CD79A chimeric antigen receptor

Abstract

The present invention provides improved compositions for adoptive cell therapy of cancers expressing CD79A.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 62 / 685,078, filed on June 14, 2018, which is incorporated herein by reference in its entirety.

[0003] Statement concerning 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 this specification. The name of the text file containing the sequence listing is BLBD_100_01WO_ST25.txt. The text file is 64KB, created on June 14, 2019, and submitted electronically through EFS-Web, submitted simultaneously with this specification. Technical Field

[0005] The present invention relates to improved compositions and methods for treating cancer. More specifically, the present invention relates to improved anti-CD79A chimeric antigen receptors (CARs), genetically modified immune effector cells, and the use of these compositions for effectively treating cancers expressing CD79A. Background Art

[0006] Cancer is a major health problem worldwide. Based on the rate of 2008-2010, 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 suffer from cancer between the ages of 50 and 70, compared to 15.30% of women. On January 1, 2010, there were approximately 13,027,914 men and women alive with a history of cancer in the United States - 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) will be diagnosed with cancer in the United States and 580,350 men and women will die of cancer nationwide. Howlader et al. 2013.

[0007] Malignant transformation of B cells leads to cancer, including but not limited to lymphomas, such as multiple myeloma and non-Hodgkin's lymphoma. Most patients with B cell malignancies, including non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), are important contributors to cancer mortality. The response of B cell malignancies to various forms of treatment is mixed. Traditional methods for treating B cell malignancies, including chemotherapy and radiotherapy, have limited effectiveness due to toxic side effects. The immunotherapy using anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD52, anti-CD80 and anti-HLA-DR therapeutic antibodies has limited success, in part due to poor pharmacokinetic profiles, rapid elimination of antibodies by serum proteases and filtration at glomeruli, and limited expression levels of infiltration to tumor sites and target antigens on cancer cells. Attempts to use genetically modified cells expressing chimeric antigen receptors (CARs) have also been successful to a limited extent. Additionally, the therapeutic efficacy of a given antigen binding domain used in a particular CAR may be unpredictable: if the antigen binding domain binds too strongly, the CAR T cell may induce massive cytokine release, leading to a potentially lethal immune response known as a "cytokine storm," and if the antigen binding domain binds too weakly, the CAR T cell may not show sufficient therapeutic efficacy in clearing cancer cells. Summary of the invention

[0008] The present invention generally provides improved vectors for producing adoptive cell therapy and methods of use thereof. More specifically, the present invention provides anti-CD79A CAR molecules and their use in treating, preventing or ameliorating cancers expressing CD79A.

[0009] In various embodiments, a chimeric antigen receptor (CAR) is provided, comprising: an extracellular domain comprising: a) an anti-CD79A antibody or an antigen-binding fragment thereof that binds to one or more epitopes of a human CD79A polypeptide, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising a CDRL1-CDRL3 sequence as set forth in SEQ ID NO: 1-3, 9-11 or 17-19, and / or and a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence as set forth in SEQ ID NO: 4-6, 12-14 or 20-22; b) a transmembrane domain; c) one or more intracellular co-stimulatory signaling domains; and / or d) a primary signaling domain.

[0010] In a specific embodiment, the anti-CD79A antibody or antigen-binding fragment that binds to the human CD79A polypeptide is 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 bifunctional antibody, a trifunctional antibody, a tetrafunctional antibody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0011] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment that binds to a human CD79A polypeptide is a scFv.

[0012] In specific embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs as set forth in any one of SEQ ID NOs: 1-3, and / or one or more heavy chain CDRs as set forth in any one of SEQ ID NOs: 4-6.

[0013] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs as set forth in any one of SEQ ID NOs: 9-11, and / or one or more heavy chain CDRs as set forth in any one of SEQ ID NOs: 12-14.

[0014] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs as set forth in any one of SEQ ID NOs: 17-19, and / or one or more heavy chain CDRs as set forth in any one of SEQ ID NOs: 20-22.

[0015] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as set forth in any one of SEQ ID NOs: 7, 15, or 23, and / or a variable heavy chain sequence as set forth in any one of SEQ ID NOs: 8, 16, or 24.

[0016] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence set forth in any one of SEQ ID NOs:7, 15 or 23, and / or a variable heavy chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence set forth in any one of SEQ ID NOs:8, 16 or 24.

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

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

[0019] In additional embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as set forth in SEQ ID NO:23 and / or a variable heavy chain sequence as set forth in SEQ ID NO:24.

[0020] In further embodiments, the transmembrane domain is 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, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, and PD1.

[0021] In other embodiments, the transmembrane domain is from a polypeptide selected from the group consisting of: CD8α, CD28, CD4, CD45, PD1, and CD152.

[0022] In some embodiments, the transmembrane domain is from CD8α.

[0023] In further embodiments, the one or more co-stimulatory signaling domains are from a co-stimulatory 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.

[0024] In certain embodiments, the one or more costimulatory signaling domains are from a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

[0025] In some embodiments, one or more co-stimulatory signaling domains are from CD137.

[0026] In specific 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.

[0027] In specific embodiments, the primary signaling domain is isolated from CD3ζ.

[0028] In other embodiments, the CAR further comprises a hinge region polypeptide.

[0029] In certain embodiments, the hinge region polypeptide comprises the hinge region of CD8α.

[0030] In other embodiments, the CAR further comprises a spacer.

[0031] In additional embodiments, the CAR further comprises a signal peptide.

[0032] In specific embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide, a CD8 alpha signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide.

[0033] In specific embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 30.

[0034] In a specific embodiment, the CAR comprises the amino acid sequence set forth in SEQ ID NO:25.

[0035] In a specific embodiment, the CAR comprises the amino acid sequence set forth in SEQ ID NO:26.

[0036] In a specific embodiment, the CAR comprises the amino acid sequence set forth in SEQ ID NO:27.

[0037] In a specific embodiment, the CAR comprises the amino acid sequence set forth in SEQ ID NO:28.

[0038] In a specific embodiment, the CAR comprises the amino acid sequence set forth in SEQ ID NO:29.

[0039] In a specific embodiment, the CAR comprises the amino acid sequence set forth in SEQ ID NO:30.

[0040] In various embodiments, a polypeptide comprising the amino acid sequence of a CAR contemplated herein is provided.

[0041] In various embodiments, polynucleotides encoding the CARs contemplated herein are provided.

[0042] In specific embodiments, a polynucleotide encoding a CAR contemplated herein comprises a sequence set forth in any one of SEQ ID NOs: 31-36.

[0043] In various embodiments, vectors comprising a polynucleotide encoding a CAR contemplated herein are provided.

[0044] In certain embodiments, the vector is an expression vector.

[0045] In certain embodiments, the vector is an episomal vector.

[0046] In further embodiments, the vector is a viral vector.

[0047] In further embodiments, the vector is a retroviral vector.

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

[0049] In further embodiments, the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2); Vesna-Medivirus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0050] In specific embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element; a promoter operably linked to a polynucleotide; and a right (3') retroviral LTR.

[0051] In additional embodiments, the vector further comprises a heterologous polyadenylation sequence.

[0052] In specific embodiments, the vector further comprises a Hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE).

[0053] In other embodiments, the promoter of the 5' LTR is replaced with a heterologous promoter.

[0054] In further embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40) promoter.

[0055] In some embodiments, the 5'LTR or 3'LTR is a lentiviral LTR.

[0056] In certain embodiments, the 3'LTR comprises one or more modifications.

[0057] In certain embodiments, the 3'LTR comprises one or more deletions.

[0058] In specific embodiments, the 3' LTR is a self-inactivating (SIN) LTR.

[0059] In specific embodiments, the polyadenylation sequence is a bovine growth hormone polyadenylation or a signal rabbit β-globin polyadenylation sequence.

[0060] In other embodiments, the polynucleotide comprises an optimized Kozak sequence.

[0061] In other embodiments, the promoter operably linked to the polynucleotide is selected from the group consisting of: cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF1-α), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer / chicken β-actin promoter (CAG), polyoma enhancer / herpes simplex thymidine kinase promoter (MC1), β-actin promoter (β-ACT), simian virus 40 promoter (SV40) and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site replaced (MND) promoter.

[0062] In various embodiments, an immune effector cell is provided, comprising a vector encoding a CAR contemplated herein.

[0063] In certain embodiments, the immune effector cells are selected from the group consisting of T lymphocytes, natural killer (NK) cells, and NKT cells.

[0064] In some embodiments, immune effector cells are transduced with a vector contemplated herein and activated and stimulated in the presence of a PI3K pathway inhibitor, thereby maintaining proliferation of the transduced immune effector cells compared to proliferation of transduced immune effector cells activated and stimulated in the absence of the PI3K pathway inhibitor.

[0065] In specific embodiments, immune effector cells activated and stimulated in the presence of a PI3K pathway inhibitor have increased expression of i) one or more markers selected from the group consisting of CD62L, CD127, CD197, and CD38, or ii) all of the markers CD62L, CD127, CD197, and CD38, compared to immune effector cells activated and stimulated in the absence of the PI3K pathway inhibitor.

[0066] In specific embodiments, immune effector cells activated and stimulated in the presence of a PI3K pathway inhibitor have increased expression of i) one or more markers selected from the group consisting of CD62L, CD127, CD27, and CD8, or ii) all of the markers CD62L, CD127, CD27, and CD8, compared to immune effector cells activated and stimulated in the absence of a PI3K pathway inhibitor.

[0067] In one embodiment, the PI3K inhibitor is ZSTK474.

[0068] In various embodiments, a composition is provided comprising the immune effector cells contemplated herein and a physiologically acceptable excipient.

[0069] In various embodiments, a method of producing a population of immune effector cells comprising a CAR encompassed herein is provided, comprising introducing into the population of immune effector cells a vector encoding a CAR encompassed herein.

[0070] In certain embodiments, the method further comprises stimulating immune effector cells by contacting the cells with an antibody that binds CD3 and an antibody that binds CD28 and inducing proliferation of the cells; thereby generating an expanded population of immune effector cells.

[0071] In certain embodiments, immune effector cells are stimulated and induced to proliferate prior to introduction of the vector.

[0072] In other embodiments, the immune effector cell population comprises T cells.

[0073] In some embodiments, the immune effector cell population comprises NK cells.

[0074] In specific embodiments, cells are activated and stimulated in the presence of a PI3K pathway inhibitor, thereby maintaining proliferation of the transduced immune effector cells compared to proliferation of immune effector cells activated and stimulated in the absence of the PI3K pathway inhibitor.

[0075] In some embodiments, immune effector cells activated and stimulated in the presence of a PI3K pathway inhibitor have increased expression of i) one or more markers selected from the group consisting of CD62L, CD127, CD197, and CD38, or ii) all of the markers CD62L, CD127, CD197, and CD38, compared to immune effector cells activated and stimulated in the absence of the PI3K pathway inhibitor.

[0076] In specific embodiments, immune effector cells activated and stimulated in the presence of a PI3K pathway inhibitor have increased expression of i) one or more markers selected from the group consisting of CD62L, CD127, CD27, and CD8, or ii) all of the markers CD62L, CD127, CD27, and CD8, compared to immune effector cells activated and stimulated in the absence of a PI3K pathway inhibitor.

[0077] In one embodiment, the PI3K inhibitor is ZSTK474.

[0078] In various embodiments, methods are provided for increasing the cytotoxicity of cancer cells expressing CD79A in a subject, comprising administering to the subject an amount of a composition encompassed herein sufficient to increase the cytotoxicity of cancer cells expressing CD79A compared to the cytotoxicity of cancer cells expressing CD79A prior to administration.

[0079] In various embodiments, methods of reducing the number of cancer cells expressing CD79A in a subject are provided, comprising administering to the subject an amount of a composition encompassed herein sufficient to reduce the number of cancer cells expressing CD79A compared to the number of cancer cells expressing CD79A prior to administration.

[0080] In various embodiments, provided is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

[0081] In certain embodiments, the cancer is a liquid cancer.

[0082] In some embodiments, the cancer is a hematological malignancy.

[0083] In further embodiments, the cancer is lung cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, adrenal cancer, melanoma, uterine cancer, testicular cancer, or bladder cancer, 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).

[0084] In specific embodiments, the non-Hodgkin's lymphoma is small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL).

[0085] In certain embodiments, the cancer is acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0086] In certain embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0087] In specific 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.

[0088] In various embodiments, a method of improving one or more symptoms associated with cancer expressing CD79A in a subject is provided, comprising administering to the subject an amount of a composition encompassed herein sufficient to improve at least one symptom associated with cancer cells expressing CD79A.

[0089] In certain embodiments, 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 bloating or pain, bone or joint pain, fractures, unplanned weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 shows CD79A expression on target cells, CAR expression, and anti-CD79A CAR T cell antigen-dependent activity in the presence of target cells expressing CD79A. A) This figure shows that CD79A is expressed on Pfeiffer and Daudi cells, but not on K562 cells. B) This figure shows representative anti-CD79A CAR expression on T cells measured using flow cytometry. C) This figure shows IFNγ secretion of anti-CD79A CAR T cells co-cultured in the absence of target cells or with K562 cells (CD79A-), Daudi cells (CD79A+, high expression) or Pfeiffer cells (CD79A+, low expression).

[0091] Fig. 2 shows CAR expression, CD79A expression on target cells and anti-CD79A CAR T cell antigen-dependent activity in the presence of target cells expressing CD79A. A) This figure shows the CD79A receptor density on Daudi, NU-DUL-1, SU-DHL-2 and Pfeiffer cells. B) This figure shows representative anti-CD79A CAR expression on T cells measured using flow cytometry. C) This figure shows IFNγ secretion of anti-CD79A CAR T cells co-cultured in the absence of target cells or with Huh7 cells (CD79A-), Huh7.CD79a cells (CD79A+), Daudi cells (CD79A+), NU-DUL-1 (CD79A+), SU-DHL-2 (CD79A+) or Pfeiffer cells (CD79A+).

[0092] A brief description of sequence identifiers

[0093] SEQ ID NOs: 1-24 set forth the amino acid sequences of exemplary light chain CDR sequences, heavy chain CDR sequences, variable domain light chains, and variable domain heavy chains of the CAR anti-CD79A contemplated herein.

[0094] SEQ ID NOs:25-30 set forth the amino acid sequences of exemplary anti-CD79A CARs.

[0095] SEQ ID NOs:31-36 set forth the nucleic acid sequences of exemplary anti-CD79A CARs.

[0096] SEQ ID NOs:37-38 set forth the amino acid sequences of exemplary human CD79A polypeptides.

[0097] SEQ ID NOs: 39-49 set forth the amino acid sequences of various linkers.

[0098] SEQ ID NOs: 50-74 set forth the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites. DETAILED DESCRIPTION

[0099] A. Overview

[0100] The present invention generally relates to improved compositions and methods for preventing or treating cancers expressing CD79A or preventing, treating or improving at least one symptom associated with cancers expressing CD79A. In a particular embodiment, the present invention relates to improved adoptive cell therapy for cancers expressing CD79A, wherein the cell therapy uses genetically modified immune effector cells. Genetic methods provide potential means for enhancing immune recognition and eliminating cancer cells. A promising strategy is to genetically engineer immune effector cells to express chimeric antigen receptors (CARs) that redirect cytotoxicity to cancer cells.

[0101] Improved compositions and methods of adoptive cell therapy contemplated herein provide genetically modified immune effector cells that can be easily expanded, exhibit long-term persistence in vivo, and exhibit antigen-dependent cytotoxicity against cells expressing CD79A, also known as B cell antigen receptor complex-associated protein alpha chain, membrane-bound immunoglobulin-associated protein (MB1, MB-1), surface IgM-associated protein, and Ig-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.

[0102] CD79 is composed of two proteins, i.e. CD79A and CD79B. CD79A is located at chromosome 19q13.2 and encodes a glycoprotein of 226 amino acids of about 47kDa. The precise molecular weight depends on the degree of glycosylation. CD79B is located at chromosome 17q23 and encodes a glycoprotein of 229 amino acids of about 37kDa. CD79A and CD79B share an exon-intron structure, both of which contain a single IgSF Ig domain (a C type of 111 residues for CD79A and a V type of 129 residues for CD79B). Each also contains a highly conserved transmembrane domain and a cytoplasmic tail region of 61 (CD79A) or 48 (CD79B) amino acids, which also exhibits significant amino acid evolutionary conservation. CD79A and CD79B are expressed by the earliest B cell progenitor cells. CD79A / B heterodimers are also observed on the surface of early B cell progenitor cells in the absence of μ heavy chains, although no protein is required to make the progenitor cells committed to the B cell lineage. Subsequently in development, CD79A and CD79B are co-expressed as mature BCR complexes together with all isotypes of Ig on the B cell surface. The CD79 protein is specific to the B lineage and expressed throughout the generation of B lymphocytes. CD79A and CD79B can be used as markers for differentiating B cell neoplasms, including acute leukemias of DLBCL, most precursor B cell types, B cell lines, B cell lymphomas, and some myelomas.

[0103] In various embodiments, the CAR comprising an anti-CD79A antibody sequence is highly potent; undergoes stable in vivo expansion; and recognizes cancer cells expressing CD79A and exhibits cytotoxic activity against cancer cells expressing CD79A.

[0104] In one embodiment, a CAR comprising an anti-CD79A antibody or antigen-binding fragment, a transmembrane domain, and one or more intracellular signaling domains is provided.

[0105] In one embodiment, the immune effector cell is genetically modified to express CAR. T cells expressing CAR are referred to herein as CAR T cells or CAR modified T cells.

[0106] In various embodiments, genetically modified immune effector cells are administered to subjects having cancer cells expressing CD79A, including but not limited to liquid tumors and hematological malignancies. In one embodiment, anti-CD79A CAR T cells are administered to subjects with DLBCL.

[0107] 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 be generally 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 the present 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 (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, Vols. I and II (Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (edited by John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober 2001 John Wiley & Sons, New York, 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 Hybridization (N. Gait, ed., 1984); Hybridization (B. Hames and S. Higgins, 1985); Transcription and Translation (B. Hames and S. Higgins, 1984); Animal Cell Culture (R. Freshney, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, 3rd edition, 2010 Humana Press); Immobilized Cells and Enzymes (IRL Press, 1986); Methods in Enzymology (Academic Press, Inc., New York); Gene Transfer Vectors for Mammalian Cells (Gene Transfer Vectors for Mammalian Cells) ... Cells) (JH Miller and MP=Calos, ed., 1987, Cold Spring Harbor Laboratory; Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. 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. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., 1991); Annual Review of Immunology, 1991. Immunology; and monographs in journals such as Advances in Immunology.

[0108] B. Definition

[0109] Before describing the present disclosure in greater detail, it may be helpful to understand the present disclosure by providing definitions of certain terms to be used herein.

[0110] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any method and material similar or equivalent to the method and material described herein may be used to practice or test a particular embodiment, preferred embodiments of compositions, methods and materials are described herein. For purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout the present disclosure.

[0111] As used herein, the articles "a," "an," and "the" refer to one or more than one (ie, 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.

[0112] The use of alternatives such as "or" should be understood to mean either one, both, or any combination of the alternatives.

[0113] The term "and / or" should be understood to mean one or both of the alternatives.

[0114] 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% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is ±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.

[0115] In one embodiment, a range, such as 1 to 5, about 1 to 5, or about 1 to about 5, refers to every 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.

[0116] As used herein, the term "substantially" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces an effect, such as a physiological effect, that is about the same as the effect produced by a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0117] Throughout this specification, unless the context requires otherwise, the words "comprise / comprises / comprising" should be understood to imply the inclusion of the 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 is intended to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and no other elements can be present. "Consisting essentially of is intended to include any element listed after the phrase, and is limited to other elements that do not interfere with or affect the activity or action of the listed elements specified in the present disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are essential or essential, and no other elements are present that substantially affect the activity or action of the listed elements.

