 113 <212> PRT <213> Artificial Seqauence <220> <223> Laboratory-made - anti-CD79A-10 light chain <400> 15 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 16 <211> 121 <212> PRT <213> Artificial Seqauence <220> <223> Laboratory - made - anti - CD79A - 10 heavy chain <400> 16 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Phe Ser 20 25 30 Trp Met Asn Trp Val Lys Trp Gly Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asn Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Trp Val Tyr Ser Gly Asn Asn Tyr Ala Val Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 17 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made anti-CD79A-01_HL CAR construct <400> 17 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Ser Thr Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn 65 70 75 80 Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 85 90 95 Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser 100 105 110 Ala Val Tyr Phe Cys Glu Arg Phe Tyr Tyr Gly Asn Thr Phe Ala Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Leu Met 145 150 155 160 Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser 165 170 175 Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr 180 185 190 Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu 195 200 205 Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu 225 230 235 240 Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro 245 250 255 Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 18 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> Laboratory - Manufactured - Anti - CD79A - 01_LH CAR Construct <400> 18 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu 20 25 30 Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln 35 40 45 Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg 65 70 75 80 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr 100 105 110 Tyr Cys Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu 145 150 155 160 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Ser Thr Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln 180 185 190 Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn 195 200 205 Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 210 215 220 Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser 225 230 235 240 Ala Val Tyr Phe Cys Glu Arg Phe Tyr Tyr Gly Asn Thr Phe Ala Met 245 250 255 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 19 <211> 493 <212> PRT <213> Artificial Sequence <220> <223> Laboratory - made - anti - CD79A - 10_HL CAR construct <400> 19 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 ​​​​Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr 35 40 45 Ala Phe Ser Phe Ser Trp Met Asn Trp Val Lys Trp Gly Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asn Gly Asp Thr Asn 65 70 75 80 Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 85 90 95 Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser 100 105 110 Ala Val Tyr Phe Cys Ala Arg Trp Val Tyr Ser Gly Asn Asn Tyr Ala 115 120 125 Val Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val 145 150 155 160 Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala 165 170 175 Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn 180 185 190 Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu 195 200 205 Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe 210 215 220 ...

Claims

1. A fusion polypeptide, comprising: a) an anti-CD79A chimeric antigen receptor (CAR), wherein the anti-CD79A CAR comprises: an anti-CD79A antibody or an antigen-binding fragment thereof, CD8α hinge domain, CD8α transmembrane domain, CD137 intracellular co-stimulatory signaling domain, and CD3ζ primary signaling domain; b) 2A self-cleaving polypeptide; and c) an anti-CD20 chimeric co-stimulatory receptor (CCR), wherein the anti-CD20 CCR consists of: An anti-CD20 antibody or an antigen-binding fragment thereof, wherein the anti-CD20 antibody or the antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO: 25, a CDRL2 sequence as shown in SEQ ID NO: 26, and a CDRL3 sequence as shown in SEQ ID NO: 27, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO: 28, a CDRH2 sequence as shown in SEQ ID NO: 29, and a CDRH3 sequence as shown in SEQ ID NO: 30, CD8α hinge domain, CD8α transmembrane domain, and CD28 intracellular co-stimulatory signaling domain; When the fusion polypeptide is expressed in cells, it can produce cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

2. The fusion polypeptide of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment thereof is selected from a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), Fv, a single-chain Fv protein ("scFv"), a double scFv, (scFv)2, a miniantibody, a double antibody, a three-antibody, a four-antibody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanoantibody).

3. The fusion polypeptide of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment thereof is a scFv.

4. The fusion polypeptide of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO: 1, a CDRL2 sequence as shown in SEQ ID NO: 2, and a CDRL3 sequence as shown in SEQ ID NO: 3, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO: 4, a CDRH2 sequence as shown in SEQ ID NO: 5, and a CDRH3 sequence as shown in SEQ ID NO: 6; or the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO: 9, a CDRL2 sequence as shown in SEQ ID NO: 10, and a CDRL3 sequence as shown in SEQ ID NO: 11, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO: 12, a CDRH2 sequence as shown in SEQ ID NO: 13, and a CDRH3 sequence as shown in SEQ ID NO:

14.

5. The fusion polypeptide of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO: 1, a CDRL2 sequence as shown in SEQ ID NO: 2, and a CDRL3 sequence as shown in SEQ ID NO: 3, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO: 4, a CDRH2 sequence as shown in SEQ ID NO: 5, and a CDRH3 sequence as shown in SEQ ID NO:

6.

6. The fusion polypeptide of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO:9, a CDRL2 sequence as shown in SEQ ID NO:10, and a CDRL3 sequence as shown in SEQ ID NO:11, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO:12, a CDRH2 sequence as shown in SEQ ID NO:13, and a CDRH3 sequence as shown in SEQ ID NO:

14.

7. The fusion polypeptide of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO: 7 and a variable heavy chain sequence as shown in SEQ ID NO: 8; or a variable light chain sequence as shown in SEQ ID NO: 15 and a variable heavy chain sequence as shown in SEQ ID NO:

16.

8. The fusion polypeptide of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO: 7 and a variable heavy chain sequence as shown in SEQ ID NO:

8.

9. The fusion polypeptide of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO: 15 and a variable heavy chain sequence as shown in SEQ ID NO:

16.

10. A fusion polypeptide comprising an anti-CD79A CAR, comprising: An anti-CD79A antibody or an antigen-binding fragment thereof, comprising: The variable light chain sequence shown in SEQ ID NO:7 and the variable heavy chain sequence shown in SEQ ID NO:8; or the variable light chain sequence shown in SEQ ID NO:15 and the variable heavy chain sequence shown in SEQ ID NO:16, CD8α hinge domain, CD8α transmembrane domain, CD137 co-stimulatory domain and CD3ζ primary signaling domain; 2A self-cleaving peptide, and An anti-CD20 CCR, wherein the anti-CD20 CCR consists of: An anti-CD20 antibody or an antigen-binding fragment thereof, wherein the anti-CD20 antibody or the antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO: 25, a CDRL2 sequence as shown in SEQ ID NO: 26, and a CDRL3 sequence as shown in SEQ ID NO: 27, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO: 28, a CDRH2 sequence as shown in SEQ ID NO: 29, and a CDRH3 sequence as shown in SEQ ID NO: 30, CD8α hinge domain, CD8α transmembrane domain, and CD28 intracellular co-stimulatory signaling domain, When the fusion polypeptide is expressed in cells, it can produce cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

11. The fusion polypeptide of claim 1 or 10, wherein the 2A self-cleaving polypeptide is a viral 2A self-cleaving polypeptide selected from the group consisting of foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis virus type A (ERAV) 2A (E2A) peptide, Tetrasomal virus (TaV) 2A (T2A) peptide, porcine Teschovirus-1 (PTV-1) 2A (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide.

12. A fusion polypeptide comprising: an anti-CD79A CAR comprising an amino acid sequence as shown in any one of SEQ ID NOs: 17-20, a T2A self-cleaving polypeptide, and an anti-CD20 CCR, wherein the anti-CD20 CCR consists of an anti-CD20 antibody or an antigen-binding fragment thereof, a CD8α hinge domain, a CD8α transmembrane domain, and a CD28 intracellular co-stimulatory signaling domain, wherein the anti-CD20 antibody or an antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO:25, a CDRL2 sequence as shown in SEQ ID NO:26, and a CDRL3 sequence as shown in SEQ ID NO:27, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO:28, a CDRH2 sequence as shown in SEQ ID NO:29, and a CDRH3 sequence as shown in SEQ ID NO:30, When the fusion polypeptide is expressed in cells, it can produce cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

13. A fusion polypeptide as described in any one of claims 1, 10 and 12, wherein the anti-CD20 antibody or its antigen-binding fragment is selected from a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), Fv, a single-chain Fv protein ("scFv"), a double scFv, (scFv)2, a miniantibody, a double antibody, a three-antibody, a four-antibody, a disulfide-stabilized Fv protein ("dsFv") and a single domain antibody (sdAb, nanobody).

14. The fusion polypeptide of any one of claims 1, 10 and 12, wherein the anti-CD20 antibody or antigen-binding fragment thereof is a scFv.