[0118] Reference throughout this specification to "one embodiment," "an 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 aforementioned phrases appearing in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner. It should also be understood that a positive recitation of a feature in one embodiment serves as a basis for excluding the feature in a particular embodiment.

[0119] C. Chimeric Antigen Receptor

[0120] In various embodiments, there is provided a genetically engineered receptor for redirecting the cytotoxicity of immune effector cells to cancer cells expressing CD79A. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs). CAR is a molecule as follows: a specific combination of antibodies based on the intracellular domain of the desired antigen (eg, CD79A) and activated T cell receptors is used to produce a chimeric protein showing specific anti-CD79A cellular immune activity. As used herein, the term "chimeric" describes being composed of different proteins or DNA portions from different sources.

[0121] In a specific embodiment, CAR comprises an extracellular domain (also referred to as a binding domain or antigen-specific binding domain) bound to CD79A, a transmembrane domain, and an intracellular signaling domain. The anti-CD79A antigen binding domain of CAR engages with CD79A on the surface of target cells to aggregate CAR, and delivers activation stimuli to cells containing CAR. The main feature of CAR is that it can redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis, or the generation of molecules that can mediate cell death of target antigen-expressing cells in a manner unrelated to major histocompatibility (MHC), using monoclonal antibodies, soluble ligands, or cell-specific co-receptors The cell-specific targeting ability.

[0122] In various embodiments, the CAR contains an extracellular binding domain comprising a CD79A-specific binding domain; a transmembrane domain; and one or more intracellular co-stimulatory signaling domains and / or primary signaling domains.

[0123] In certain embodiments, the CAR contains 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 comprising; and one or more intracellular co-stimulatory signaling domains and / or primary signaling domains.

[0124] 1. Combined domain

[0125] In a particular embodiment, CAR comprises an extracellular binding domain, which comprises an anti-CD79A antibody or its antigen-binding fragment specifically bound to a human CD79A polypeptide expressed on a target cell, such as a cancer cell. 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 that can specifically bind to a target antigen of interest (e.g., CD79A). The binding domain may be derived from a natural, synthetic, semisynthetic or recombinant source.

[0126] As used herein, the term "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specific targeting" describes that an anti-CD79A antibody or antigen-binding fragment thereof (or a CAR comprising the same) binds to CD79A with a binding affinity greater than background binding. If the binding domain (or a CAR comprising the binding domain or a fusion protein containing the binding domain) is greater than or equal to about 10 5 M -1 Affinity or K a(i.e., the equilibrium association constant for a specific binding interaction in units of 1 / M) binds to or associates with CD79A, then it "specifically binds" to a CD79A polypeptide. In certain embodiments, a binding domain (or fusion protein thereof) binds to or associates with CD79A at a specific equilibrium association constant of greater than or equal to about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 10 13 M -1 K a A "high affinity" binding domain (or a single-chain fusion protein thereof) is one that has at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 or larger K a of those binding domains.

[0127] Alternatively, affinity can be defined as the equilibrium dissociation constant (K) for a specific binding interaction. d ) and in units of M (e.g. 10 -5 M to 10 -13M, or lower). The affinity of the binding domain polypeptides and CAR proteins according to the present invention can be readily determined using conventional techniques, such as by competitive enzyme-linked immunosorbent assay (ELISA), or by binding association, or displacement analysis using labeled ligands, or using a surface plasmon resonance device, such as the Biacore T100 available from Biacore, Inc., Piscataway, NJ, or optical biosensor technology, such as the EPIC system or EnSpire available from Corning and Perkin Elmer, respectively (see also, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614 or equivalents).

[0128] In one embodiment, the affinity of specific binding is about 2-fold greater than background binding, about 5-fold greater than background binding, about 10-fold greater than background binding, about 20-fold greater than background binding, about 50-fold greater than background binding, about 100-fold greater than background binding, or about 1000-fold greater than background binding or more.

[0129] In a specific embodiment, the extracellular binding domain of CAR comprises an antibody or an antigen binding fragment thereof. "Antibody" refers to a binding agent, which is a polypeptide, at least comprising a light chain 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. "Isolated antibody or antigen binding fragment thereof" is identified and separated and / or recovered from a component of its natural environment.

[0130] "Antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including compositions injected or inhaled into an animal (such as compositions including cancer-specific proteins). Antigens react with specific humoral or cellular immune products, including products induced by heterologous antigens (such as the disclosed antigens). In specific embodiments, the target antigen is an epitope of a CD79A polypeptide.

[0131] "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 juxtaposed by the tertiary folding of a 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 generally includes at least 3, and more typically at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0132] Antibodies include antigen-binding fragments thereof, such as Camel Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins ("scFv"), double scFv, (scFv)2, mini antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, 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 mouse antibodies), heterojunction 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.

[0133] As will be understood by the skilled person 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 consists of the second and third constant regions (for IgE and IgM, the fourth constant region) of the two heavy chains combined together and a disulfide bond (interchain) is formed in the hinge. Heavy chains γ, α and δ have a constant region consisting of three tandem (in a row) Ig domains, and a hinge region for increasing flexibility; heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are respectively referred to as "CH2 domains" and "CH3 domains". Each arm of the Y includes the variable region of a single heavy chain and a first constant region bound to 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.

[0134] The light and heavy chain variable regions contain a "framework" region interspersed with three hypervariable regions (also called "complementarity determining regions" or "CDRs"). CDRs can be defined or identified by conventional methods, such as by sequences 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, U.S. Department of Health and Human Services, 1991, which are incorporated herein by reference), or by structures according to Chothia et al. (Chothia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917 (1987), Chothia, C. et al., Nature, 342:877-883 (1989)).

[0135] Illustrative examples of rules for predicting light chain CDRs include: CDR-L1 begins at approximately residue 24, is preceded by Cys, is about 10-17 residues, and is followed by Trp (usually Trp-Tyr-Gln, and Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 begins at approximately 16 residues after the end of CDR-L1, is generally preceded by Ile-Tyr, and Val-Tyr, Ile-Lys, Ile-Phe, and is 7 residues; and CDR-L3 begins at 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:76) (XXX is any amino acid).

[0136] An illustrative example of a rule for predicting a heavy chain CDR includes: CDR-H1 begins approximately at residue 26, is preceded by Cys-XXX-XXX-XXX (SEQ ID NO: 77), is 10-12 residues long and is followed by a Trp (usually Trp-Val, as well as Trp-Ile, Trp-Ala); CDR-H2 begins approximately 15 residues after the end of CDR-H1, is generally preceded by Leu-Glu-Trp-Ile-Gly (SEQ ID NO: NO:78), or multiple variants, is 16-19 residues, 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 (usually Cys-Ala-Arg), is 3 to 25 residues, and is followed by Trp-Gly-XXX-Gly (SEQ ID NO:79).

[0137] In one embodiment, the light chain CDRs and heavy chain CDRs are determined according to the Kabat method.

[0138] In one embodiment, the light chain CDR 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 included 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, such as AbYsis (www.bioinf.org.uk / abysis / ).

[0139] The sequences of the framework regions of different light or heavy chains are relatively conserved within species (such as humans). The framework region of an antibody (which is the combined framework region of the component light and heavy chains) is used to locate and align CDRs in three-dimensional space. CDRs are primarily responsible for binding to the epitope of an antigen. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3, which are numbered sequentially from the N-terminus, and are also commonly identified by the chain in which the specific CDR is located. Therefore, the CDRs located in the variable domain of the heavy chain of an antibody are referred to as CDRH1, CDRH2, and CDRH3, while the CDRs located in the variable domain of the light chain of an antibody are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different combination sites for different antigens) have different CDRs. Although the CDRs between antibodies are different, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. These positions in the CDRs are referred to as specific determinants (SDRs). Illustrative examples of light chain CDRs suitable for constructing anti-CD79A CARs encompassed in specific embodiments include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 1-3, 9-11, and 17-19. Illustrative examples of heavy chain CDRs suitable for constructing anti-CD79A CARs encompassed in specific embodiments include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 4-6, 12-14, and 20-22.

[0140] The "V L " or "VL" refers to the variable region of an immunoglobulin light chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab or other antibody fragment as contemplated herein. Illustrative examples of light chain variable regions suitable for constructing the anti-CD79A CARs contemplated in specific embodiments include, but are not limited to, the light chain variable region sequences set forth in SEQ ID NOs: 7, 15 and 23.

[0141] The "V H " or "VH" refers to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab or other antibody fragment as contemplated herein. Illustrative examples of heavy chain variable regions suitable for constructing the anti-CD79A CARs contemplated in specific embodiments include, but are not limited to, the heavy chain variable region sequences set forth in SEQ ID NOs: 8, 16 and 24.

[0142] 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 made by methods known to those skilled in the art, such as by preparing antibody-forming hybrid cells from a fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0143] A "chimeric antibody" has framework residues from one species (such as humans) and CDRs (generally conferring antigen binding) from another species (such as a mouse). In certain preferred embodiments, the CAR comprises an antigen-specific binding domain that is a chimeric antibody or antigen-binding fragment thereof.

[0144] In a preferred embodiment, the antibody is a human antibody (e.g., a human monoclonal antibody) or a fragment thereof that specifically binds to a human CD79A polypeptide. Human antibodies can be constructed by combining Fv clone variable domain sequences selected from a human phage display library with known human constant domain sequences as described above. Alternatively, human monoclonal antibodies can be prepared by a fusion tumor method. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described, for example, by Kozbor, Journal of Immunology (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., Journal of Immunology (J. Immunol.), 147: 86 (1991). In addition, transgenic animals (e.g., mice) can be used to produce a complete spectrum of human antibodies in the absence of endogenous immunoglobulin production. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993). Gene shuffling can also be used to derive human antibodies from non-human, such as rodent antibodies, wherein 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 fully human antibodies that do not have FR or CDR residues of non-human origin.

[0145] In one embodiment, CAR comprises a "humanized" antibody. "Humanized" antibodies are immunoglobulins including human framework regions and one or more CDRs from non-human (e.g., mouse, rat, or synthetic) immunoglobulins. The non-human immunoglobulins providing CDRs are referred to as "donors," and the human immunoglobulins providing frameworks are referred to as "acceptors." In one embodiment, all CDRs are from donor immunoglobulins in humanized immunoglobulins. The constant region may not exist, but if it does, it must be substantially consistent with the human immunoglobulin constant region, i.e., at least about 85 to 90%, such as about 95% or more consistent. Therefore, all parts of humanized immunoglobulins (possibly excluding CDRs) are substantially consistent with the corresponding parts of natural human immunoglobulin sequences. Humanization or other monoclonal antibodies may have other conservative amino acid substitutions that have little effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by means of genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0146] In specific embodiments, anti-CD79A antibodies or antigen-binding fragments thereof include, but are not limited to, Camel Ig (camel antibody (VHH)), Ig NAR, Fab fragment, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2, miniantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, disulfide-stabilized Fv proteins ("dsFv") and single-domain antibodies (sdAb, nanoantibodies).

[0147] As used herein, "Camel Ig" or "camel VHH" refers to the known smallest antigen-binding unit of a heavy chain antibody (Koch-Nolte et al., Journal of the Federation of American Societies for Experimental Biology (FASEB J.), 21:3490-3498 (2007)). "Heavy chain antibody" or "camel antibody" refers to an antibody containing two VH domains and no light chain (Riechmann L. et al., Journal of Immunology Methods (J. Immunol. Methods) 231:25-38 (1999); WO94 / 04678; WO94 / 25591; U.S. Pat. No. 6,005,079).

[0148] "IgNAR" for "immunoglobulin new antigen receptor" refers to a class of antibodies from the shark immune system repertoire consisting of a homodimer of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains. IgNARs represent some of the smallest known protein backbones based on immunoglobulins and are highly stable and have potent binding characteristics. The intrinsic stability can be attributed to (i) the underlying Ig scaffold, which presents a significantly larger number of residues exposed to charged and hydrophilic surfaces compared to conventional antibody VH and VL domains found in murine antibodies; and (ii) stabilizing structural features in the complementarity determining region (CDR) loops, including patterns of inter-loop disulfide bridges, and intra-loop hydrogen bonding.

[0149] 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 a F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0150] "Fv" is the smallest antibody fragment containing a complete antigen binding site. In one embodiment, a double-chain Fv species is composed of a dimer formed by a heavy chain variable domain and a light chain variable domain tightly associated via non-covalent bonding. In a 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 the light chain and the heavy chain can be similar to the "dimerization" structure association of a double-chain Fv species. In this configuration, 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 collectively confer antigen binding specificity to the antibody. However, although a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) can recognize and bind to an antigen, the affinity is lower than the entire binding site.

[0151] The Fab fragment contains the variable domains of the heavy and light chains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that the carboxyl terminus of the heavy chain CH1 domain has several residues added, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the name used for Fab' herein, in which the cysteine ​​residues of the constant domain carry free thiol groups. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The bispecific Fab dimer (Fab2) has two Fab' fragments, each of which binds to a different antigen. The trispecific Fab trimer (Fab3) has three Fab' fragments, each of which binds to a different antigen.

[0152] The term "bifunctional antibody" 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 (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen binding sites. Bifunctional antibodies can be bivalent or bispecific. Bifunctional antibodies are more fully described in, for example, EP404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proceedings of the National Academy of Sciences of the United States of America 90:6444-6448 (1993). Trifunctional antibodies and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0153] "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).

[0154] "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). In general, the scFv polypeptide also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0155] In preferred embodiments, the anti-CD79A antigen binding fragment is a scFv. In specific embodiments, the scFv is a murine, human or humanized scFv. Single-chain antibodies can be cloned from the V region genes of a hybridoma specific for the desired target. The generation of such hybridomas has become routine. It can be used to clone the variable region heavy chain (V H ) and variable region light chain (V L ) have been described, for example, in Orlandi et al., Proc. Natl. Acad. Sci. USA, 1989;86:3833-3837.

[0156] In various embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising a CDRL1-CDRL3 sequence as set forth in SEQ ID NO: 1-3, 9-11, or 17-19, and / or a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence as set forth in SEQ ID NO: 4-6, 12-14. In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence as set forth in any one of SEQ ID NO: 7, 15, or 23, and / or a variable heavy chain sequence as set forth in any one of SEQ ID NO: 8, 16, or 24.

[0157] In certain embodiments, the antigen-specific binding domain is a scFv that binds to a human CD79A polypeptide.

[0158] An exemplary CD79A-specific binding domain is an immunoglobulin variable region that is specific for CD79A and comprises at least one human framework region. "Human framework region" refers to a wild-type (i.e., naturally occurring) framework region of a human immunoglobulin variable region; an altered framework region of a human immunoglobulin variable region, wherein less than about 50% (e.g., preferably less than about 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) of the amino acids in the region are deleted or substituted (e.g., with one or more amino acid residues of a non-human immunoglobulin framework region at a corresponding position); or an altered framework region of a non-human immunoglobulin variable region, wherein less than about 50% (e.g., less than 45%, 40%, 30%, 25%, 20%, 15%, 10% or 5%) of the amino acids in the region are deleted or substituted (e.g., at an exposed residue position and / or with one or more amino acid residues of a human immunoglobulin framework region at a corresponding position), so that in one aspect, immunogenicity is reduced.

[0159] In certain embodiments, the human framework region is a wild-type framework region of a human immunoglobulin variable region. In certain other embodiments, the human framework region is an altered framework region of a human immunoglobulin variable region having an amino acid deletion or substitution at one, two, three, four, five, six, seven, eight, nine, ten or more positions. In other embodiments, the human framework region is an altered framework region of a non-human immunoglobulin variable region having an amino acid deletion or substitution at one, two, three, four, five, six, seven, eight, nine, ten or more positions.

[0160] In specific embodiments, the CD79A-specific binding domain comprises at least one, two, three, four, five, six, seven or eight human framework regions (FRs) selected from the group consisting of human light chain FR1, human heavy chain FR1, human light chain FR2, human heavy chain FR2, human light chain FR3, human heavy chain FR3, human light chain FR4 and human heavy chain FR4.

[0161] Human FRs that may be present in a CD79A-specific binding domain also include variants of the exemplary FRs provided herein, wherein one, two, three, four, five, six, seven, eight, nine, ten or more amino acids of the exemplary FRs have been substituted or deleted.

[0162] In certain embodiments, the CD79A-specific binding domain comprises (a) a humanized light chain variable region comprising human light chain FR1, human light chain FR2, human light chain FR3, and human light chain FR4, and (b) a humanized heavy chain variable region comprising human heavy chain FR1, human heavy chain FR2, human heavy chain FR3, and human heavy chain FR4.

[0163] The CD79A specific binding domains provided herein also comprise one, two, three, four, five or six CDRs. Such CDRs may be non-human CDRs or altered non-human CDRs selected from the following: CDRL1, CDRL2 and CDRL3 of the light chain, and CDRH1, CDRH2 and CDRH3 of the heavy chain. In certain embodiments, the CD79A specific binding domain comprises (a) a light chain variable region comprising light chain CDRL1, light chain CDRL2 and light chain CDRL3, and (b) a heavy chain variable region comprising heavy chain CDRH1, heavy chain CDRH2 and heavy chain CDRH3.

[0164] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising a CDRL1-CDRL3 sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 1-3, 9-11 or 17-19, and / or a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 4-6, 12-14 or 20-22.

[0165] In one embodiment, the CD79A-specific binding domain comprises a light chain CDR sequence set forth in SEQ ID NO: 1-3, 9-11 or 17-19. In a particular embodiment, the CD79A-specific binding domain comprises a light chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to a light chain CDR sequence set forth in SEQ ID NO: 1-3, 9-11 or 17-19.

[0166] In one embodiment, the CD79A-specific binding domain comprises a heavy chain CDR sequence set forth in SEQ ID NO: 4-6, 12-14, or 20-22. In a specific embodiment, the CD79A-specific binding domain comprises a heavy chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to a heavy chain CDR sequence set forth in SEQ ID NO: 4-6, 12-14, or 20-22.

[0167] In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof comprises a variable light chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in any of SEQ ID NOs: 7, 15, or 23, and / or a variable heavy chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in any of SEQ ID NOs: 8, 16, or 24.

[0168] 2. Linker

[0169] In certain embodiments, the anti-CD79A CAR comprises linker residues between different domains, for example, added for proper spacing and conformation of the molecule. In a specific embodiment, the linker is a variable region linker sequence. A "variable region linker sequence" is an amino acid sequence that connects V H and V LDomain and provide a spacer function compatible with the interaction of the two sub-binding domains, so that the resulting polypeptide retains the specific binding affinity for the target molecule identical to the antibody comprising the same light chain and heavy chain variable region. In a particular embodiment, CAR comprises one, two, three, four or five or more connexons. In a particular embodiment, the length of the connexon 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 connexon 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.

[0170] Illustrative examples of linkers include glycine polymers (G) n ; Glycine-serine polymer (G 1-5 S 1-5 ) n , wherein n is an integer of at least one, two, three, four, or five; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and thus may serve as neutral tethers between domains of fusion proteins, such as the CARs described herein. Glycine enters even more tracts than alanine. Space, and much less restricted than residues with longer side chains (see Scheraga, "Rev. Computational Chem. "11173-142 (1992)). One of ordinary skill in the art will recognize that the design of the CAR in a particular embodiment may include a fully or partially flexible linker, such that the linker may include a flexible linker and one or more portions that confer a less flexible structure to obtain the desired CAR structure.