15. The fusion polypeptide of any one of claims 1, 10 and 12, wherein the anti-CD20 antibody or antigen-binding fragment thereof has a variable light chain sequence as shown in SEQ ID NO: 31 and a variable heavy chain sequence as shown in SEQ ID NO:

32.

16. A fusion polypeptide comprising an anti-CD79A CAR, a T2A self-cleaving polypeptide and an anti-CD20 CCR, wherein the amino acid sequence of the anti-CD79A CAR is as shown in any one of SEQ ID NOs: 17-20, wherein the amino acid sequence of the anti-CD20 CCR is as shown in SEQ ID NO: 33 or SEQ ID NO: 35, and wherein the fusion polypeptide is capable of producing cytotoxic activity against single CAR and CCR-positive target cells, and dual CAR and CCR-positive target cells when expressed in a cell.

17. A fusion polypeptide comprising an anti-CD79A CAR and an anti-CD20 CCR, wherein the fusion polypeptide consists of the amino acid sequence shown in SEQ ID NO: 37 or SEQ ID NO: 39, and wherein the fusion polypeptide is capable of producing cytotoxic activity against single CAR and CCR-positive target cells, and dual CAR and CCR-positive target cells when expressed in a cell.

18. The fusion polypeptide of any one of claims 1, 10 and 12, wherein the anti-CD20 CCR does not comprise a primary signaling domain.

19. A polynucleotide encoding an anti-CD79A CAR and an anti-CD20 CCR as described in any one of claims 1, 10, 12, 16 and 17.

20. A polynucleotide encoding the fusion polypeptide of any one of claims 1, 10, 12, 16 and 17.

21. A polynucleotide comprising the sequence shown in SEQ ID NO: 38 or SEQ ID NO:

40.

22. A vector comprising the polynucleotide encoding the fusion polypeptide of any one of claims 1, 10, 12, 16 and 17.

23. The vector of claim 22, wherein the vector is an expression vector.

24. The vector of claim 22, wherein the vector is an episomal vector.

25. The vector of claim 22, wherein the vector is a viral vector.

26. The vector of claim 22, wherein the vector is a retroviral vector.

27. The vector of claim 22, wherein the vector is a lentiviral vector.

28. A cell expressing the fusion polypeptide of any one of claims 1, 10, 12, 16 and 17.

29. A cell comprising a polynucleotide encoding the fusion polypeptide of any one of claims 1, 10, 12, 16 and 17.

30. A cell comprising one or more polynucleotides encoding: (a) an anti-CD79A CAR comprising an anti-CD79A antibody or an antigen-binding fragment thereof, a CD8α hinge domain, a CD8α transmembrane domain, a CD137 intracellular co-stimulatory domain, and a CD3ζ primary signaling domain; and (b) an anti-CD20 CCR, wherein the anti-CD20 CCR consists of an anti-CD20 antibody or an antigen-binding fragment thereof, a CD8α hinge domain, a CD8α transmembrane domain, and a CD28 intracellular co-stimulatory signaling domain, wherein the anti-CD20 antibody or antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO:25, a CDRL2 sequence as shown in SEQ ID NO:26, and a CDRL3 sequence as shown in SEQ ID NO:27, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO:28, a CDRH2 sequence as shown in SEQ ID NO:29, and a CDRH3 sequence as shown in SEQ ID NO:30, and The cells are capable of producing cytotoxic activity against single CAR and CCR-positive target cells, as well as dual CAR and CCR-positive target cells.

31. The cell of claim 30, wherein the anti-CD79A antibody or antigen-binding fragment thereof and the anti-CD20 antibody or antigen-binding fragment thereof are 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), Fv, a single-chain Fv protein ("scFv"), a double scFv, (scFv)2, a mini-antibody, a double antibody, a three-antibody, a four-antibody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nano-antibody).

32. The cell of claim 30, wherein the anti-CD79A antibody or antigen-binding fragment thereof and the anti-CD20 antibody or antigen-binding fragment thereof are both scFvs.