[0171] Other exemplary linkers include, but are not limited to, the following amino acid sequences: DGGGS (SEQ ID NO: 39); TGEKP (SEQ ID NO: 40) (see, e.g., Liu et al., Proc. Natl. Acad. Sci. USA, 5525-5530 (1997)); GGRR (SEQ ID NO: 41) (Pomerantz et al. 1995, supra); (GGGGS) n, wherein = 1, 2, 3, 4 or 5 (SEQ ID NO: 42) (Kim et al., Proc. Natl. Acad. Sci. USA 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: 43) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 44) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO: 45); LRQRDGERP (SEQ ID NO: 46); LRQKDGGGSERP (SEQ ID NO: 47); LRQKD(GGGS)2ERP (SEQ ID NO: 48). NO:48). Alternatively, the flexible linker can be rationally designed using computer programs that can model the DNA-binding site and the peptide itself (Desjarlais and Berg, Proc. Natl. Acad. Sci. USA 90:2256-2260 (1993), Proc. Natl. Acad. Sci. USA 91:11099-11103 (1994)) or by phage display methods. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO:49) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0172] 3. Spacer subdomain

[0173] In certain embodiments, the binding domain of the anti-CD79A CAR is followed by one or more "spacer domains", which refer to moving the antigen binding domain away from the effector cell surface so that appropriate cell / cell contact, antigen binding and activation can be achieved (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. In certain embodiments, the spacer domain is part of an immunoglobulin, which includes but is not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may include a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region amino acid sequence.

[0174] In one embodiment, the spacer domain comprises CH2 and CH3 of IgG1, IgG4 or IgD.

[0175] 4. Hinge domain

[0176] The binding domain of anti-CD79A CAR is generally followed by one or more "hinge domains", which play a role in positioning the antigen binding domain away from the effector cell surface so as to achieve appropriate cell / cell contact, antigen binding and activation. Anti-CD79A CARs typically include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0177] "Altered hinge region" refers to (a) a naturally occurring hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions); (b) a portion of a naturally occurring hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions) of at least 10 amino acids (e.g., at least 12, 13, 14 or 15 amino acids); or (c) a portion of a naturally occurring hinge region comprising a core hinge region (which may 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). In certain embodiments, one or more cysteine ​​residues in a naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue (eg, a serine residue).

[0178] Exemplary hinge domains suitable for use in CAR described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α and CD4, which may be wild-type hinge regions from these molecules or may be altered. In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge. In another embodiment, the hinge domain comprises a CD8α hinge region.

[0179] 5. Transmembrane (TM) domain

[0180] "Transmembrane domain" is a part of the plasma membrane of fused extracellular binding part and intracellular signaling domain in anti-CD79A CAR and anchoring CAR to immune effector cells.TM domains can be derived from natural, synthetic, semi-synthetic or recombinant sources.TM domains can be derived from (that is, at least comprising the transmembrane region of the α or β chain of T cell receptor, 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 particular embodiment, TM domains are synthetic and mainly include hydrophobic residues, such as leucine and valine.

[0181] In one embodiment, CAR comprises a TM domain derived from PD1, CD152, CD28 or CD8α. In another embodiment, 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 CAR and the intracellular signaling domain. Glycine-serine based linkers provide particularly suitable linkers.

[0182] 6. Intracellular signaling domain

[0183] In certain embodiments, the anti-CD79A CAR comprises one or more intracellular signal transduction domains. "Intracellular signal transduction domain" refers to the part of the CAR that is involved in transducing information of effective anti-CD79A CAR binding to human CD79A polypeptide to the interior of immune effector cells to induce effector cell functions, such as activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors into target cells bound to the CAR, or other cellular responses triggered by antigen binding to the extracellular CAR domain.

[0184] The term "effector function" refers to a specialized function of an immune effector cell. The effector function of a T cell may be, for example, a cytolytic activity or a help or activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain may 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 may 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.

[0185] As is well known, the signal generated only via TCR is not enough to fully activate T cells and a secondary or costimulatory signal is also required. Therefore, T cell activation can be referred to as being mediated by two different categories of intracellular signal transduction domains: a primary signaling domain that initiates antigen-dependent primary activation via TCR (e.g., TCR / CD3 complex) and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary or costimulatory signal. In a preferred embodiment, CAR comprises an intracellular signaling domain, the intracellular signaling domain comprising one or more "costimulatory signaling domains" and a "primary signaling domain".

[0186] The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0187] Illustrative examples of ITAM-containing primary signaling domains suitable for use in specific embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a specific preferred embodiment, the anti-CD79A CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. 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.

[0188] In a specific embodiment, CAR includes one or more costimulatory signaling domains to enhance the efficacy and amplification of T cells expressing CAR receptors. 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, which provide a second signal required for the effective activation and function of T lymphocytes when bound to an antigen. Illustrative examples of such costimulatory molecules include 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. In one embodiment, the CAR comprises one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137 and CD134, and a CD3ζ primary signaling domain.

[0189] In another embodiment, the CAR comprises CD28 and CD137 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0190] In another embodiment, the CAR comprises CD28 and CD134 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0191] In one embodiment, the CAR comprises CD137 and CD134 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0192] In one embodiment, the CAR comprises a CD137 co-stimulatory signaling domain and a CD3ζ primary signaling domain.

[0193] In one embodiment, the CAR comprises a CD134 co-stimulatory signaling domain and a CD3ζ primary signaling domain.

[0194] In one embodiment, the CAR comprises a CD28 co-stimulatory signaling domain and a CD3ζ primary signaling domain.

[0195] In certain embodiments, the CAR comprises an anti-CD79A antibody or an antigen-binding fragment thereof that specifically binds to a CD79A polypeptide expressed on a cancer cell.

[0196] In one embodiment, the CAR comprises an anti-CD79A scFv 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 134, CD137, CD152, CD154, AMN1 and PD1; and one or more intracellular co-stimulatory signaling domains from a co-stimulatory 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; and a primary signaling domain from each of the following: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

[0197] In one embodiment, the 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 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; and one or more intracellular co-stimulatory signaling domains from a co-stimulatory 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; and a primary signaling domain from: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

[0198] In one embodiment, the 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 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 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 and the intracellular signaling domain of CAR; and one or more intracellular co-stimulatory signaling domains from a co-stimulatory molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD1 1, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70; and primary signaling domains from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0199] In a specific embodiment, the 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 polypeptide; a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD137 intracellular co-stimulatory signaling domain; and a CD3ζ primary signaling domain.

[0200] In a specific embodiment, the CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide; a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD134 intracellular co-stimulatory signaling domain; and a CD3ζ primary signaling domain.

[0201] In a specific embodiment, the CAR comprises an anti-CD79A scFv that binds to a CD79A polypeptide; a hinge domain comprising a CD8α polypeptide; a CD8α transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD28 intracellular co-stimulatory signaling domain; and a CD3ζ primary signaling domain.

[0202] The design of the CARs contemplated in particular embodiments enables improved expansion, long-term persistence, and cytotoxic properties in T cells expressing the CAR compared to unmodified T cells or T cells modified to express other CARs.

[0203] D. Peptide

[0204] Various polypeptides, fusion polypeptides and polypeptide variants encompassed herein include, but are not limited to, CAR polypeptides and fragments thereof. In a preferred embodiment, a polypeptide comprising one or more CARs is provided. In a specific embodiment, CAR is an anti-CD79A CAR comprising an amino acid sequence as described in any one of SEQ ID NO:25-30.

[0205] Unless otherwise specified, "polypeptide", "peptide" and "protein" can be used interchangeably and in the conventional sense, i.e., as an amino acid sequence. The polypeptide is not limited to a specific length, for example, it may include 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, etc., as well as other naturally occurring and non-naturally occurring modifications known in the art. In various embodiments, the CAR polypeptide comprises a signal (or leader) sequence at the N-terminus of the protein, which co-translationally or post-translationally guides the transfer of the protein. Illustrative examples of suitable signal sequences for CARs covered in specific embodiments include, but are not limited to, IgG1 heavy chain signal polypeptides, CD8α signal polypeptides, or human GM-CSF receptor α signal polypeptides. The polypeptide can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides covered herein cover CARs of the present disclosure, or sequences having deletions, additions and / or substitutions of one or more amino acids of CARs covered herein.

[0206] As used herein, "isolated polypeptide" and the like refer to a peptide or polypeptide molecule that is synthesized in vitro, separated and / or purified from the cellular environment and from association with other components in the cell, i.e., it is not significantly associated with in vivo substances. In specific embodiments, the isolated polypeptide is a synthetic polypeptide, a semisynthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0207] Polypeptides include "polypeptide variants". Polypeptide variants may differ from naturally occurring polypeptides in one or more substitutions, deletions, additions and / or insertions. Such variants may occur naturally or may be produced synthetically, for example, by modifying one or more of the above polypeptide sequences. For example, in certain embodiments, it may be necessary to improve the binding affinity and / or other biological properties of CAR by introducing one or more substitutions, deletions, additions and / or insertions into the binding domain, hinge, TM domain, co-stimulatory signaling domain, or primary signaling domain of the CAR polypeptide. In specific embodiments, polypeptides include polypeptides 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 specific embodiments, the biological activity is binding affinity. In specific embodiments, the biological activity is cytolytic activity.

[0208] Polypeptides include "polypeptide fragments". A polypeptide fragment refers to a polypeptide that may be a monomer or polymer 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 term "biologically active fragment" or "minimum biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of a naturally occurring polypeptide. In a preferred embodiment, the biological activity is binding affinity for a unique type. In certain embodiments, a polypeptide fragment may comprise an amino acid chain having a length of at least 5 to about 500 amino acids. It will be appreciated that in certain embodiments, the fragment length is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids. Particularly useful polypeptide fragments include functional domains, including antigen binding domains or antibody fragments. In the case of anti-CD79A antibodies, useful fragments include, but are not limited to: CDR regions; CDR3 regions of heavy or light chains; variable regions of heavy or light chains; a portion of an antibody chain or variable region, including two CDRs; etc.

[0209] The polypeptides may also be fused or conjugated in-frame to linkers or other sequences to facilitate synthesis, purification, or identification of the polypeptide (eg, poly-His), or to facilitate binding of the polypeptide to a solid support.

[0210] As described above, in certain 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 DNA. Methods for mutagenesis and nucleotide sequence changes are well known in the art. See, for example, Kunkel (1985, Proceedings of the National Academy of Sciences of the United States of America 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, 4th edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidance as to appropriate amino acid substitutions that do not affect the biological activity of a 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).

[0211] In certain embodiments, polypeptide variants include one or more conservative substitutions. A "conservative substitution" is a substitution of an amino acid with another amino acid having similar properties, such that a person skilled in the art of peptide chemistry would expect the secondary structure and hydrophilic properties of the polypeptide to remain substantially unchanged. The structures of the polynucleotides and polypeptides covered in the specific embodiments can be modified and still obtain functional molecules encoding variants or derived polypeptides with desired characteristics. When it is desired to change the amino acid sequence of a polypeptide to produce an equivalent or even improved variant polypeptide, for example, one skilled in the art may change one or more of the codons of the coding DNA sequence, for example, according to Table 1.

[0212] Table 1 - Amino acid codons

[0213]

[0214]

[0215] Computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, MacVector or Vector NTI software, can be used to find guidance for determining amino acid residues that can be substituted, inserted or deleted without eliminating biological activity. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, that is, substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of one of the amino acid families associated with side chains. Naturally occurring amino acids are generally divided into four families: 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 substitutions of amino acids are known to those skilled in the art and are generally performed without changing the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not significantly alter the biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0216] As outlined 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.

[0217] Polypeptide variants further 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 obtained by attaching a functional group to a group found at an amino acid chain or at an N- or C-terminal residue. Variants also include allelic variants, species variants, and mutant proteins. Truncations or deletions in regions that do not affect the functional activity of a protein are also variants.

[0218] In one embodiment, where expression of two or more polypeptides is desired, the polynucleotide sequences encoding the polypeptides may be separated by an IRES sequence as discussed elsewhere herein. In another embodiment, two or more polypeptides may be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences.

[0219] The polypeptides covered in the specific embodiments include fusion polypeptides. In a preferred embodiment, a fusion polypeptide and a polynucleotide encoding a fusion polypeptide are provided, such as a CAR. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine or ten or more polypeptide fragments. Fusion polypeptides are usually C-terminal connected to N-terminal, but it can also be C-terminal connected to C-terminal, N-terminal connected to N-terminal or N-terminal connected to C-terminal. The polypeptides of the fusion protein can be in any order or specified order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutant variants, subsequences and interspecies homologs, as long as the desired transcriptional activity of the fusion polypeptide is preserved. Fusion polypeptides can be prepared by chemical synthesis methods or by chemical connection between two parts or can generally be prepared using other standard techniques. The connected DNA sequence comprising the fusion polypeptide is operably connected to a suitable transcription or translation control element as discussed elsewhere herein.

[0220] In one embodiment, the fusion partner comprises a sequence that facilitates expression of the protein (expression enhancer) at a higher yield than the native recombinant protein. Other fusion partners may be selected to increase the solubility of the protein or to target the protein to a desired intracellular compartment or to facilitate transport of the fusion protein across the cell membrane.

[0221] The fusion polypeptide may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. In addition, the polypeptide cleavage site may be placed in any connecting peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribonuclease recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0222] 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 Pl (P35) protease, byovirus NIa protease, byovirus RNA-2-encoded protease, aphthous virus L protease, enterovirus 2A protease, rhinovirus 2A protease, picornavirus 3C protease, comovirus 24K protease, nematode-transmitted polyhedron virus 24K protease, RTSV (rice tungro virus) 3C-like protease, PYVF (parsnip yellow spot virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. In one embodiment, the tobacco etch virus (TEV) protease cleavage site is preferred due to its higher cleavage stringency, such as EXXYXQ(G / S) (SEQ ID NO:50), such as ENLYFQG (SEQ ID NO:51) and ENLYFQS (SEQ ID NO:52), where X represents any amino acid (TEV cleavage occurs between Q and G or Q and S).

[0223] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0224] 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 particular embodiments, the viral 2A peptide is an aphthous virus 2A peptide, a potyvirus 2A peptide or a cardiovirus 2A peptide.

[0225] In one embodiment, the viral 2A peptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, Thosea asigna (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, Theilovirus 2A peptide and encephalomyocarditis virus 2A peptide.

[0226] Illustrative examples of 2A sites are provided in Table 2.

[0227] Table 2

[0228] SEQ ID NO:53 GSGATNFSLLKQAGDVEENPGP SEQ ID NO:54 ATNFSLLKQAGDVEENPGP SEQ ID NO:55 LLKQAGDVEENPGP SEQ ID NO:56 GSGEGRGSLLTCGDVEENPGP SEQ ID NO:57 EGRGSLLTCGDVEENPGP SEQ ID NO:58 LLTCGDVEENPGP SEQ ID NO:59 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO:60 QCTNYALLKLAGDVESNPGP SEQ ID NO:61 LLKLAGDVESNPGP SEQ ID NO:62 GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:63 VKQTLNFDLLKLAGDVESNPGP SEQ ID NO:64 LLKLAGDVESNPGP SEQ ID NO:65 LLNFDLLKLAGDVESNPGP SEQ ID NO:66 TLNFDLLKLAGDVESNPGP SEQ ID NO:67 LLKLAGDVESNPGP SEQ ID NO:68 NFDLLKLAGDVESNPGP SEQ ID NO:69 QLLNFDLLKLAGDVESNPGP SEQ ID NO:70 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:71 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO:72 LNFDLLKLAGDVESNPGP SEQ ID NO:73 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:74 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

[0229] In a preferred embodiment, the polypeptide comprises an anti-CD79A CAR polypeptide.

[0230] E. Polynucleotide

[0231] In a preferred embodiment, a polynucleotide encoding one or more CAR polypeptides is provided. 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, synthetic or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA or recombinant DNA. A polynucleotide refers to 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, e.g., 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.

[0232] Illustrative examples of polynucleotides include, but are not limited to, SEQ ID NOs: 31-36 and polynucleotides encoding SEQ ID NOs: 1-30.

[0233] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from the sequences that flank it in its naturally occurring state, such as a DNA fragment removed from the sequences that are normally adjacent to the fragment. In certain embodiments, an "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and have been made artificially. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semisynthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0234] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0235] In certain embodiments, the polynucleotide may undergo codon optimization. As used herein, the term "codon optimization" 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) changes in codon preference between two or more organisms or genes or synthetically constructed preference tables; (ii) changes in the degree of codon preference within an organism, gene, or gene set; (iii) systematic changes in codons, including the environment; (iv) codon changes based on decoding tRNA; (v) codon changes based on GC% in one position of a triplet; (vi) changes in the degree of similarity to a reference sequence, such as a naturally occurring sequence; (vii) changes 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 as the basis for designing a codon substitution set; (x) systematic changes in the codon set for each amino acid; and / or (xi) isolated and removed pseudo translation start sites.

[0236] 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 that have one or more nucleotides added or deleted or replaced with different nucleotides compared to a reference polynucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0237] Polynucleotide variants include polynucleotide fragments that encode biologically active polypeptide fragments or variants. As used herein, the term "polynucleotide fragment" refers to a polynucleotide fragment that is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 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 50, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides, encoding 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 refers to a polynucleotide that encodes a polypeptide having an amino terminal deletion, a carboxyl terminal deletion and / or one or more internal deletions or substitutions of an amino acid of a naturally occurring or recombinantly produced polypeptide.

[0238] As used herein, the description of "sequence identity" or, for example, "a sequence that is 50% identical to ..." refers to the degree of sequence identity measured nucleotide by nucleotide or amino acid by amino acid over the comparison window. Therefore, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions where the same nucleic acid bases (e.g., A, T, C, G, I) or the same 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) exist 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. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein, typically wherein the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0239] Terms used to describe the sequence relationships of two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". The length of a "reference sequence" is at least 12, but typically 15 to 18, and often at least 25 monomer units, 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 a sequence is compared to a reference sequence having the same number of contiguous positions after the two sequences are optimally aligned. For the two sequences of the best alignment, the comparison window can include the addition or deletion (that is, room) of about 20% or lower percentage compared with the reference sequence (not including the sequence of addition or deletion). The sequence optimal comparison for comparing the comparison window can be performed by computerized execution algorithm (Wisconsin Genetics Software Package 7.0 version in Wisconsin, USA, genetics computer group, 575 Science Drive Madison (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575Science Drive Madison, WI, USA) GAP, BESTFIT, FASTA and TFASTA) or by the inspection and optimal comparison (that is, causing the highest homology percentage on the comparison window) produced by any one of the multiple selection methods. It can also be referred to as such as Altschul et al., 1997, " Nucleic Acids Research (Nucl.Acids Res.) " 25: 3389 disclosed BLAST program family. A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0240] Terms describing the orientation of a polynucleotide include: 5' (generally the end of the polynucleotide with a free phosphate group) and 3' (generally the end of the polynucleotide with a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in either 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," "positive," or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except that it is uracil (U) in RNA, rather than thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand (which is the strand transcribed by RNA polymerase) is designated as the "template," "antisense," "negative," 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.

[0241] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) that are related by the base pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG 3' is 3'TCAGTAC 5'. The latter sequence is usually written as the reverse complementary sequence 5'CATGACT 3' with a 5' end on the left and a 3' end on the right. A sequence that is equal 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 "full" complementarity between nucleic acids.

[0242] In addition, it will be appreciated by those skilled in the art that, as described herein, due to the degeneracy of the genetic code, there are multiple nucleotide sequences of fragments of coded polypeptides or their variants. Some of these polynucleotides carry minimal homology with the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, such as polynucleotides optimized for human and / or primate codon selection. In addition, alleles of genes comprising the polynucleotide sequences provided herein can also be used. Alleles are endogenous genes that change due to one or more mutations (e.g., deletions, additions, and / or substitutions) of nucleotides.

[0243] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a gene sequence within a vector that can express RNA and subsequently express a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, such as a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, such as a promoter, an enhancer, a poly (A) sequence, and one or more genes of interest, such as one or more polynucleotides of interest. 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 positioned and sequentially oriented 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, undergoes appropriate post-translational modifications required for the activity of the transformed cell, and is translocated to an appropriate compartment by targeting an appropriate intracellular compartment or secreting into an extracellular compartment to obtain biological activity. Preferably, the cassette makes its 3' and 5' ends suitable for easy insertion into the vector, for example, it has a restriction endonuclease site at each end. In a preferred embodiment, the nucleic acid cassette contains a sequence of a chimeric antigen receptor for increasing the cytotoxicity of cancer cells expressing CD79A. The cassette can be removed as a single unit and inserted into a plasmid or viral vector.

[0244] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide encoding a polypeptide, a polypeptide variant, or a fusion polypeptide. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In certain embodiments, the polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in treating or preventing a disease or condition. The polynucleotide of interest and the polypeptide encoded therefrom include polynucleotides encoding wild-type polypeptides and both 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 with 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% biological activity of the corresponding wild-type polypeptide.

[0245] As disclosed elsewhere herein or as known in the art, the polynucleotides encompassed herein (regardless of the length of the coding sequence itself) can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, other restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, so that their overall length can vary significantly. It is therefore contemplated that polynucleotide fragments of virtually any length can be employed in particular embodiments, with the overall length preferably being limited by the ease of preparation and use of the intended recombinant DNA protocol.

[0246] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of well-established techniques known and available in the art. To express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0247] 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.

[0248] Other 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.

[0249] 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 (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (eg, SV40).

[0250] 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 specific embodiments, the coding sequence of the polypeptides disclosed herein can be joined to such expression vectors for expression of the polypeptides in mammalian cells.

[0251] In a specific embodiment, the vector is an episomal vector or a vector maintained outside the chromosome. As used herein, the term "episomal" means that the vector can replicate without being integrated into the host's chromosomal DNA and is not gradually lost by dividing host cells, and also means that the vector replicates outside the chromosome or free.

[0252] "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 - which interact with host cell proteins to carry out transcription and translation. Such 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.

[0253] In certain embodiments, vectors, including but not limited to expression vectors and viral vectors, will include exogenous, endogenous or heterologous control sequences, such as promoters and / or enhancers. An "endogenous" control sequence is a control sequence that is naturally associated with a given gene in a genome. An "exogenous" control sequence is a control sequence that is positioned in conjunction with a gene by means of genetic manipulation (i.e., molecular biotechnology) so that transcription of the gene is directed by the associated enhancer / promoter. A "heterologous" control sequence is an exogenous sequence from a species different from the cell being genetically manipulated.

[0254] As used herein, the term "promoter" refers to a recognition site of 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 operable in a mammalian cell comprises an AT-rich region located about 25 to 30 bases upstream of the transcription start site, and / or another sequence found at 70 to 80 bases upstream of the transcription start site, and N can be a CNCAAT region of any nucleotide.

[0255] The term "enhancer" refers to a stretch 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 cooperatively or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a stretch of DNA that contains sequences that can provide both promoter and enhancer functions.

[0256] The term "operably linked" refers to a juxtaposition of the components described in a relationship that permits 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.

[0257] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that constantly or continuously causes transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows expression in a 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 allows expression in a variety of defined cell and tissue types, respectively.

[0258] Illustrative ubiquitous expression control sequences suitable for use in specific embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the simian virus 40 (SV40) (e.g., early or late), the Moloneymurine leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5 and P11 promoters from vaccinia virus, the elongation factor 1-alpha (EF1a) promoter, the 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, dl587rev primer binding site replaced (MND) U3 promoter (Haas et al., Journal of Virology. 2003; 77(17): 9439-9450).

[0259] In one embodiment, the vector comprises the MNDU3 promoter.

[0260] In one embodiment, the vector comprises an EF1a promoter comprising the first intron of the human EF1a gene.

[0261] In one embodiment, the vector comprises the EF1a promoter lacking the first intron of the human EF1a gene.

[0262] In certain embodiments, it may be desirable to express the polynucleotide comprising the CAR from a T cell-specific promoter.

[0263] 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, such as controlling expression by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide or causes increased or decreased expression of a polynucleotide encoded by the polynucleotide of interest.

[0264] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters for genes encoding glucocorticoids or estrogen receptors (which can be induced by treatment with the corresponding hormones); metallothionein promoters (which can be induced by treatment with various heavy metals); MX-1 promoters (which can be induced by interferon); the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67); cumate-inducible gene switch (WO 2002 / 088346); tetracycline-dependent regulatory system; etc.

[0265] Conditional expression can also be achieved by using site-specific DNA recombinase. According to certain embodiments, the vector comprises at least one (usually two) site for the reorganization mediated by the site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes excision-type or integrated protein, enzyme, cofactor or associated protein that participates in the recombination reaction involving one or more (for example, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) recombination sites, which can be wild-type protein (see Landy, "Current Opinion in Biotechnology" 3:699-707 (1993)) or its mutant, derivative (for example, fusion protein containing recombinant protein sequence or its fragment), fragment and variant. 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.

[0266] The vector may include one or more recombination sites for any of a variety of site-specific recombinases. It should be understood that, in addition to any sites required for integration of a vector such as a retroviral vector or a lentiviral vector, a target site for a site-specific recombinase is also required. As used herein, the term "recombination sequence," "recombination site," or "site-specific recombination site" refers to a specific nucleic acid sequence that a recombinase recognizes and binds.

[0267] 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 a recombinase binding site) (see Figure 1 in Sauer B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0268] 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).

[0269] Other examples of recognition sequences are attB, attP, attL and attR sequences, which are recognized by the recombinase lambda integrase, e.g. Identification. SSR mediates recombination only between the heterotypic sites attB (length 34 bp) and attP (length 39 bp) (Groth et al., 2000). Both attB and attP, named for the phage integrase attachment sites on the bacterial and phage genomes, respectively, contain potential The product sites attL and attR are further The mediated recombination is inert in nature (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 attP sites into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, the typical strategy places an attP-carrying "docking site" into a given locus by homologous recombination, which is then paired with an incoming sequence carrying attB for insertion.

[0270] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into the start codon (such as ATG) of a cistron (protein coding region), thereby causing 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 sequences 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 EMCV IRES.

[0271] 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 common Kozak sequence is (GCC) RCCATGG (SEQ ID NO: 75), where R is a purine (A or G) (Kozak, 1986. Cell. 44 (2): 283-92; and Kozak, 1987. Nucleic Acids Research 15 (20): 8125-48). In a specific embodiment, the vector comprises a polynucleotide having a common Kozak sequence and encoding a desired polypeptide (eg, CAR).

[0272] Elements that guide efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are usually found downstream of polyadenylation signals. In a specific embodiment, the vector comprises a polyadenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that guides both termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. The polyadenylation sequence can enhance mRNA stability by adding a polyA tail to the 3' end of the coding sequence, and thus contributes to increased translation efficiency. Cleavage and polyadenylation are guided by polyadenylic acid sequences in RNA. The core poly (A) sequence of mammalian pre-mRNA has two recognition elements flanking cleavage-polyadenylation sites. Typically, the almost invariant AAUAAA hexamer is located 20-50 nucleotides upstream of a highly variable element rich in U or GU residues. The 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 a desired 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), or another suitable heterologous or endogenous poly(A) sequence known in the art.

[0273] In some embodiments, the polynucleotide or a cell having the polynucleotide utilizes a suicide gene (including an inducible suicide gene) to reduce the risk of direct toxicity and / or uncontrolled proliferation. In particular aspects, the suicide gene is not immunogenic to a host having the polynucleotide or cell. An example of a suicide gene that can be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical dimerization inducer (CID).

[0274] In certain embodiments, one or more polynucleotides encoding an anti-CD79A CAR are introduced into a cell (e.g., an immune effector cell) via a non-viral or viral vector.

[0275] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting 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 replicates directly and autonomously in a cell, or may include a sequence sufficient to allow integration into host cell DNA. In a specific embodiment, a non-viral vector is used to deliver one or more polynucleotides covered herein to a T cell. In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding an anti-CD79A CAR.

[0276] 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.

[0277] Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycationic or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene guns, and heat shock.

[0278] Illustrative examples of polynucleotide delivery systems suitable for use in particular embodiments include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Liposomal transfection reagents are commercially available (e.g., Transfectam TM and Lipofectin TM ). Cationic and neutral lipids suitable for efficient receptor recognition liposome transfection 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 delivery, bacterial-derived delivery, and delivery based on non-living nanounits are also covered in specific embodiments.

[0279] 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 a non-integrated transgene for a period of hours, days, or weeks, wherein the period of expression is less than the period of expression of the polynucleotide if it is integrated into the genome or contained within a stable plasmid replicon in the cell.

[0280] 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. Generally speaking, the in vitro transcribed RNA is produced by an in vitro transcribed vector. The in vitro transcribed vector comprises a template for producing the in vitro transcribed RNA.

[0281] In certain embodiments, the mRNAs may further comprise a 5' cap or a modified 5' cap and / or a poly(A) sequence. As used herein, a 5' cap (also referred to as an RNA cap, an RNA 7-methylguanosine cap, or an RNA m 7G The 5' cap is a modified guanine nucleotide that is added to the "front end" or 5' end of a eukaryotic messenger RNA shortly after transcription begins. The 5' cap comprises an end group that is connected 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 about 50 to about 5000 adenines. In one embodiment, the mRNA comprises a poly (A) sequence of about 100 to about 1000 bases, about 200 to about 500 bases, or about 300 to about 400 bases. In one embodiment, the mRNA comprises a poly (A) sequence of about 65 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1000 or more bases. The poly (A) sequence can be chemically or enzymatically modified to modulate mRNA functionality, such as localization, stability, or translation efficiency.

[0282] Viral vectors comprising polynucleotides encompassed in specific embodiments can be delivered in vivo by administration to individual patients, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or local administration, as described below. Alternatively, the vector can be delivered to ex vivo cells, such as cells removed from individual patients (e.g., fluid peripheral blood, lymphocytes, bone marrow puncture fluid, tissue biopsy samples, etc.) or hematopoietic stem cells of a universal donor, which are then reimplanted into the patient.

[0283] In one embodiment, a viral vector comprising a polynucleotide encoding an anti-CD79A CAR is directly administered to an organism to transduce cells in vivo. Alternatively, naked DNA may be administered. Administration is performed by any of the approaches commonly used to introduce molecules to ultimately contact blood or tissue cells, including but not limited to injection, infusion, surface administration, and electroporation. Suitable methods for administering such nucleic acids are available and well-known to those skilled in the art, and although more than one approach may be used to administer a specific composition, a specific approach may generally provide a more rapid and more effective response than another approach.

[0284] 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.

[0285] In various embodiments, the one or more polynucleotides are introduced into immune effector cells (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.

[0286] AAV is a small (about 26nm) replication-deficient, mainly free-type, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can merge its genome into the genome of the host cell. Recombinant AAV (rAAV) is usually composed of a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITR) at a minimum. The length of the ITR sequence is about 145bp. In a specific embodiment, rAAV comprises ITR and a capsid sequence separated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10.

[0287] In some embodiments, chimeric rAAV uses ITR sequences isolated from one AAV serotype and capsid sequences isolated from different AAV serotypes. For example, rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2 / AAV6. In specific embodiments, the rAAV vector may comprise 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, rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV6. In a preferred embodiment, rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV2.

[0288] In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more readily transduce cells of interest.

[0289] The construction of rAAV vectors, their production and purification have been disclosed in, for example, U.S. Pat. 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.

[0290] In various embodiments, the one or more polynucleotides are introduced into immune effector cells by transducing the cells with a retrovirus (e.g., a lentivirus) comprising one or more polynucleotides encoding an anti-CD79A CAR.

[0291] As used herein, the term "retrovirus" refers to an RNA virus whose genomic RNA is reverse transcribed into a linear double-stranded DNA copy and whose genomic DNA is subsequently covalently integrated into the host genome. Illustrative 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), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV) and lentivirus.

[0292] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedivirus (VMV) virus; 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.

[0293] In various embodiments, the lentiviral vectors contemplated herein comprise one or more LTRs, and one or more or all of the following auxiliary elements: cPPT / FLAP, Psi(Ψ) packaging signal, export element, poly(A) sequence, and may optionally comprise a WPRE or HPRE, insulator elements, selectable markers, and cellular suicide genes as discussed elsewhere herein.

[0294] In certain embodiments, the lentiviral vectors contemplated herein may be integrating or non-integrating or integration-defective lentiviruses. As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus that has an integrase that is unable to integrate the viral genome into the genome of a host cell. Integration-incompetent viral vectors have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety.

[0295] Illustrative 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.

[0296] In one embodiment, the HIV-1 integrase-deficient pol gene comprises D64V, D116I, D116A, E152G or E152A mutations; D64V, D116I and E152G mutations; or D64V, D116A and E152A mutations.

[0297] In one embodiment, the HIV-1 integrase-deficient pol gene comprises a D64V mutation.

[0298] The term "long terminal repeat (LTR)" refers to the base pair domain located at the end of retroviral DNA, which in its native sequence context is a direct repeat and contains the U3, R and U5 regions.

[0299] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence includes a central polypurine stretch and a central termination sequence (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and Zennou et al., 2000, Cell, 101:173. In another embodiment, the lentiviral vector contains a FLAP element having one or more mutations in the cPPT and / or CTS element. In yet another embodiment, the lentiviral vector comprises a cPPT or CTS element. In yet another embodiment, the lentiviral vector does not contain a cPPT or CTS element.

[0300] 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 the 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.

[0301] The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus of a cell to the cytoplasm. Examples of RNA export elements include, but are not limited to, 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 hepatitis B virus post-transcriptional regulatory element (HPRE).

[0302] In a specific embodiment, the 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 the expression of heterologous nucleic acids at the protein site, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, Journal of Virology, 73: 2886); the post-transcriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5: 3864); etc. (Liu et al., 1995, Genes Dev., 9: 1766).

[0303] Lentiviral vectors preferably contain several safety enhancements caused by modified LTRs. "Self-inactivating" (SIN) vectors refer to replication-defective vectors, for example, in which the right (3') LTR enhancer-promoter region, called the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter 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.

[0304] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus in which a viral envelope protein has been replaced by a viral envelope protein of another virus having preferred characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope protein, which enables 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.

[0305] In certain embodiments, the lentiviral vector is prepared 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.

[0306] According to certain specific embodiments covered herein, most or all of the viral vector backbone sequences are derived from lentiviruses, such as HIV-1. However, it should be understood that retroviral and / or lentiviral sequences from a variety of different sources can be used, or combinations and numerous substitutions and changes can be provided to certain lentiviral sequences without impairing 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 are suitable for producing viral vectors or transfer plasmids covered herein.

[0307] In various embodiments, one or more polynucleotides encoding an anti-CD79A CAR are introduced into immune effector cells by transducing the cells with an adenovirus comprising the one or more polynucleotides.

[0308] Adenovirus-based vectors are able to achieve extremely high transduction efficiencies in many cell types without the need for cell division. Using this type of vector, high titers and high levels of expression have been achieved. This vector can be prepared in large quantities in a relatively simple system. Most adenovirus vectors are engineered to replace the Ad E1a, E1b and / or E3 genes with transgenes; subsequently, replication-defective vectors are propagated in human 293 cells that supply trans-deleted gene function. Ad vectors can transduce various types of tissues in vivo, including non-dividing differentiated cells, such as those found in the liver, kidneys, and muscles. Conventional Ad vectors have a large carrying capacity.

[0309] The production and delivery of current replication-deficient adenoviral vectors can utilize a unique helper cell line called 293, which utilizes Ad5 DNA fragments to transform human embryonic kidney cells and constitutively express the E1 protein (Graham et al., 1977). Since the E3 region is dispensable in the adenoviral genome (Jones and Shenk, 1978), current adenoviral vectors carry foreign DNA in the E1, D3, or both regions with the aid of 293 cells (Graham and Prevec, 1991). Adenoviral vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine research (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Studies of administering 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 and Gerard, 1993), and stereotaxic inoculation into the brain (Le Gal La Salle et al., 1993). Examples of the use of Ad vectors in clinical trials include polynucleotide therapy for anti-tumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0310] In various embodiments, one or more polynucleotides encoding an anti-CD79A CAR are introduced into immune effector cells by transducing the cells with a herpes simplex virus, e.g., HSV-1, HSV-2, comprising the one or more polynucleotides.

[0311] Mature HSV virions consist of an icosahedral capsid encapsidating a viral genome consisting of a 152kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector lacks one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-deficient. Most replication-deficient 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 early genes selected from the group consisting of: ICP4, ICP22, ICP27, ICP47 and combinations thereof. The advantage of the HSV vector is that it can enter a latent period to cause long-term DNA expression and it has a larger viral DNA genome, which can accommodate exogenous DNA insertion sequences of up to 25kb. HSV-based vectors are described, for example, in U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0312] F. Genetically modified cells

[0313] In various embodiments, cells genetically modified to express CARs as contemplated herein are provided for the treatment of cancer. As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cells", "modified cells" and "redirected cells" are used interchangeably. As used herein, the term "gene therapy" refers to the introduction of additional genetic material in the form of DNA or RNA into the total genetic material in a cell, which restores, corrects or modifies the expression of a gene or is used to express a therapeutic polypeptide (eg, CAR) for purposes.

[0314] In a specific embodiment, the anti-CD79A CARs covered herein are introduced and expressed in immune effector cells to redirect their specificity to the target antigen of interest, such as a CD79A polypeptide. "Immune effector cells" are any cells in the immune system with one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, ADCC and / or CDC induction). Illustrative immune effector cells covered herein are T lymphocytes, including but not limited to cytotoxic T cells (CTL; ​​CD8 + T cells), TILs, and helper T cells (HTL; CD4 +T cells. In certain embodiments, the cells comprise αβ T cells. In certain embodiments, the cells comprise γδ T cells. In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0315] Immune effector cells can be autologous / autogeneic ("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, "syngeneic" 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 allogeneic.

[0316] Illustrative immune effector cells for anti-CD79A CARs covered in specific embodiments include T lymphocytes. The term "T cell" or "T lymphocyte" is recognized in the field 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 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other T cell subset. Other illustrative T cell populations suitable for use in specific 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 ).

[0317] As will be understood by technicians, other cells can also be used as immune effector cells with anti-CD79A CAR as covered herein. Specifically, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include progenitor cells of effector cells, wherein the progenitor cells can be induced in vivo or in vitro to differentiate into immune effector cells. Therefore, in a particular embodiment, immune effector cells include progenitor cells of immune effector cells, such as hematopoietic stem cells (HSC) contained in CD34+ cell populations derived from umbilical cord blood, bone marrow or flowing peripheral blood, which differentiate into mature immune effector cells after being given to a subject, or it can be induced in vitro to differentiate into mature immune effector cells.

[0318] As used herein, immune effector cells genetically engineered to contain a CD79A-specific CAR may be referred to as "CD79A-specific redirected immune effector cells."

[0319] As used herein, the term "CD34 + As used herein, "CD34" refers to a cell that expresses the CD34 protein on its cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin) that normally acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes. + The cell population contains hematopoietic stem cells (HSCs), which, upon administration to a patient, differentiate and contribute to all hematopoietic lineages (including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineages).