33. The cell of claim 30, wherein the anti-CD79A scFv comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO: 1, a CDRL2 sequence as shown in SEQ ID NO: 2, and a CDRL3 sequence as shown in SEQ ID NO: 3, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO: 4, a CDRH2 sequence as shown in SEQ ID NO: 5, and a CDRH3 sequence as shown in SEQ ID NO: 6; or the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO: 9, a CDRL2 sequence as shown in SEQ ID NO: 10, and a CDRL3 sequence as shown in SEQ ID NO: 11, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO: 12, a CDRH2 sequence as shown in SEQ ID NO: 13, and a CDRH3 sequence as shown in SEQ ID NO:

14.

34. The cell of claim 30, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO: 1, a CDRL2 sequence as shown in SEQ ID NO: 2, and a CDRL3 sequence as shown in SEQ ID NO: 3, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO: 4, a CDRH2 sequence as shown in SEQ ID NO: 5, and a CDRH3 sequence as shown in SEQ ID NO:

6.

35. The cell of claim 30, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence and a variable heavy chain sequence, wherein the variable light chain sequence has a CDRL1 sequence as shown in SEQ ID NO:9, a CDRL2 sequence as shown in SEQ ID NO:10, and a CDRL3 sequence as shown in SEQ ID NO:11, and the variable heavy chain sequence has a CDRH1 sequence as shown in SEQ ID NO:12, a CDRH2 sequence as shown in SEQ ID NO:13, and a CDRH3 sequence as shown in SEQ ID NO:

14.

36. The cell of claim 30, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO:7 and a variable heavy chain sequence as shown in SEQ ID NO:8; or a variable light chain sequence as shown in SEQ ID NO:15 and a variable heavy chain sequence as shown in SEQ ID NO:

16.

37. The cell of claim 30, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO:7 and a variable heavy chain sequence as shown in SEQ ID NO:

8.

38. The cell of claim 30, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO: 15 and a variable heavy chain sequence as shown in SEQ ID NO:

16.

39. The cell of claim 30, wherein the anti-CD20 antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO:31 and the variable heavy chain sequence shown in SEQ ID NO:

32.

40. The cell of claim 30, wherein the anti-CD20 CCR does not comprise a primary signaling domain.

41. The cell of claim 30, wherein the cell expresses an anti-CD79A CAR having an amino acid sequence as shown in any one of SEQ ID NOs: 17-20 and an anti-CD20 CCR having an amino acid sequence as shown in SEQ ID NO: 33 or SEQ ID NO:

35.

42. The cell of claim 30, wherein the cell comprises a first polynucleotide encoding the anti-CD79A CAR and a second polynucleotide encoding the anti-CD20 CCR.

43. The cell of claim 30, wherein the polynucleotide encodes the anti-CD79A CAR and the anti-CD20 CCR.

44. The cell of claim 43, wherein the polynucleotide encodes the anti-CD79A CAR, the IRES sequence and the anti-CD20 CCR.

45. The cell of claim 43, wherein the polynucleotide encodes the anti-CD79A CAR, the 2A self-cleaving polypeptide and the anti-CD20 CCR.

46. ​​The cell of claim 45, wherein the 2A self-cleaving polypeptide is a viral self-cleaving polypeptide.

47. A cell as described in claim 45, wherein the viral self-cleaving polypeptide is selected from: foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis virus type A (ERAV) 2A (E2A) peptide, beta-tetrasomal virus (TaV) 2A (T2A) peptide, porcine teschovirus-1 (PTV-1) 2A (P2A) peptide, Theileria virus 2A peptide and encephalomyocarditis virus 2A peptide.

48. The cell of claim 30, 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 in the casitas B lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene.

49. The cell of claim 48, wherein the HE variant introduces one or more insertions or deletions into the HE target site in the CBLB gene shown in SEQ ID NO:

55.

50. The cell of claim 48, wherein the megaTAL introduces one or more insertions or deletions into the megaTAL target site in the CBLB gene shown in SEQ ID NO:

56.

51. The cell of claim 48, wherein the insertion or deletion in the CBLB gene reduces CBLB expression, function and / or activity.

52. The cell of claim 30, wherein the cell comprises one or more modified CBLB alleles.

53. The cell of claim 30, wherein the cell comprises one or more modified CBLB alleles that do not express or produce CBLB, or express or produce non-functional CBLB.

54. The cell of claim 30, 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 in a programmed cell death 1 (PDCD-1) gene.