[0320] In a particular embodiment, a method for preparing an immune effector cell is provided, and the immune effector cell expresses the anti-CD79A CAR covered herein. In one embodiment, the method includes transfection or transduction of immune effector cells separated from an individual, so that the immune effector cells express one or more anti-CD79A CARs covered herein. In certain embodiments, immune effector cells are separated from an individual and genetically modified without further in vitro manipulation. The cell can then be directly given to the individual again. In other embodiments, immune effector cells are first activated and stimulated in vitro to proliferate, and then genetically modified to express anti-CD79A CAR. In this regard, immune effector cells can be cultured before and / or after genetic modification (i.e., transduction or transfection to express anti-CD79A CAR covered herein).

[0321] In a specific embodiment, before in vitro manipulation or genetic modification of the immune effector cells described herein, the cell source is obtained from a subject. In a specific embodiment, the immune effector cells modified by CAR include T cells.

[0322] In a particular embodiment, PBMC can be directly genetically modified to express anti-CD79A CAR using the methods covered herein. In certain embodiments, after separating PBMC, T lymphocytes are further separated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into original, memory and effector T cell subsets before or after genetic modification and / or amplification.

[0323] Immune effector cells, such as T cells, can be genetically modified using known methods after separation, or immune effector cells can be activated and amplified in vitro before genetic modification (or differentiated in the case of progenitor cells). In a particular embodiment, immune effector cells, such as T cells, are genetically modified using chimeric antigen receptors as contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding an anti-CD79A CAR), and then activated and amplified in vitro. In various embodiments, T cells may be activated and expanded before or after being genetically modified to express a CAR using methods such as 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.

[0324] In one embodiment, CD34 + Cells are transduced with nucleic acid constructs as contemplated herein. In certain embodiments, the transduced CD34 + The cells differentiate into mature immune effector cells in vivo following administration to a subject, generally the subject from which the cells were initially isolated. + The cells can be stimulated in vitro 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) according to previously described methods prior to exposure to a CAR as described herein or after genetic modification with a CAR as described herein.

[0325] In certain embodiments, the modified immune effector cell population for treating cancer comprises a CAR as contemplated herein. For example, the modified immune effector cell population is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient (autologous donor) diagnosed with a B cell malignancy described herein. The PBMC formation may be CD4+ 、CD8 + or CD4 + and CD8 + Heterogeneous T lymphocyte population.

[0326] PBMC may also include other cytotoxic lymphocytes, such as NK cells or NKT cells. The expression vector carrying the coding sequence of the CAR covered in a specific embodiment is introduced into a population of human donor T cells, NK cells or NKT cells. 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 propagated to increase the number of T cells expressing these CAR proteins, plus anti-CD3 antibodies and or anti-CD28 antibodies and IL-2 for cell activation, or any other method known in the art as described elsewhere herein. Standard procedures are used to freeze T cells expressing CAR protein T cells to store and / or prepare for human subjects. In one embodiment, in the absence of non-human animal derived products, such as fetal bovine serum (fetal calfserum / fetal bovine serum), T cells are transduced in vitro, cultured and / or amplified. Because the heterogeneous PBMC population is genetically modified, the resulting transduced cells are heterogeneous modified cell populations comprising anti-CD79A CAR as covered herein.

[0327] In another embodiment, for example, a mixture of 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 modified cell population, wherein a certain proportion of modified cells express more than one different CAR protein.

[0328] GT cell production method

[0329] In various embodiments, genetically modified T cells are expanded by contacting an agent that stimulates CD3 TCR complex-associated signaling with a ligand that stimulates a co-stimulatory molecule on the surface of the T cell.

[0330] In specific embodiments, PBMCs or isolated T cells are contacted with stimulatory and co-stimulatory agents attached to beads or other surfaces (e.g., soluble anti-CD3 and anti-CD28 antibodies) in culture medium with appropriate cytokines (e.g., IL-2, IL-7, and / or IL-15).

[0331] In specific embodiments, PBMCs or isolated T cells are contacted with stimulatory and co-stimulatory agents (e.g., soluble anti-CD3 and anti-CD28 antibodies) attached to beads or other surfaces in culture medium with appropriate cytokines (e.g., IL-2, IL-7 and / or IL-15) and / or one or more agents that modulate the PI3K / Akt / mTOR cell signaling pathway.

[0332] In preferred embodiments, T cells manufactured by the methods covered 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 covered herein have excellent properties, including increased survival, expansion in the relative absence of differentiation, and in vivo retention. In one embodiment, the method for manufacturing T cells comprises contacting cells with one or more agents that regulate the PI3K cell signaling pathway. In one embodiment, the method for manufacturing T cells comprises contacting cells with one or more agents that regulate the PI3K / Akt / mTOR cell signaling pathway. In various embodiments, T cells may be obtained from any source and contacted with agents during the activation and / or expansion phase of the manufacturing method. The resulting T cell composition is rich in T cells that develop powerfully, and the T cells are capable of proliferating and expressing one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, CD38, and CD8. In one embodiment, a cell population comprising T cells treated with one or more PI3K inhibitors is rich in CD8 that co-expresses one or more or all of the following biomarkers. + T cell populations: CD62L, CD127, CD197 and CD38.

[0333] In one embodiment, a cell population comprising T cells treated with one or more PI3K inhibitors is enriched for CD8 T cells that co-express one or more or all of the following biomarkers: + T cell populations: CD62L, CD127, CD27, and CD8.

[0334] In one embodiment, modified T cells comprising maintained proliferation levels and reduced differentiation are manufactured. In a specific embodiment, T cells are manufactured by stimulating T cells to become activated and proliferate in the presence of one or more stimulatory signals and an agent that is an inhibitor of the PI3K cell signaling pathway.

[0335] The T cells may then be modified to express anti-CD79A CAR. In one embodiment, T cells are modified by transducing T cells with a viral vector comprising an anti-CD79A CAR as contemplated herein. In a certain embodiment, T cells are modified before stimulation and activation in the presence of an inhibitor of the PI3K cell signaling pathway. In another embodiment, T cells are modified after stimulation and activation in the presence of an inhibitor of the PI3K cell signaling pathway. In a specific embodiment, T cells are modified within 12 hours, 24 hours, 36 hours, or 48 hours of stimulation and activation in the presence of an inhibitor of the PI3K cell signaling pathway.

[0336] After activating the T cells, the cells are cultured to allow them 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.

[0337] In various embodiments, T cell compositions are manufactured in the presence of one or more PI3K / Akt / mTOR cell signaling pathway inhibitors. The inhibitors can target one or more activities or a single activity in the pathway. Without wishing to be bound by any particular theory, it is expected that treating T cells with one or more inhibitors of the PI3K pathway or contacting T cells with the one or more inhibitors during the stimulation, activation and / or amplification phase of the manufacturing method will preferentially increase young T cells, thereby producing an excellent therapeutic T cell composition.

[0338] In a specific embodiment, a method of increasing the proliferation of T cells expressing an engineered T cell receptor is provided. Such methods may include, for example, collecting a T cell source from a subject, stimulating and activating T cells in the presence of one or more PI3K pathway inhibitors, modifying T cells to express anti-CD79A CAR, and expanding cultured T cells.

[0339] In a certain embodiment, a method for generating a population of T cells is contemplated, wherein the population of T cells is enriched to express one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, CD38, and CD8. In one embodiment, the immature T cells comprise one or more, or all, of the following biomarkers: CD62L, CD127, CD197, and CD38.

[0340] In one embodiment, the naive T cells comprise one or more or all of the following biomarkers: CD62L, CD127, CD27, and CD8.

[0341] In one embodiment, a young T cell that does not express CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3 is provided. As discussed elsewhere herein, the expression level of young T cell biomarkers is correlated with the expression level of such markers in a more differentiated T cell or immune effector cell population.

[0342] In one embodiment, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell manufacturing methods contemplated herein. PBMCs form a heterogeneous population of T lymphocytes, which can be CD4 + 、CD8 + or CD4 + and CD8 + , and may include other mononuclear cells, such as monocytes, B cells, NK cells, and NKT cells. An expression vector comprising a polynucleotide encoding an engineered TCR or CAR encompassed in a specific embodiment is introduced into a population of human donor T cells, NK cells, or NKT cells. In a specific embodiment, successfully transduced T cells carrying an expression vector can be sorted using flow cytometry to isolate CD3-positive T cells, and then further propagated to increase the number of modified T cells, plus cell activation using anti-CD3 antibodies and or anti-CD28 antibodies and IL-2, IL-7, and / or IL-15.

[0343] The manufacturing methods covered herein may further include freezing modified T cells for storage and / or preparation for human subjects. In one embodiment, a method for storing genetically modified murines, humans or humanized CAR proteins expressing immune effector cells (which target cells expressing CD79A) includes freezing immune effector cells so that the cells remain viable when thawed. A portion of immune effector cells expressing CAR proteins can be frozen by methods known in the art to provide a permanent source of such cells for future treatment of patients suffering from cancer cells expressing CD79A. T cells are frozen so that the cells remain viable when thawed. If necessary, frozen transformed immune effector cells can be thawed, grown and amplified to obtain more such cells. As used herein, "freezing" refers to preserving cells by cooling to subzero temperatures, such as (usually) 77K or -196°C (the boiling point of liquid nitrogen). Cryoprotectants are usually used at subzero temperatures to prevent the stored 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, polyvinylpyrrolidine (Rinfret, Annals of the New York Academy of Sciences, 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 1°C to 3°C per minute. After at least two hours, the T cells have reached a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage, for example in a long-term cryogenic storage vessel.

[0344] 1. T cells

[0345] In a particular embodiment, the manufacture of improved CAR T cell compositions is provided. The T cells used for CAR T cell preparation can be self / autologous ("self") or non-self ("non-self", such as allogeneic, isogenic or allogenic). In a preferred embodiment, the T cells are obtained from a mammalian subject. In a more preferred embodiment, the T cells are obtained from a primate subject. In a most preferred embodiment, the T cells are obtained from a human subject.

[0346] T cells can be obtained from a variety of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, T cells can be obtained using a variety of techniques known to the skilled artisan (e.g., sedimentation, e.g., FICOLL TMA blood unit collected from a subject (separation) is obtained by apheresis. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma portion and the cells are placed in an appropriate buffer or culture medium for subsequent processing. The cells can be washed with PBS or with another suitable solution without calcium, magnesium and most (if not all) other divalent cations. As will be understood by a person skilled in the art, the washing step can be achieved by methods known to those skilled in the art (e.g., by using a semi-automated flow-through centrifuge). For example, Cobe 2991 cell processor, Baxter CytoMate, etc. After washing, the cells can be resuspended in a variety of biocompatible buffers or other physiological saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample can be removed in the culture medium in which the cells are directly resuspended.

[0347] In certain embodiments, a population of cells (e.g., PBMCs) comprising T cells is used in the manufacturing methods contemplated herein. In other embodiments, an isolated or purified population of T cells is used in the manufacturing methods contemplated herein. Cells can be isolated from peripheral blood mononuclear cells (PBMCs) by, for example, PERCOLL TM Gradient centrifugation lyses red blood cells and depletes monocytes. In some embodiments, following isolation of PBMCs, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets either before or after activation, expansion, and / or genetic modification.

[0348] In certain embodiments, a population of cells (e.g., PBMCs) comprising T cells is used in the manufacturing methods contemplated herein. In other embodiments, an isolated or purified population of T cells is used in the manufacturing methods contemplated herein. Cells can be isolated from peripheral blood mononuclear cells (PBMCs) by, for example, PERCOLL TM Gradient centrifugation lyses red blood cells and depletes monocytes. In some embodiments, following isolation of PBMCs, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets either before or after activation, expansion, and / or genetic modification.

[0349] In a specific embodiment, immune effector cell colony is genetically modified to express CAR using the method covered herein, but is not subjected to positive or negative selection PBMC made.In certain embodiments, after separating PBMC, T lymphocytes are further separated, and in certain embodiments, cytotoxic and helper T lymphocytes can be sorted into original, memory and effector T cell subsets before or after genetic modification and / or amplification.

[0350] In certain embodiments, a specific subset of T cells expressing one or more of the following markers can be further separated by positive or negative selection techniques: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR. In one embodiment, a specific subset of T cells expressing one or more of the markers selected from the group consisting of: i) CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; ii) CD62L, CD127, CD197, and CD38; or iii) CD62L, CD127, CD27, and CD8. In various embodiments, the manufactured T cell composition does not express or substantially does not express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0351] In one embodiment, the expression of one or more of the markers selected from the group consisting of i) CD62L, CD127, CD197, and CD38 or ii) CD62L, CD127, CD27, and CD8 is increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 25-fold, or more, compared to a population of T cells activated and expanded in the absence of a PI3K inhibitor. In one embodiment, the T cells comprise CD8 + T cells.

[0352] In one embodiment, compared to a population of T cells activated and expanded without a PI3K inhibitor, the expression of one or more of the markers selected from the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3 is reduced by at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, or more: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3. In one embodiment, the T cells comprise CD8 + T cells.

[0353] In one embodiment, the manufacturing methods encompassed herein increase the number of CAR T cells containing one or more markers of naive or developmentally potent T cells. Without wishing to be bound by any particular theory, the inventors believe that treating a cell population containing T cells with one or more PI3K inhibitors results in increased expansion of developmentally potent T cells, and provides a more stable and effective adoptive CAR T cell immunotherapy compared to existing CAR T cell therapies.

[0354] Illustrative examples of markers of naive or developing powerful T cells increased in T cells made using the methods encompassed in specific embodiments include, but are not limited to, i) CD62L, CD127, CD197, and CD38, or ii) CD62L, CD127, CD27, and CD8. In specific embodiments, naive T cells do not express or do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, BTLA, CD45RA, CTLA4, TIM3, and LAG3.

[0355] With respect to T cells, the T cell populations produced by the various expansion methods encompassed herein can have a variety of specific phenotypic properties depending on the conditions utilized. In various embodiments, the expanded T cell populations comprise one or more of the following phenotypic markers: CD62L, CD27, CD127, CD197, CD38, CD8, and HLA-DR.

[0356] In one embodiment, such phenotypic markers include enhanced expression of one or more or all of CD62L, CD127, CD197, and CD38. In a specific embodiment, the expression of CD8 T cells characterized by phenotypic markers of naive T cells, including CD62L, CD127, CD197, and CD38, is enhanced. + T lymphocyte expansion.

[0357] In one embodiment, such phenotypic markers include enhanced expression of one or more or all of CD62L, CD127, CD27, and CD8. In a specific embodiment, the expression of phenotypic markers characterized by naive T cells, including CD62L, CD127, CD27, and CD8, is enhanced. + T lymphocyte expansion.

[0358] In specific embodiments, T cells are expanded that are characterized by expression of phenotypic markers of central memory T cells, including CD45RO, CD62L, CD127, CD197, and CD38, and that are negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells.

[0359] In certain embodiments, the + CD4 T cells that express phenotypic markers including CD62L and are negative for expression of CD45RA and / or CD45RO + T lymphocyte expansion. In some embodiments, it is characterized by central memory CD4 + Expression of phenotypic markers of cells (including CD62L and CD45RO) + In some embodiments, the effector CD4 + Cells were CD62L positive and CD45RO negative.

[0360] In certain embodiments, T cells are separated from an individual, and activated and stimulated in vitro to proliferate, and then genetically modified to express anti-CD79A CAR. In this regard, T cells can be cultured before and / or after genetic modification (i.e., transduction or transfection to express anti-CD79A CARs encompassed herein).

[0361] 2. Activation and amplification

[0362] In order to achieve a sufficient therapeutic dose of T cell compositions, T cells are generally subjected to one or more rounds of stimulation, activation and / or expansion. T cells can be generally activated and expanded using methods such as, 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. T cells modified to express anti-CD79A CAR can be activated and expanded before and / or after T cells are modified. In addition, T cells can be contacted with one or more agents that modulate the PI3K / Akt / mTOR cell signaling pathway before, during, and / or after activation and / or expansion. In one embodiment, T cells made by the methods contemplated herein undergo one, two, three, four, or five or more rounds of activation and expansion, each of which may include one or more agents that modulate the PI3K / Akt / mTOR cell signaling pathway.

[0363] Artificial antigen presenting cells (aAPCs) support functional human CD8 +In vitro growth and long-term expansion of T cells without the addition of exogenous cytokines. In specific embodiments, PBMCs or isolated T cells are contacted with stimulatory agents and co-stimulatory agents (such as anti-CD3 and anti-CD28 antibodies) generally attached to beads or other surfaces in culture medium with appropriate cytokines (such as IL-2, IL-7 and / or IL-15).

[0364] In other embodiments, artificial APC (aAPC) is prepared by engineering K562, U937, 721.221, T2 and C1R cells to guide the stable expression and secretion of multiple co-stimulatory molecules and cytokines. In a specific embodiment, K32 or U32 aAPC is used to guide the presentation of one or more antibody-based stimulatory molecules on the surface of AAPC cells. T cell populations can be expanded by aAPC expressing multiple co-stimulatory molecules (including but not limited to CD137L (4-1BBL), CD134L (OX40L) and / or CD80 or CD86). aAPC provides an effective platform for expanding genetically modified T cells and maintaining CD8 + CD28 expression on T. aAPC provided in WO 03 / 057171 and US 2003 / 0147869 are incorporated herein by reference in their entirety.

[0365] In one embodiment, the co-stimulatory ligand is presented on the antigen presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) of the cognate co-stimulatory molecule on the specific binding T cell, thereby in addition to the primary signal provided by, for example, binding to the TCR / CD3 complex, a signal for mediating the required T cell response is also provided. Suitable co-stimulatory ligands include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, agonists or antibodies binding to Toll ligand receptors, and ligands specifically binding to B7-H3.

[0366] In certain embodiments, the co-stimulatory ligand comprises an antibody or antigen-binding fragment thereof that specifically binds to a co-stimulatory molecule present on T cells, including but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0367] Suitable co-stimulatory ligands further include target antigens, which can be provided in a soluble form or expressed on an APC or aAPC that binds to an engineered TCR or CAR expressed on a modified T cell.

[0368] In various embodiments, the methods of manufacturing T cells encompassed herein include activating a cell population comprising T cells and expanding the T cell population. T cell activation can be achieved by providing a primary stimulation signal via a T cell TCR / CD3 complex or via stimulation of CD2 surface proteins and by providing a secondary costimulatory signal via an auxiliary molecule (e.g., CD28).

[0369] The TCR / CD3 complex can be stimulated by contacting the T cell with a suitable CD3 binding agent (e.g., a CD3 ligand or an anti-CD3 monoclonal antibody). Illustrative examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1.

[0370] In another embodiment, CD2 binding agents can be used to provide primary stimulation signals to T cells. Illustrative examples of CD2 binding agents include, but are not limited to, CD2 ligands and anti-CD2 antibodies, such as a combination of T11.3 antibodies with T11.1 or T11.2 antibodies (Meuer, SC et al. (1984) Cell 36: 897-906) and a combination of 9.6 antibodies (which recognize the same epitope as TI 1.1) with 9-1 antibodies (Yang, SY et al. (1986) Journal of Immunology 137: 1097-1100). Other antibodies that bind to the same epitope as any of the antibodies described above can also be used. Other antibodies or antibody combinations can be prepared and identified by standard techniques as disclosed elsewhere herein.

[0371] In addition to the primary stimulation signal provided via the TCR / CD3 complex or via CD2, inducing T cell responses requires a second costimulatory signal. In a particular embodiment, a CD28 binding agent can be used to provide a costimulatory signal. Illustrative examples of CD28 binding agents include, but are not limited to, natural CD 28 ligands, such as natural ligands of CD28 (e.g., members of the B7 protein family, such as B7-1 (CD80) and B7-2 (CD86)); and anti-CD28 monoclonal antibodies or fragments thereof capable of cross-linking CD28 molecules, such as monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0372] In one embodiment, a molecule that provides a primary stimulation signal (eg, a molecule that provides stimulation via the TCR / CD3 complex or CD2) is coupled to the same surface as a co-stimulatory molecule.

[0373] In certain embodiments, the binding agent that provides the stimulation and co-stimulation signal is localized on the cell surface. This can be achieved by transfecting or transducing the cell with a nucleic acid encoding the binding agent (in a form suitable for its expression on the cell surface), or by coupling the binding agent to the cell surface.

[0374] In another embodiment, a molecule that provides a primary stimulation signal (eg, a molecule that provides stimulation via the TCR / CD3 complex or CD2) and a co-stimulatory molecule are presented on an antigen presenting cell.

[0375] In one embodiment, the molecule that provides the primary stimulation signal (eg, a molecule that provides stimulation via the TCR / CD3 complex or CD2) and the co-stimulatory molecule are provided on separate surfaces.

[0376] In a certain embodiment, one of the binding agents that provide the stimulatory and co-stimulatory signals is soluble (provided in solution) and the other binding agent is provided on one or more surfaces.

[0377] In certain embodiments, both the binding agents that provide the stimulatory and co-stimulatory signals are provided in soluble form (provided in solution).

[0378] In various embodiments, the methods of making T cells contemplated herein comprise activating T cells with anti-CD3 and anti-CD28 antibodies.

[0379] The T cell compositions manufactured by the methods covered in the specific embodiments include T cells activated and / or expanded in the presence of one or more agents that inhibit the PI3K cell signaling pathway. T cells modified to express anti-CD79A CAR can be activated and expanded before and / or after the T cells are modified. In a specific embodiment, the T cell population is activated, modified to express anti-CD79A CAR, and then cultured for expansion.

[0380] In one embodiment, the T cells manufactured by the methods contemplated herein comprise an increased number of T cells expressing markers indicating high proliferative potential and capable of self-renewal but not expressing or substantially expressing undetectable T cell differentiation markers. These T cells can be repeatedly activated and expanded in a stable manner and thus provide improved therapeutic T cell compositions.

[0381] In one embodiment, a population of T cells activated and expanded in the presence of one or more agents that inhibit the PI3K cell signaling pathway expands at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1000-fold, or more, compared to a population of T cells activated and expanded in the absence of a PI3K inhibitor.

[0382] In one embodiment, a population of T cells characterized by expression of a naive T cell marker is activated and expanded in the presence of one or more agents that inhibit the PI3K cell signaling pathway by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1000-fold, or more, compared to a population of T cells activated and expanded in the absence of a PI3K inhibitor.

[0383] In one embodiment, the T cells activated by the methods encompassed herein are expanded further comprising culturing a cell population comprising T cells for a few hours (about 3 hours) to about 7 days to about 28 days or any integer value per hour in between. In another embodiment, the T cell composition can be cultured for 14 days. In a specific embodiment, the T cells are cultured for about 21 days. In another embodiment, the T cell composition is cultured for about 2-3 days. It may also be necessary to have several stimulation / activation / amplification cycles so that the culture time of the T cells can be 60 days or longer.

[0384] In a specific embodiment, conditions suitable for T cell culture include an appropriate culture medium (e.g., Minimal Essential Medium or RPMI medium 1640 or X-Vivo 15 (Lonza)); and one or more factors required for proliferation and survival, including but not limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or any other additives suitable for cell growth known to the skilled artisan.

[0385] Other illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 5, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in amounts sufficient for the growth and expansion of T cells.

[0386] Illustrative examples of other additives for T cell expansion include, but are not limited to, surfactants, human plasma protein preparations, pH buffers (eg, HEPES), and reducing agents (eg, N-acetyl-cysteine ​​and 2-mercaptoethanol).

[0387] Antibiotics (eg, penicillin and streptomycin) are included only in experimental cultures, not in cell cultures to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as appropriate temperature (eg, 37° C.) and atmosphere (eg, air plus 5% CO 2 ).

[0388] 3. Reagents

[0389] In various embodiments, a method for manufacturing T cells that expands undifferentiated or powerful T cells is provided, comprising contacting T cells with agents that regulate the PI3K pathway in cells. In various embodiments, a method for manufacturing T cells that expands undifferentiated or powerful T cells is provided, comprising contacting T cells with agents that regulate the PI3K / AKT / mTOR pathway in cells. Cells can be contacted before, during, and / or after activation and expansion. The T cell composition retains sufficient T cell potency so that it can undergo multiple rounds of expansion without substantially increasing differentiation.

[0390] As used herein, the term "modulate," "modulator," or "modulatory agent," or similar terms, refers to the ability of an agent to induce a change in a cell signaling pathway. A modulator can increase or decrease the amount, activity of a pathway component, or increase or decrease a desired effect or outcome of a cell signaling pathway. In one embodiment, a modulator is an inhibitor. In another embodiment, a modulator is an activator.

[0391] An "agent" refers to a compound, small molecule (eg, small organic molecule), nucleic acid, polypeptide, or fragment, isoform, variant, analog, or derivative thereof, that is used to modulate the PI3K / AKT / mTOR pathway.

[0392] "Small molecule" refers to a composition having a molecular weight of less than about 5 kD, less than about 4 kD, less than about 3 kD, less than about 2 kD, less than about 1 kD, or less than about .5 kD. Small molecules can include nucleic acids, peptides, polypeptides, peptide mimetics, peptoids, carbohydrates, lipids, components thereof, or other organic or inorganic molecules. Chemical and / or biological mixture libraries (e.g., fungal, bacterial, or algal extracts) are known in the art and can be screened with any assay. Examples of methods for synthesizing molecular libraries can be found in (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et al., 1994).

[0393] "Analog" refers to a small organic compound, nucleotide, protein or polypeptide that has an activity or function similar or identical to that of a compound, nucleotide, protein or polypeptide or a compound, or a compound that has the desired activity but does not necessarily contain a sequence or structure similar or identical to that of the preferred embodiments.

[0394] "Derivative" refers to a compound, protein or polypeptide comprising an amino acid sequence of a parent protein or polypeptide that has been altered by the introduction of amino acid residue substitutions, deletions or additions, or a nucleic acid or nucleotide that has been modified by the introduction of nucleotide substitutions or deletions, additions or mutations. The derivative nucleic acid, nucleotide, protein or polypeptide has a similar or identical function to the parent polypeptide.

[0395] In various embodiments, agents that modulate the PI3K pathway activate pathway components. "Activators" or "agonists" refer to agents that promote, increase, or induce one or more activities of molecules in the PI3K / AKT / mTOR pathway, including but not limited to molecules that activate one or more activities of PI3K.

[0396] In various embodiments, agents that regulate the PI3K pathway inhibit pathway components. "Inhibitors" or "antagonists" refer to agents that inhibit, reduce or decrease one or more activities of molecules in the PI3K / AKT / mTOR pathway, including but not limited to molecules that inhibit one or more activities of PI3K. In one embodiment, the inhibitor is a dual molecule inhibitor. In a particular embodiment, the inhibitor can inhibit a class of molecules with the same or substantially similar activity (pan-inhibitors) or can specifically inhibit the activity of a molecule (selective or specific inhibitors). Inhibition can also be irreversible or reversible.

[0397] In one embodiment, the IC50 of the inhibitor is at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 μM, at least 10 μM, at least 50 μM, or at least 100 μM. IC50 determinations can be achieved using any conventional techniques known in the art. For example, IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of a research inhibitor. The experimentally obtained enzyme activity values ​​are then plotted relative to the inhibitor concentration used. The concentration of the inhibitor that exhibits 50% enzyme activity (compared to the activity in the absence of any inhibitor) is considered an "IC50" value. Similarly, other inhibitory concentrations can be defined by appropriate determination of activity.

[0398] In various embodiments, T cells are contacted, treated or cultured with one or more PI3K / AKT / mTOR pathway regulators at a concentration of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 μM, at least 10 μM, at least 50 μM, at least 100 μM or at least 1 M.

[0399] In certain embodiments, T cells can be contacted with, treated with, or cultured with one or more PI3K / AKT / mTOR pathway modulators for at least 12 hours, 18 hours, 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, and for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.

[0400] Phosphatidyl-inositol-3 kinase / Akt / mammalian target of rapamycin pathway serves as a channel for integrating growth factor signaling with cell proliferation, differentiation, metabolism and survival. PI3K is a highly conserved family of intracellular lipid kinases. Class IA PI3K is activated by growth factor receptor tyrosine kinases (RTKs) directly or via interactions with adapter molecules of the insulin receptor substrate family. This activity leads to the production of 3,4,5-phosphatidylinositol triphosphate (PIP3), a regulator of serine / threonine kinase Akt. mTOR acts through a typical PI3K pathway via 2 unique complexes of different binding partners each characterized by conferring unique activity. mTORC1 (a complex of mTOR with PRAS40, raptor and mLST8 / GbL) serves as a downstream effector of PI3K / Akt signaling, correlating growth factor signals with protein translation, cell growth, proliferation and survival. mTORC2 (a complex of mTOR with rictor, mSIN1, protor and mLST8) serves as an upstream activator of Akt.

[0401] After growth factor receptor-mediated PI3K activation, Akt is recruited to the membrane via the interaction of its pleckstrin homology domain with PIP3, thus exposing its activation loop and achieving phosphorylation at threonine 308 (Thr308) by the constitutively active phosphoinositide-dependent protein kinase 1 (PDK1). For maximum activation, Akt is also phosphorylated at serine 473 (Ser473) at its C-terminal hydrophobic motif by mTORC2. DNA-PK and HSP have also been shown to be important in regulating Akt activity. Akt activates mTORC1 via inhibitory phosphorylation of TSC2, which, together with TSC1, negatively regulates mTORC1 by inhibiting Rheb GTPase, a positive regulator of mTORC1. mTORC1 has two well-defined substrates, p70S6K (hereinafter referred to as S6K1) and 4E-BP1, both of which critically regulate protein synthesis. Thus, mTORC1 is an important downstream effector of PI3K, linking growth factor signaling to protein translation and cell proliferation.

[0402] a. PI3K inhibitors

[0403] As used herein, the term "PI3K inhibitor" refers to a nucleic acid, peptide, compound or small organic molecule that is bound to PI3K and inhibits at least one of its activities. PI3K proteins can be divided into three categories, class 1 PI3K, class 2 PI3K and class 3 PI3K. Class 1 PI3K exists in the form of a heterodimer consisting of one of four p110 catalytic subunits (p110α, p110β, p110δ and p110γ) and one of two regulatory subunit families. PI3K inhibitors preferably target class 1 PI3K inhibitors. In one embodiment, PI3K inhibitors will present selectivity for one or more isoforms of class 1 PI3K inhibitors (i.e., selectivity for one or more of p110α, p110β, p110δ and p110γ or p110α, p110β, p110δ and p110γ). On the other hand, PI3K inhibitors will not present isoform selectivity and are considered as "pan-PI3K inhibitors". In one embodiment, the PI3K inhibitor will compete with ATP for binding to the PI3K catalytic domain.

[0404] In certain embodiments, the PI3K inhibitor can, for example, target PI3K and other proteins in the PI3K-AKT-mTOR pathway. In a specific embodiment, a PI3K inhibitor targeting both mTOR and PI3K can be referred to as an mTOR inhibitor or a PI3K inhibitor. A PI3K inhibitor that only targets 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 of at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 1000 times or more about PI3K than an inhibitor IC50 for mTOR and / or other proteins in the pathway.

[0405] In certain embodiments, exemplary PI3K inhibitors inhibit PI3K with an IC50 (concentration that inhibits 50% 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.

[0406] 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).

[0407] Other illustrative examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145.

[0408] Other illustrative examples of pan-PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

[0409] In a preferred embodiment, the PI3K inhibitor is ZSTK474.

[0410] b. AKT inhibitors

[0411] As used herein, the term "AKT inhibitor" refers to a nucleic acid, peptide, compound or small organic molecule that inhibits at least one activity of AKT. AKT inhibitors can be grouped into several classes, including lipid-based inhibitors (e.g., inhibitors that prevent AKT from being localized to the pleckstrin homology domain of the plasma membrane), ATP competitive inhibitors and allosteric inhibitors. In one embodiment, the AKT inhibitor acts by binding to the AKT catalytic site. In a specific embodiment, the Akt inhibitor acts by inhibiting the phosphorylation of downstream AKT targets (e.g., mTOR). In another embodiment, AKT activity is inhibited by inhibiting the input signal of activating Akt by inhibiting, for example, DNA-PK activation of AKT, PDK-1 activation of AKT and / or mTORC2 activation of Akt.

[0412] AKT inhibitors can target all three AKT isoforms AKT1, AKT2, AKT3, or can be isoform selective and only target one or two AKT isoforms. In one embodiment, AKT inhibitors can target other proteins in AKT and PI3K-AKT-mTOR pathways. Only AKT inhibitors targeting AKT can be referred to as selective AKT inhibitors. In one embodiment, selective AKT inhibitors can be understood to refer to agents that exhibit a 50% inhibitory concentration of at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 1000 times or more about AKT than inhibitor IC50s for other proteins in pathways.

[0413] In certain embodiments, exemplary AKT inhibitors inhibit AKT with an IC50 (concentration that inhibits 50% 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, AKT inhibits AKT 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.

[0414] Illustrative examples of AKT inhibitors for use in combination with auristatin-based antibody-drug conjugates include, for example, perifosine (Keryx), MK2206 (Merck), VQD-002 (VioQuest), XL418 (Exelixis), GSK690693, GDC-0068, and PX316 (PROLX Pharmaceuticals).

[0415] An illustrative, non-limiting example of a selective Akt1 inhibitor is A-674563.

[0416] An illustrative, non-limiting example of a selective Akt2 inhibitor is CCT128930.

[0417] In specific embodiments, the Akt inhibitor is DNA-PK activation of Akt, PDK-1 activation of Akt, mTORC2 activation of Akt, or HSP activation of Akt.

[0418] Illustrative examples of DNA-PK inhibitors include, but are not limited to, NU7441, PI-103, NU7026, PIK-75, and PP-121.

[0419] c. mTOR inhibitors

[0420] The term "mTOR inhibitor" or "agent that inhibits mTOR" refers to a nucleic acid, peptide, compound or small organic molecule that inhibits at least one activity (e.g., serine / threonine protein kinase activity) of the mTOR protein on at least one of its substrates (e.g., p70S6 kinase 1, 4E-BP1, AKT / PKB and eEF2). The mTOR inhibitor can directly bind to and inhibit mTORC1, mTORC2, or both mTORC1 and mTORC2.

[0421] Inhibition of mTORC1 and / or mTORC2 activity can be determined by a reduction in signal transduction of the PI3K / Akt / mTOR pathway. A variety of readouts can be used to establish a reduction in the output of the signaling pathway. Some non-limiting exemplary readouts include (1) a reduction in Akt phosphorylation at residues including but not limited to 5473 and T308; (2) a reduction in Akt activation, as evidenced by, for example, a reduction in phosphorylation of Akt substrates (including but not limited to FoxO1 / O3a T24 / 32, GSK3a / β, S21 / 9, and TSC2 T1462); (3) a reduction in phosphorylation of signaling molecules downstream of mTOR (including but not limited to ribosomal S6 S240 / 244, 70S6K T389, and 4EBP1 T37 / 46); and (4) inhibition of proliferation of cancer cells.

[0422] In one embodiment, the mTOR inhibitor is an active site inhibitor. These are mTOR inhibitors that bind to the ATP binding site (also referred to as the ATP binding pocket) of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2. A class of active site inhibitors suitable for use in the T cell manufacturing methods covered in specific embodiments is a dual specific inhibitor that targets and directly inhibits both PI3K and mTOR. Dual specific inhibitors bind to the ATP binding sites of both mTOR and PI3K. Illustrative examples of the inhibitor include, but are not limited to: imidazoquinazoline, wortmannin, LY294002, PI-103 (Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765 (Exelixis) and NVP-BEZ235 (Novartis).

[0423] Another class of mTOR active site inhibitors suitable for use in the methods contemplated in particular embodiments selectively inhibits mTORC1 and mTORC2 activity relative to one or more type I phosphatidylinositol 3-kinases (e.g., PI3 kinase α, β, γ, or δ). These active site inhibitors bind to the active site of mTOR but not to the active site of PI3K. Illustrative examples of such inhibitors include, but are not limited to, pyrazolopyrimidine, Torin1 (Guertin and Sabatini), PP242 (2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth), and AZD8055 (Liu et al., Nature Review, 8, 627-644, 2009).

[0424] In one embodiment, a selective mTOR inhibitor refers to an agent that exhibits a 50% inhibitory concentration (IC50) for mTORC1 and / or mTORC2 that is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more lower than the IC50 for an inhibitor of one, two, three or more type I PI3 kinases, or all type I PI3 kinases.

[0425] Another class of mTOR inhibitors is referred to herein as "rapamycin analogs". As used herein, the term "rapamycin analogs" refers to compounds that specifically bind to the mTOR FRB domain (FKBP rapamycin binding domain), are structurally related to rapamycin and retain mTOR inhibitory properties. The term rapamycin analogs excludes rapamycin. Rapamycin analogs include esters, ethers, oximes, hydrazones and hydroxylamines of rapamycin and compounds in which the functional groups on the rapamycin core structure have been modified by reduction or oxidation. Pharmaceutically acceptable salts of the compounds are also considered to be rapamycin derivatives. Illustrative examples of rapamycin analogs suitable for the methods covered in specific embodiments include, but are not limited to, temsirolimus (CC1779), everolimus (everolimus) (RAD001), deforolimus (deforolimus) (AP23573), AZD8055 (AstraZeneca) and OSI-027 (OSI).

[0426] In one embodiment, the agent is the mTOR inhibitor rapamycin (sirolimus).

[0427] In certain embodiments, exemplary mTOR inhibitors inhibit mTORC1, mTORC2, or both mTORC1 and mTORC2 with an IC50 (concentration that inhibits 50% 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 aspect, the mTOR inhibitor inhibits mTORC1, mTORC2, or both mTORC1 and mTORC2 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.

[0428] In one embodiment, exemplary mTOR inhibitors inhibit PI3K and mTORC1 or mTORC2 or both mTORC1 and mTORC2 and PI3K with an IC50 (concentration that inhibits 50% activity) of about 200nM or less, preferably about 100nM or less, even more preferably about 60nM or less, about 25nM, about 10nM, about 5nM, about 1nM, 100μM, 50μM, 25μM, 10μM, 1μM or less. In one aspect, mTOR inhibitors inhibit PI3K and mTORC1 or mTORC2 or both mTORC1 and mTORC2 and PI3K with an IC50 of about 2nM to about 100nM, more preferably about 2nM to about 50nM, even more preferably about 2nM to about 15nM.

[0429] Other illustrative examples of mTOR inhibitors suitable for use in particular embodiments include, but are not limited to, AZD8055, INK128, rapamycin, PF-04691502, and everolimus.

[0430] mTOR was shown to exhibit stable and specific catalytic activity towards the physiological substrate proteins p70 S6 ribosomal protein kinase I (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphorylation-specific antibodies in Western blotting.

[0431] In one embodiment, the inhibitor of the PI3K / AKT / mTOR pathway is an s6 kinase inhibitor selected from the group consisting of: BI-D1870, H89, PF-4708671, FMK and AT7867.

[0432] H. Compositions and Formulations

[0433] The compositions contemplated herein may include one or more CAR polypeptides, polynucleotides, vectors comprising them, genetically modified immune effector cells, etc. as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals alone or in combination with one or more other treatment modes. It should also be understood that, if necessary, the composition may also be administered in combination with other agents (e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents). There are almost no restrictions on other components that may also be included in the composition, as long as other agents do not adversely affect the ability of the composition to deliver the intended treatment.

[0434] 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 human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0435] As used herein, "pharmaceutically acceptable carriers, diluents or excipients" include, but are 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 United States Food and Drug Administration as acceptable 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; gum tragacanth; 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 solutions; and any other compatible substances employed in pharmaceutical formulations.