55. The cell of claim 54, wherein the HE variant introduces one or more insertions or deletions into the HE target site in the PDCD-1 gene shown in SEQ ID NO:

51.

56. The cell of claim 54, wherein the megaTAL introduces one or more insertions or deletions into the megaTAL target site in the PDCD-1 gene shown in SEQ ID NO:

52.

57. The cell of claim 54, wherein the insertion or deletion in the PDCD-1 gene reduces PDCD-1 expression, function and / or activity.

58. The cell of claim 30, wherein the cell comprises one or more modified PDCD-1 alleles.

59. The cell of claim 30, wherein the cell comprises one or more modified PDCD-1 alleles that do not express or produce PDCD-1 or express or produce non-functional PDCD-1.

60. The cell of any one of claims 30-59, wherein the cell is a hematopoietic cell.

61. The cell of claim 30, wherein the cell is a hematopoietic stem or progenitor cell.

62. The cell of claim 30, wherein the cell is a CD34+ hematopoietic stem or progenitor cell.

63. The cell of claim 30, wherein the cell is an immune effector cell.

64. The cell of claim 30, wherein the cell is a T cell.

65. The cell of any one of claims 30-59, wherein the cell is a CD3 + 、CD4 + and / or CD8 + cell.

66. The cell of any one of claims 30-59, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

67. The cell of any one of claims 30-59, wherein the cell is a Natural Killer (NK) cell or a Natural Killer T (NKT) cell.

68. A cell population comprising a plurality of cells as described in claim 30.

69. A cell population comprising one or more hematopoietic stem or progenitor cells as described in claim 61 and one or more immune effector cells as described in claim 63.

70. A cell population comprising one or more CD34+ hematopoietic stem or progenitor cells as described in claim 62 and one or more T cells as described in claim 64.

71. A composition comprising the cells of claim 30 and a physiologically acceptable excipient.

72. A composition comprising the cell population of claim 68 and a physiologically acceptable excipient.

73. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for killing cancer cells expressing CD79A or CD20 in a subject, wherein the cancer cells are from a cancer selected from the group consisting of 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 myeloid leukemia (AML) or chronic myeloid leukemia. CML, 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

74. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for killing cancer cells expressing CD79A and CD20 in a subject, wherein the cancer cells are from a cancer selected from the group consisting of 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 myeloid leukemia (AML) or chronic myeloid leukemia. CML, 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

75. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for killing cancer cells expressing CD79A and / or CD20 in a subject, wherein the cancer cells are from a cancer selected from the group consisting of 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 myeloid leukemia (AML) or chronic myeloid leukemia (CLL). CML, 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

76. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for reducing the number of cancer cells expressing CD79A or CD20 in a subject, the number being reduced compared to the number of cancer cells expressing CD79A or CD20 before administration of the medicament, wherein the cancer cells are from a cancer selected from the group consisting of osteosarcoma or Ewing's sarcoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), M), acute myeloid leukemia (AML) or chronic myeloid leukemia (CML), 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

77. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for reducing the number of cancer cells expressing CD79A and CD20 in a subject, the number being reduced as compared to the number of cancer cells expressing CD79A and CD20 before administration of the medicament, wherein the cancer cells are from a cancer selected from the group consisting of osteosarcoma or Ewing's sarcoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), M), acute myeloid leukemia (AML) or chronic myeloid leukemia (CML), 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

78. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for reducing the number of cancer cells expressing CD79A and / or CD20 in a subject, the number being reduced compared to the number of cancer cells expressing CD79A and / or CD20 before administration of the medicament, wherein the cancer cells are from a cancer selected from the group consisting of 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 myeloid leukemia (AML) or chronic myeloid leukemia (CML), 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

79. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the cancer cells are from a cancer selected from the group consisting of 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 myeloid leukemia (AML) or chronic myeloid leukemia (CML), 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

80. The method of claim 79, 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 myeloid leukemia (AML) or chronic myeloid leukemia (CML), 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

81. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for treating a subject suffering from DLBCL.