[0436] In a particular embodiment, compositions include a certain amount of immune effector cells expressing CARs encompassed herein. As used herein, the term "amount" refers to "effective amount" or "effective amount" that genetically modified therapeutic cells (e.g., T cells) achieve beneficial or desired preventive or therapeutic results (including clinical results).

[0437] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve the desired preventive outcome. Typically, but not necessarily, the prophylactically effective amount is less than the therapeutically effective amount because a prophylactic dose is used in subjects prior to disease or at an earlier stage of disease.

[0438] A "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on factors such as: disease state; age, sex, and weight of the individual; and the ability of stem cells and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the virus or transduced therapeutic cells are outweighed by the therapeutic beneficial effects. The term "therapeutically effective amount" encompasses an amount that is effective to "treat" a subject (e.g., a patient). When specifying a therapeutic amount, the precise amount of the composition to be administered can be determined by the physician taking into account individual differences in age, weight, tumor size, degree of infection or cancer metastasis, and the condition of the patient (subject). It can generally be specified that a pharmaceutical composition comprising T cells described herein can be administered in 10 2Up to 10 10 cells / kg body weight, preferably 10 5 Up to 10 6 The dosage of 10 cells / kg body weight (including all integer values ​​within those ranges) is given. The number of cells will depend on the end use for which the composition is intended and the type of cells included therein. For use as provided herein, the volume of cells is generally one liter or less, and can be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the density of the desired cells is generally 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 into multiples that are 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 of the infused cells will be redirected to a specific target antigen, a lower number of cells may be administered, in the range of 10 6 / kg (10 per patient 6 -10 11 The cell composition expressing CAR can be given multiple times at doses within these ranges. The cells can be allogeneic, isogenic, allogeneic or self-for patients undergoing treatment. If necessary, treatment may also include administering mitogens (eg, PHA) or lymphokines, cytokines and / or chemokines (eg, 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 immune responses.

[0439] In general, compositions comprising cells activated and amplified as described herein can be used to treat and prevent diseases produced in immunocompromised individuals. In a particular embodiment, compositions comprising CAR-modified T cells encompassed herein are used to treat cancer. CAR-modified T cells can be administered alone or in the form of a pharmaceutical composition combined with a carrier, diluent, excipient and / or other components (eg, IL-2 or other cytokines or cell colonies). In a particular embodiment, the pharmaceutical composition comprises a certain amount of genetically modified T cells, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0440] The pharmaceutical composition comprising an immune effector cell colony (eg, T cell) expressing CAR may include a buffer, 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 (eg, aluminum hydroxide); And preservative. The composition is preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

[0441] Liquid pharmaceutical compositions (which are in the form of 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 solutions, isotonic sodium chloride; fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerol, propylene glycol or other solvents that may serve as solvents or suspension media; 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 preparations may be sealed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0442] In one embodiment, the T cell compositions contemplated 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.

[0443] Compared with the culture medium containing serum, serum-free culture medium has several advantages, including simplified and more specific composition, reduced contamination, elimination of potential infectious sources and lower cost. In various embodiments, serum-free culture medium is free of animal components and can be optionally 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 animal-free culture medium and nutrition is obtained from synthetic, plant or microbial sources. In contrast, "protein-free" culture medium is defined as being substantially protein-free.

[0444] Illustrative examples of serum-free culture media for use in a particular composition include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0445] In a preferred embodiment, the compositions comprising the immune effector cells contemplated herein are formulated in a solution comprising PlasmaLyte A.

[0446] In another preferred embodiment, the composition comprising the immune effector cells contemplated herein is formulated in a solution comprising a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservation agent can be used to maintain high cell viability results after thawing. Illustrative examples of cryopreservation medium used in a particular composition include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0447] In a more preferred embodiment, the composition comprising immune effector cells contemplated herein is formulated in a solution comprising 50:50 PlasmaLyte A:CryoStor CS10.

[0448] In a particular embodiment, the composition includes an effective amount of immune effector cells expressing CAR alone or in combination with one or more therapeutic agents. Therefore, the immune effector cell composition expressing CAR can be given alone or in combination with other known cancer treatments (such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc.). The composition can also be given in combination with antibiotics. Such therapeutic agents can be accepted as standard treatments for specific disease states (such as specific cancers) as described herein in the art. The exemplary therapeutic agents covered in a particular embodiment include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutics, radiotherapeutics, therapeutic antibodies or other active and auxiliary agents.

[0449] In certain embodiments, the compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered in combination with a variety of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN TM); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trishydroxymethylmelamine; nitrogen mustards, such as chlorambucil, naphthyl mustard, chlorphosphamide, estramustine, ifosfamide, dichloromethane, dichloromethane oxide hydrochloride, melphalan, novembichin, cholesterol-containing Phenylacetate mustard, prednisone mustard, trolofosamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclarubicin, actinomycin, oslamectin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, carmomycin, chromomycin, dactinomycin, daunomycin, detorubicin, 6-diazo-5-oxo-L- Norleucine, doxorubicin, epirubicin, esorubicin, idarucizumab, mexilomycin, mitomycin, mycophenolic acid, nogamycin, olivomycin, peplomycin, sphaminomycin, puromycin, quinamycin, rodorubicin, streptozocin, streptozotocin, tuberculocidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as Such as fludarabine, 6-mercaptopurine, thiabendazole, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as caprotestosterone, drostanolone propionate, cyclothiodine, melastosane, testolactone; anti-adrenal, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; acetoglucuronide; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; colchicine; diazocone; elformithine; elenium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidarol; nitroacidine; pentostatin; phenamine; pirarubicin; podophyllic acid; 2-acetylhydrazine; procarbazine; Razoxane; sizoran; spirogermanium; tricosporic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannochloridone; dibromomannitol; dibromodulanol; pipobroman; metoclopramide; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel ( Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid derivatives such as Targretin TM (Bexarotene), Panretin TM (alitretinoin);ONTAK TM(denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents used to modulate or inhibit the effects of hormones on cancer, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, troxifene, raloxifene hydrochloride, LY117018, onapristone and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0450] A variety of other therapeutic agents can be used in combination with the compositions described herein. In one embodiment, the composition comprising the immune effector cells expressing CAR is administered together with an anti-inflammatory agent. Anti-inflammatory agents or drugs include but are not limited to steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide and mycophenolate.

[0451] Other exemplary NSAIDs are selected from the group consisting of: ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors (e.g. (rofecoxib) and (celecoxib) and sialic acid esters. Exemplary analgesics are selected from the group consisting of acetaminophen, oxycodone, propoxyphene hydrochloride of tramadol. Exemplary glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone or prednisone. Exemplary biological response modifiers include molecules directed to cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors (e.g., TNF antagonists (e.g., etanercept) Adalimumab and infliximab )), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular) and minocycline.

[0452] Illustrative examples of therapeutic antibodies suitable for combination with the CAR-modified T cells contemplated in particular embodiments include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (avastin), bivatuzumab, blinatumomab, conatumumab, crizotinib, daratumumab, duligotumab, dacetuzumab, dalotuzumab, durvalumab, elotuzumab (HuLuc63) , gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0453] In certain embodiments, the compositions described herein are administered in conjunction with cytokines. As used herein, "cytokine" means a generic term for proteins released by a cell population that act on another cell in the form of an intercellular mediator. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and β; mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor, such as NGF-β; platelet growth factor; transforming growth factor (TGF), such as TGF- α and TGF-β; insulin-like growth factor-I and II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, β and γ; colony stimulating factors (CSF), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines.

[0454] In a particular embodiment, the composition comprises CAR T cells as contemplated herein, which are cultured in the presence of a PI3K inhibitor as contemplated herein and express one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, and HLA-DR, which can be further separated by positive or negative selection techniques. In one embodiment, the composition comprises a specific T cell subset expressing one or more of the markers selected from the group consisting of: i) CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; ii) CD62L, CD127, CD197, CD38; and iii) CD62L, CD27, CD127, and CD8, which are further separated by positive or negative selection techniques. In various embodiments, the composition does not express or does not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0455] In one embodiment, expression of one or more of the markers selected from the group consisting of CD62L, CD127, CD197, and CD38 is increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 25-fold, or more, compared to a population of T cells activated and expanded in the absence of a PI3K inhibitor.

[0456] In one embodiment, the expression of one or more of the markers selected from the group consisting of CD62L, CD127, CD27 and CD8 is increased by at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times or more compared to a population of T cells activated and expanded in the absence of a PI3K inhibitor.

[0457] In one embodiment, the expression of one or more of the markers selected from the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3 is reduced by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 25-fold or more compared to a population of T cells activated and expanded in the absence of a PI3K inhibitor.

[0458] I. Target Cells and Antigens

[0459] In a particular embodiment, a genetically modified immune effector cell is provided that is redirected to a target cell, such as a cancer cell, and comprises a CAR having a binding domain that binds to CD79A on the target cell. As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade neighboring tissues.

[0460] As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibits one or more of the following: uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., infiltration and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via 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 "metastases" or "cancer metastases." Metastatic tumors contain cells that are similar to those in the original (primary) tumor.

[0461] As used herein, the term "benign" or "non-malignant" refers to a tumor that can grow but does not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0462] "Cancer cell" refers to a single cell of a cancerous growth or tissue. Cancer cells include both solid cancers and liquid cancers. "Tumor" or "tumor cell" generally refers to a swelling or lesion formed by an abnormal growth of cells, which may be benign, precancerous or malignant. Most cancers form tumors, but liquid cancers, such as leukemias, do not necessarily form tumors. For cancers that form tumors, the terms "cancer (cell)" and "tumor (cell)" are used interchangeably. The amount of tumor in an individual is the "tumor burden", which can be measured in terms of the number, volume or weight of the tumor.

[0463] The term "relapse" refers to the diagnosis of cancer return, or signs and symptoms of cancer return, after a period of improvement or remission.

[0464] "Remission" is also called "clinical remission" and includes both partial and complete remission. In a partial remission, some but not all signs and symptoms of cancer disappear. In a complete remission, all signs and symptoms of cancer disappear, but cancer may still be present in the body.

[0465] "Refractory" refers to a cancer that is resistant or unresponsive to therapy with a particular therapeutic agent. A cancer can be refractory from the outset of treatment (i.e., unresponsive to initial exposure to the therapeutic agent), or become refractory over the course of a first treatment period or during subsequent treatment periods due to the development of resistance to the therapeutic agent.

[0466] In one embodiment, the target cell expresses an antigen, eg, a target antigen that is not substantially found on the surface of other normal (desired) cells.

[0467] In some embodiments, the target cell is a hematopoietic cell, a lymphocyte, or a myeloid cell.

[0468] In certain embodiments, the target cell is part of blood, lymphatic tissue, or bone marrow tissue.

[0469] In certain embodiments, the target cell is a cancer cell or cancer stem cell expressing CD79A.

[0470] In certain embodiments, the target cell is a liquid cancer cell or a blood cancer cell expressing CD79A.

[0471] Illustrative examples of liquid or blood cancers that may be prevented, treated, or ameliorated by the compositions contemplated by particular embodiments include, but are not limited to, leukemia, lymphoma, and multiple myeloma.

[0472] Illustrative examples of cells that can be targeted by the anti-CD79A CARs encompassed in particular embodiments include, but are not limited to, cells of the following leukemias: 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.

[0473] Illustrative examples of cells that can be targeted by the compositions and methods encompassed in particular embodiments include, but are not limited to, cells of 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 lymphoblastic lymphoma, and mantle cell lymphoma; and T-cell non-Hodgkin lymphomas: mycosis fungoides, polymorphic large cell lymphoma, Sézary syndrome, and precursor T lymphoblastic lymphoma.

[0474] Illustrative examples of cells that can be targeted by the compositions and methods encompassed in particular embodiments include, but are not limited to, cells of the following multiple myeloma: overt multiple myeloma, smoldering multiple myeloma (MGUS), plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0475] In a preferred embodiment, the CD79A expressing target cells are DLBCL cancer cells.

[0476] J. Treatment methods

[0477] The genetically modified immune effector cells contemplated herein provide an improved adoptive immunotherapy approach for preventing, treating, and ameliorating cancers expressing CD79A or preventing, treating, or ameliorating at least one symptom associated with cancers expressing CD79A.

[0478] In various embodiments, the genetically modified immune effector cells contemplated herein provide an improved adoptive immunotherapy approach for increasing cytotoxicity in CD79A-expressing cancer cells in a subject or for reducing the number of CD79A-expressing cancer cells in a subject.

[0479] In a particular embodiment, the specificity of primary immune effector cells is redirected to cells expressing CD79A, such as cancer cells, by genetically modifying primary immune effector cells with CARs covered herein. In various embodiments, viral vectors are used to genetically modify immune effector cells with specific polynucleotides, and the polynucleotides encode the following CARs: anti-CD79A antigen binding domains binding CD79A polypeptides; hinge domains; transmembrane (TM) domains; short oligopeptides or polypeptide linkers connecting TM domains to the intracellular signaling domains of CAR; and one or more intracellular costimulatory signaling domains; and primary signaling domains.

[0480] In one embodiment, a class of cell therapy is provided in which T cells are genetically modified to express an anti-CD79A CAR targeting cancer cells expressing CD79A, and the CAR T cells are infused into recipients in need. The infused cells are able to kill the disease-causing cells in the recipient. Unlike antibody therapy, CAR T cells are able to replicate in vivo, resulting in long-term retention, which can lead to continued cancer therapy.

[0481] In one embodiment, anti-CD79A CAR T cells can undergo stable in vivo T cell expansion and can persist for a long time. In another embodiment, anti-CD79A CAR T cells evolve into specific memory T cells or stem cell memory T cells that can be reactivated to inhibit any additional tumor formation or growth.

[0482] In certain embodiments, compositions containing immune effector cells comprising a CAR contemplated herein are used to treat conditions associated with cancer cells or cancer stem cells expressing CD79A.

[0483] Illustrative Examples of Conditions That Can Be Treated, Prevented, or Ameliorated Using Immune Effector Cells Comprising the CARs Contemplated in Specific Embodiments

[0484] In certain embodiments, compositions comprising CAR-modified T cells contemplated herein are used to treat liquid or blood cancers.

[0485] In certain embodiments, the liquid or blood cancer is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.

[0486] 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's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, polymorphic large cell lymphoma, Sayre's 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.

[0487] 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).

[0488] In a preferred embodiment, the liquid or blood cancer is DLBCL.

[0489] In a preferred embodiment, the liquid or blood cancer is relapsed / refractory DLBCL.

[0490] In a particular embodiment, a method is provided, which comprises administering to a patient in need thereof, alone or in combination with one or more therapeutic agents, a therapeutically effective amount of an immune effector cell expressing an anti-CD79A CAR as contemplated herein or a composition comprising the same. In certain embodiments, the cell is used to treat a patient at risk of a condition associated with a cancer cell expressing CD79A. Therefore, in a particular embodiment, a method for treating or preventing or improving at least one symptom of cancer comprises administering to a subject in need thereof a therapeutically effective amount of a CAR modified cell as contemplated herein.

[0491] As used herein, the terms "individual" and "subject" are generally used interchangeably and refer to any animal that exhibits symptoms of a disease, disorder or condition that can be treated with gene therapy vectors, cell-based therapeutic agents and methods covered elsewhere herein. In a preferred embodiment, the subject includes any animal that exhibits symptoms of a disease, disorder or condition associated with cancer, which can be treated with gene therapy vectors, cell-based therapeutic agents and methods covered elsewhere herein. Suitable subjects (e.g., patients) include experimental animals (e.g., mice, rats, rabbits or guinea pigs), farm animals and livestock or pets (e.g., cats or dogs). Non-human primates are included, and preferably human patients. Typical subjects include human patients with cancers expressing CD79A, diagnosed with cancers expressing CD79A, or at risk of cancers expressing CD79A (e.g., DLBCL).

[0492] As used herein, the term "patient" refers to a subject who has been diagnosed with a particular disease, disorder or condition that can be treated with the gene therapy vectors, cell-based therapeutics and methods disclosed elsewhere herein.

[0493] As used herein, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even 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 delaying the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms.

[0494] As used herein, "prevent" and similar words, such as "prevented", "preventing", etc., refer to methods for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It 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, "prevent" 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.

[0495] As used herein, the phrase "improving at least one symptom of..." refers to reducing one or more symptoms of the disease or condition being treated in the subject. In specific embodiments, the disease or condition being treated is cancer, wherein the one or more symptoms improved include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal bloating or pain (due to enlarged abdominal organs), bone or joint pain, bone fractures, unplanned weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).

[0496] "Enhancement" or "promotion" or "increase" or "amplification" generally refers to the compositions covered herein (e.g., genetically modified T cells or vectors encoding CAR) can produce, induce or cause a greater physiological response (i.e., downstream effect) than the response caused by the vehicle or control molecule / composition. Measurable physiological responses may include, in particular, an increase in T cell expansion, activation, retention, and / or an increase in cancer cell killing ability, as understood in the art and described herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount, and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimal points in the middle and exceeding 1, such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the vehicle or control composition.

[0497] "Reduce" or "lower" or "mitigate" or "reduce" or "attenuate" generally refers to the ability of a composition encompassed herein to produce, elicit or cause a lesser physiological response (i.e., downstream effect) than the response elicited by a vehicle or control molecule / composition. A "reduced" or "reduced" amount is typically a "statistically significant" amount, and can include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimal points in between and exceeding 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a vehicle, a control composition (reference response), or a response in a particular cell lineage.

[0498] "Maintain" or "keep" or "maintenance" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of a composition encompassed 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.

[0499] 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, but the appropriate dose can be determined by clinical trials.

[0500] In one embodiment, the amount of immune effector cells (e.g., T cells) in the composition administered to a subject is at least 0.1×105 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 9 cells, at least 5×10 9 cells, or at least 1×10 10 cells.

[0501] 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.

[0502] In one embodiment, the amount of immune effector cells (e.g., T cells) 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 1×107 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.

[0503] In certain embodiments, about 1×10 6 T cells / kg body weight is about 1×10 8 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.

[0504] One 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 may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more over time intervals 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.

[0505] In certain embodiments, it may be necessary to administer activated immune effector cells to a subject, and then subsequently draw blood (or perform apheresis), activate immune effector cells therefrom, and reinfuse these activated and amplified immune effector cells to the patient. This process can be performed multiple times every few weeks. In certain embodiments, immune effector cells can be activated by drawing blood from 10cc to 400cc. In certain embodiments, immune effector cells are activated by drawing blood from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc or 400cc or more. Without being bound by theory, this multiple blood draw / multiple reinfusion scheme can be used to select certain immune effector cell colonies.

[0506] The administration of the compositions encompassed herein can be performed in any suitable manner, including by aerosol inhalation, injection, intake, infusion, implantation or transplantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrase "parenteral administration (parenteral administration / administered parenterally)" refers to the administration mode except enteral and topical administration, usually by injection and including but not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumor, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intravertebral and intrasternal injection and infusion. In one embodiment, the compositions encompassed herein are administered to the subject by direct injection into a tumor, lymph node or infection site.

[0507] In one embodiment, an effective amount of a composition is administered to a subject in need to increase a cellular immune response to a B cell-related condition in the subject. The immune response may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. The humoral immune response mediated mainly by helper T cells that can activate B cells and thus cause antibody production can also be inductive. A variety of techniques can be used to analyze the type of immune response induced by the composition, and the techniques are fully described in the art; for example, Current Protocols in Immunology, John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, New York, New York.