82. Use of the composition of claim 71 or claim 72 in the preparation of a medicament for ameliorating one or more symptoms associated with a cancer expressing CD79A and / or CD20 in a subject, wherein the cancer cells are from a cancer selected from the group consisting of 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 myeloid leukemia (AML) or chronic myeloid leukemia (CML), 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), diffuse large B-cell lymphoma (DLBCL), overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

83. The use of claim 82, wherein the one or more symptoms improved are selected from the group consisting of: weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal swelling or pain, bone or joint pain, fractures, unexpected weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination.

84. A method for producing a cell population expressing a fusion polypeptide, the method comprising introducing the polynucleotide of claim 19 or the vector of claim 22 into the cell population, The cell population is capable of producing cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

85. A method for producing a cell population expressing anti-CD79A CAR and anti-CD20 CCR, the method comprising introducing one or more polynucleotides encoding the anti-CD79A CAR and anti-CD20 CCR of any one of claims 1, 10, 12, 16 and 17 into the cell population, The cell population is capable of producing cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

86. A method for producing a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR, the method comprising introducing into the cell population a polynucleotide encoding the fusion polypeptide sequence shown in any one of SEQ ID NOs: 37 and 39, The cell population is capable of producing cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

87. A method for producing a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR, the method comprising introducing into the cell population a first polynucleotide encoding the anti-CD79A CAR shown in any one of SEQ ID NOs: 17-20 and a second polynucleotide encoding the anti-CD20 CCR sequence shown in SEQ ID NO: 33 or SEQ ID NO: 35, The cell population is capable of producing cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

88. A method for producing a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR, the method comprising introducing the polynucleotide sequence shown in SEQ ID NO: 38 or SEQ ID NO: 40 into the cell population, The cell population is capable of producing cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

89. A method for producing a cell population expressing an anti-CD79A CAR and an anti-CD20 CCR, the method comprising introducing into the cell population a first polynucleotide sequence shown in any one of SEQ ID NOs: 21 to 24 and a second polynucleotide sequence shown in SEQ ID NO: 34 or SEQ ID NO: 36, The cell population is capable of producing cytotoxic activity against single CAR and CCR positive target cells, as well as dual CAR and CCR positive target cells.

90. The method of any one of claims 86-89, wherein one or more cells in the cell population comprise one or more insertions or deletions in the PDCD-1 gene at the polynucleotide sequence shown in SEQ ID NO:51, which reduce or eliminate PDCD-1 expression and / or function.

91. The method of claim 90, wherein a polynucleotide encoding a HE variant that binds to and cleaves the polynucleotide sequence shown in SEQ ID NO:51 is introduced into the cell population.

92. The method of any one of claims 86-89, wherein one or more cells in the cell population comprise one or more insertions or deletions in the PDCD-1 gene at the polynucleotide sequence shown in SEQ ID NO:52, which reduce or eliminate PDCD-1 expression and / or function.

93. The method of claim 92, wherein a megaTAL encoding a protein that binds and cleaves the polynucleotide sequence shown in SEQ ID NO:52 is introduced into the cell population.

94. The method of any one of claims 86-89, wherein one or more cells in the cell population comprise one or more insertions or deletions in the CBLB gene at the polynucleotide sequence shown in SEQ ID NO:55, which reduce or eliminate CBLB expression and / or function.

95. The method of claim 94, wherein a polynucleotide encoding a HE variant that binds to and cleaves the polynucleotide sequence shown in SEQ ID NO:55 is introduced into the cell population.

96. The method of any one of claims 86-89, wherein one or more cells in the cell population comprise one or more insertions or deletions in the CBLB gene at the polynucleotide sequence shown in SEQ ID NO:56, which reduce or eliminate CBLB expression and / or function.

97. The method of claim 96, wherein a megaTAL encoding a protein that binds and cleaves the polynucleotide sequence shown in SEQ ID NO:56 is introduced into the cell population.

98. The method of any one of claims 86-89, wherein the cell population comprises hematopoietic stem or progenitor cells.

99. The method of any one of claims 86-89, wherein the cell population comprises CD34+ hematopoietic stem or progenitor cells.

100. The method of any one of claims 86-89, wherein the cell population comprises immune effector cells.

101. The method of any one of claims 86-89, wherein the cell population comprises T cells, NK cells and / or NKT cells.

102. The method of any one of claims 86-89, wherein the cell population comprises T cells.

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