[0508] In the case of T cell-mediated killing, CAR-ligand binding triggers CAR signaling to T cells, resulting in activation of multiple T cell signaling pathways that induce T cells to produce or release proteins that can induce target cell apoptosis through various mechanisms. These T cell-mediated mechanisms include, but are not limited to, the transfer of intracellular cytotoxic granules from T cells to target cells; T cell secretion of proinflammatory cytokines that can directly (or indirectly via the recruitment of other killer effector cells) induce target cell killing; and the upregulation of death receptor ligands (e.g., FasL) on the surface of T cells that induce target cell apoptosis after binding to their cognate death receptors (e.g., Fas) on target cells.

[0509] In one embodiment, a method for treating a subject diagnosed with a cancer expressing CD79A is provided, comprising removing immune effector cells from a subject diagnosed with a cancer expressing CD79A, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a CAR as contemplated herein, thereby producing a modified immune effector cell colony, and administering the modified immune effector cell colony to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0510] In certain embodiments, a method of stimulating an immune effector cell-mediated immunomodulator response to a target cell population in a subject is provided, comprising the step of administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding a CAR molecule.

[0511] The method of administering the cell composition covered in a specific embodiment includes any method as follows: effectively reintroducing to the subject an ex vivo genetically modified immune effector cell that directly expresses CAR, or reintroducing a genetically modified progenitor cell of an immune effector cell, which differentiates into a mature immune effector cell expressing CAR when introduced into the subject. A method includes ex vivo transduction of peripheral blood T cells with a nucleic acid construct covered herein, and returning the transduced cells to the subject.

[0512] 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.

[0513] Although the foregoing embodiments have been described in considerable detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill 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 limiting. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results.

[0514] Examples

[0515] Example 1

[0516] Construction of anti-CD79A CAR

[0517] CAR containing humanized anti-CD79A scFv antibody is designed to contain MND promoter, hinge and transmembrane domain (from CD8α) and CD137 costimulatory domain operably connected to anti-CD79A scFv, followed by intracellular signaling domain of CD3ζ chain. Anti-CD79A CAR includes CD8α signal peptide (SP) sequence for surface expression on immune effector cells. Table 3 shows the identification, Genbank reference, source name and citation file of different nucleotide segments of exemplary anti-CD79A CAR lentiviral vectors. Exemplary CD79A CAR polypeptide sequences are described in SEQ ID NO:25-30, and exemplary CD79A CAR polynucleotide sequences are described in SEQ ID NO:31-36.

[0518] Table 3.

[0519]

[0520]

[0521] Example 2

[0522] Evaluation of human anti-CD79A CAR T cells

[0523] Chimeric antigen receptors (CARs) specific for CD79A (e.g., SEQ ID NOs: 25, 27, 29, and 30) were evaluated for CAR expression and bioactivity against cells expressing CD79A.

[0524] Target antigen expression

[0525] In one experiment, K562 cells, Pfeiffer cells, and Daudi target cells were interrogated for CD79A expression using anti-CD79A antibodies. Cells were incubated with CD79A antibodies and expression was assessed by flow cytometry. CD79A expression was undetectable on K562 cells, moderately expressed on Pfeiffer cells, and highly expressed on Daudi cells. Figure 1A .

[0526] In another experiment, CD79A expression was measured on Daudi, NU-DUL-1, SU-DHL-2, and Pfeiffer target cells using anti-CD79A antibodies. Cells were incubated with CD79A antibodies and expression was assessed by flow cytometry. CD79A expression was also highest on Daudi cells, followed by a gradual decrease on NU-DUL-1, SU-DHL-2, and Pfeiffer cells. Figure 2A .

[0527] Anti-CD79A CAR expression

[0528] CAR T cells are produced using a system that can be directly expanded to a large clinical manufacturing method. In short, peripheral blood mononuclear cells (PBMC) are cultured in a culture medium containing IL-2 (CellGenix) and antibodies (MiltenyiBiotec) specific to CD3 and CD28. The lentivirus encoding anti-CD79A CAR is added one day after the start of culture. Anti-CD79A CAR T cells are maintained in the logarithmic phase by adding fresh culture medium containing IL-2 in a total of ten days of culture. At the end of the culture, flow cytometry is used to inquire about the expression of anti-CD79A CAR T cells. In an experiment, the original human T cells engineered with anti-CD79A CAR expressing lentivirus are stained with goat anti-mouse (GaM) bound to biotin and detected with streptavidin bound to PE. Figure 1B In another experiment, anti-CD79A CAR expression on T cells was assessed with goat anti-mouse (GaM) staining, and binding of soluble CD79A antigen to anti-CD79A CAR-T cells was assessed by staining with CD79a extracellular domain-Fc fusion protein labeled with phycoerythrin (PE). Figure 2B These reagents specifically identify T cells expressing the anti-CD79A CAR.

[0529] Antigen-dependent anti-CD79A CAR T cell activity

[0530] In one experiment, an interferon-γ (IFNγ) release assay was used to evaluate the biological activity of anti-CD79A CAR T cells against CD79A-positive (Pfeiffer and Daudi) and CD79A-negative (K562) cell lines. Anti-CD79A CAR T cells were co-cultured for 24 hours in the absence of target cells or with K562 (CD79A-), Pfeiffer cells (CD79A-low), and Daudi cells (CD79A-high). Anti-CD79A CAR T cells released IFNγ only in the presence of CD79A-positive cell lines. Figure 1C .

[0531] In another experiment, interferon-γ (IFNγ) release analysis was used to evaluate the biological activity of anti-CD79A CAR T cells against CD79A positive (Huh7.CD79A, Daudi, NU-DUL-1, SU-DHL-2 and Pfeiffer) and CD79A negative (Huh7) cell lines. Anti-CD79A CAR T cells were co-cultured in the absence of target cells (T cells alone) or with Huh7 cells (CD79A-), Huh7.CD79a cells (CD79A+), Daudi cells (CD79A+), NU-DUL-1 (CD79A+), SU-DHL-2 (CD79A+) or Pfeiffer cells (CD79A+). Figure 2C .

[0532] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments and the full scope of equivalents to which the claims are entitled. Therefore, the claims should not be limited by this disclosure. Sequence Listing <110> bluebird bio, Inc. FRIEDMAN, Kevin PERKINS, Molly Reed <120> CD79A chimeric antigen receptor <130> BLBD-100 / 01WO 315698-2947 <150> US 62 / 685,078 <151> 2018-06-14 <160> 79 <170> PatentIn Version 3.5 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - light chain CDR1 <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> <221> <222> <223> Laboratory preparation - light chain CDR2 <400> 2 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - light chain CDR3 <400> 3 Phe Gln Gly Ser His Val Pro Phe Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - heavy chain CDR1 <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> <221> <222> <223> Laboratory preparation - heavy chain CDR2 <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> <221> <222> <223> Laboratory preparation - heavy chain CDR6 <400> 6 Phe Tyr Tyr Gly Asn Thr Phe Ala Met Asp Tyr 1 5 10 <210> 7 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - variable 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 Sequence <220> <221> <222> <223> Laboratory-prepared variable 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> <221> <222> <223> Laboratory preparation - Light chain CDR1 <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> <221> <222> <223> Laboratory preparation - light chain CDR2 <400> 10 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - light chain CDR3 <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> <221> <222> <223> Laboratory preparation - heavy chain CDR1 <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> <221> <222> <223> Laboratory preparation - heavy chain CDR2 <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> <221> <222> <223> Laboratory preparation - heavy chain CDR6 <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 Sequence <220> <221> <222> <223> Laboratory preparation - variable 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 Sequence <220> <221> <222> <223> Laboratory preparation - variable 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> 14 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - light chain CDR1 <400> 17 Thr Gly Thr Ser Ser Asp Phe Gly Ser Tyr Asn Leu Val Ser 1 5 10 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - light chain CDR2 <400> 18 Glu Gly Ser Lys Arg Pro Ser 1 5 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - light chain CDR3 <400> 19 Cys Ser Tyr Ala Gly Gly Ser Thr Trp Val 1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - heavy chain CDR1 <400> 20 Gly Leu Thr Phe Thr Ser Tyr Ala Met Asn 1 5 10 <210> twenty one <211> 17 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - heavy chain CDR2 <400> twenty one Gly Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> twenty two <211> 7 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - heavy chain CDR6 <400> twenty two Leu Gly Gly Trp Ser Asp Tyr 1 5 <210> 23 <211> 110 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Variable Light Chain <400> 23 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Phe Gly Ser Tyr 20 25 30 Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Gly 85 90 95 Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 24 <211> 116 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory-prepared variable heavy chain <400> twenty four Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Thr Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Gly Gly Trp Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 25 <211> 492 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti-CD79A-01_HL CAR Construct <400> 25 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> 26 <211> 492 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - anti-CD79A-01_LH CAR construct <400> 26 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> 27 <211> 493 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - anti - CD79A - 10_HL CAR construct <400> 27 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 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 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val 225 230 235 240 Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val 245 250 255 Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 340 345 350 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 355 360 365 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 370 375 380 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 385 390 395 400 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 405 410 415 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 420 425 430 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 435 440 445 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 450 455 460 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 465 470 475 480 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 28 <211> 493 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - anti-CD79A-10_LH CAR construct <400> 28 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 Val Met Thr Gln Thr 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 Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 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 Phe Cys Ser Gln Ser Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly 115 120 125 Thr Lys Leu Glu Ile Lys 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 Thr Ser Gly Tyr 165 170 175 Ala Phe Ser Phe Ser Trp Met Asn Trp Val Lys Trp Gly Pro Gly Gln 180 185 190 Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asn 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 Ala Arg Trp Val Tyr Ser Gly Asn Asn Tyr Ala 245 250 255 Val Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 340 345 350 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 355 360 365 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 370 375 380 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 385 390 395 400 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 405 410 415 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 420 425 430 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 435 440 445 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 450 455 460 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 465 470 475 480 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 29 <211> 485 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti-CD79A-02_HL CAR Construct <400> 29 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 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu 35 40 45 Thr Phe Thr Ser Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ser Gly Ile Ser Gly Ser Gly Gly Gly Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Lys Leu Gly Gly Trp Ser Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala 145 150 155 160 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly 165 170 175 Thr Ser Ser Asp Phe Gly Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln 180 185 190 His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg 195 200 205 Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr 210 215 220 Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Cys Ser Tyr Ala Gly Gly Ser Thr Trp Val Phe Gly Gly Gly 245 250 255 Thr Lys Leu Thr Val Leu Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 305 310 315 320 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 325 330 335 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 340 345 350 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 355 360 365 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 30 <211> 485 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti - CD79A - 02_LH CAR Construct <400> 30 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 Ser Ala Leu Thr Gln Pro Ala Ser Val Ser 20 25 30 Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser 35 40 45 Asp Phe Gly Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly 50 55 60 Lys Ala Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly 65 70 75 80 Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu 85 90 95 Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys 100 105 110 Ser Tyr Ala Gly Gly Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu 115 120 125 Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Thr 165 170 175 Ser Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 180 185 190 Trp Val Ser Gly Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 210 215 220 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 225 230 235 240 Tyr Cys Ala Lys Leu Gly Gly Trp Ser Asp Tyr Trp Gly Gln Gly Thr 245 250 255 Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 305 310 315 320 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 325 330 335 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 340 345 350 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 355 360 365 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 31 <211> 1479 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti-CD79A-01_HL CAR Polynucleotide Construct <400> 31 atggcacttc cagtgactgc attgttgttg cccctggccc tgctgcttca cgccgcaagg 60 ccccaggtcc agcttcagca gtccggacca gagttggtga aacctggggc atcagtcaag 120 attagctgca aggcatcagg gtacaccttc agcactagtt ggatgaattg ggtgaagcag 180 cggcctggcc aggggttgga gtggatcgga cgcatctacc caggggatgg cgatacaaac 240 tacaacggaa agtttaaagg taaggctact ttgaccgctg acaaatcatc aaacacggca 300 tatatgcaac tgtctagcct cacatctgtc gactctgccg tctacttttg cgagcgattc 360 tactatggca acacttttgc catggactac tgggggcagg ggacatcagt aacagttagt 420 tctggagggg gcggttccgg gggcggggga tccggagggg gaggcagtga tgtgctgatg 480 acccagatac cgttgtcact cccggtctca ctgggtgacc aggcaagcat cagctgcagg 540 agcagccagt ccatcgtgca ctctaacgga aatacctacc tggagtggta tctgcagaaa 600 cctggccagt cccctaagtt gttgattac aaagtttcaa accgatttag tggagttccc 660 gataggtttt ccggttccgg gagcggtaca gacttcaccc tgaagataag tcgggttgag 720 gctgaagacc tgggagtata ttattgcttt cagggcagcc acgtaccatt cacatttggg 780 agtggaacca aactggagat caaaagaacc acaacacctg ctccaaggcc ccccacacccc 840 gctccaacta tagccagcca accattgagc ctcagacctg aagcttgcag gcccgcagca 900 ggaggcgccg tccatacgcg aggcctggac ttcgcgtgtg atatttatat ttgggcccct 960 ttggccggaa catgtggggt gttgcttctc tcccttgtga tcactctgta ttgtaagcgc 1020 gggagaaaga agctcctgta catcttcaag cagcctttta tgcgacctgt gcaaaccact 1080 caggaagaag atgggtgttc atgccgcttc cccgaggagg aagaaggagg gtgtgaactg 1140 agggtgaaat tttctagaag cgccgatgct cccgcatatc agcagggtca gaatcagctc 1200 tacaatgaat tgaatctcgg caggcgagaa gagtacgatg ttctggacaa gagacggggc 1260 agggatcccg agatgggggg aaagccccgg agaaaaaatc ctcaggaggg gttgtacaat 1320 gagctgcaga aggacaagat ggctgaagcc tatagcgaga tcggaatgaa aggcgaaaga 1380 cgcagaggca aggggcatga cggtctgtac cagggtctct ctacagccac caaggacact 1440 tatgatgcgt tgcatatgca agccttgcca ccccgctaa 1479 <210> 32 <211> 1479 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti - CD79A - 01_LH CAR Polynucleotide Construct <400> 32 atggctctgc ctgtgacggc cctgcttttg cccctcgccc tgcttctgca tgccgcgaga 60 cccgacgtgt tgatgaccca aataccgctt agtctgcctg tatctcttgg ggaccaggct 120 agcatctcat gccgcagcag tcaatccatt gtgcactcaa atgggaacac ctatttggag 180 tggtatctgc aaaaaccggg acagtctccg aaactgctga tatacaaagt aagcaacagg 240 ttcagcggag ttcctgacag attcagcgga agcggttctg gaactgactt tacacttaag 300 atctctcgcg ttgaggcgga ggacctgggc gtgtattact gttttcaagg atcccacgtc 360 ccgtttacat tcggatcagg caccaagctg gagatcaagc gcggtggcgg gggttctggc 420 ggggggcggat ccggaggcgg cggatcccag gtgcagctgc agcagtctgg accagaactg 480 gttaagcccg gagcttcagt taagatttcc tgtaaggctt caggctatac attttccact 540 tcttggatga actgggtgaa acagcgccct ggccaggggc tggaatggat cggacggatc 600 tatcccggcg atggagacac taattataac ggtaagttca aagggaaggc caccctcacg 660 gccgacaagt cctccaatac agcgtacatg caactcagtt ccctgaccag cgttgatagc 720 gcagtttact tctgtgagcg cttctattac ggaaacacct tcgctatgga ttactggggt 780 caggggacct ccgtgaccgt gtcctctacc acaacacctg ctccaaggcc ccccacaccc 840 gctccaacta tagccagcca accattgagc ctcagacctg aagcttgcag gcccgcagca 900 ggaggcgccg tccatacgcg aggcctggac ttcgcgtgtg atatttatat ttgggcccct 960 ttggccggaa catgtggggt gttgcttctc tcccttgtga tcactctgta ttgtaagcgc 1020 gggagaaaga agctcctgta catcttcaag cagcctttta tgcgacctgt gcaaaccact 1080 caggaagaag atgggtgttc atgccgcttc cccgaggagg aagaaggagg gtgtgaactg 1140 agggtgaaat tttctagaag cgccgatgct cccgcatatc agcagggtca gaatcagctc 1200 tacaatgaat tgaatctcgg caggcgagaa gagtacgatg ttctggacaa gagacggggc 1260 agggatcccg agatgggggg aaagccccgg agaaaaaatc ctcaggaggg gttgtacaat 1320 gagctgcaga aggacaagat ggctgaagcc tatagcgaga tcggaatgaa aggcgaaaga 1380 cgcagaggca aggggcatga cggtctgtac cagggtctct ctacagccac caaggacact 1440 tatgatgcgt tgcatatgca agccttgcca ccccgctaa 1479 <210> 33 <211> 1482 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti-CD79A-10_HL CAR Polynucleotide Construct <400> 33 atggctctgc cagtgactgc gctgctgctg cccctcgctc ttctgctgca cgccgctcgg 60 ccacaggtcc aactgcaaca gagcggcccc gagctggtaa aacccggggc ctccgtaaaa atatcctgca agaccagcgg ctatgccttt tcattctcct ggatgaactg ggtgaagtgg ggacccggtc agggacttga gtggatcggg cgaatctatc ccggaaacgg ggacacgaat 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300 360. tacatgcagt tgtcctcttt gacatcagta gactctgcag tgtatttttg cgcccggtgg gtttactccg gaataacta cgcggttgac tattggggac agggcacctc cgtgacagtg 420 tcttctggcg gcgggggatc aggtggcggc gggtctgggg gtggagggag cgacgtggtt 480 atgacccaga cccctttgag cctgcccgtg agccttggcg accaagcctc catctcttgc 540 cggtcctctc aatcactggt gcacagtaac gggaatactt atctccactg gtatttgcaa aagcccggtc agtctcctaa gcttctgatc tataaggtgt ccaaccgctt ttctggagtg cccgagat tttccggatc tggatccgga acagacttca cattgaagat tagtaggtc gaagcggagg acttgggtgt ttttttgt tcccagagta cccacgtgcc tccatacacg 780 tttggaggtg gaaccaaact tgaaattaag accacaacac ctgctccaag gccccccaca 840 cccgctccaa ctatagccag ccaaccattg agcctcagac ctgaagcttg caggcccgca 900 gcaggaggcg ccgtccatac gcgaggcctg gacttcgcgt gtgatattta tatttgggcc 960 cctttggccg gaacatgtgg ggtgttgctt ctctcccttg tgatcactct gtattgtaag 1020 cgcgggagaa agaagctcct gtacatcttc aagcagcctt ttatgcgacc tgtgcaaacc 1080 actcaggaag aagatgggtg ttcatgccgc ttccccgagg aggaagaagg agggtgtgaa 1140 ctgagggtga aattttctag aagcgccgat gctcccgcat atcagcaggg tcagaatcag 1200 ctctacaatg aattgaatct cggcaggcga gaagtacg atgttctgga caagacgg 1260 ggcagggatc ccgagatggg gggaaagccc cggagaaaaa atcctcagga ggggttgtac 1320 aatgagctgc agaaggacaa gatggctgaa gcctatagcg agatcggaat gaaaggcgaa 1380 agacgcagag gcaaggggca tgacggtctg taccagggtc tctctacagc caccaaggac 1440 acttatgatg cgttgcatat gcaagccttg ccaccccgct aa 1482 <210> 34 <211> 1482 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti-CD79A-10_LH CAR Polynucleotide Construct <400> 34 atggccctgc cagtaacagc actgctcctt cctctcgcac tcctcctgca cgcagcaaga 60 ccagatgtcg tgatgactca gacacctctt agcttgcccg tctccttggg tgatcaggct 120 agcatttcat gtagatcaag ccagagcttg gtacattcaa atggcaatac ctacctgcac 180 tggtatctcc agaaacctgg ccagagccca aaattgctta tctataaagt tagtaacaga 240 ttctccgggg tcccagatcg attttccggc tcaggctcag gcacggattt taccctgaag 300 atctcacgag tcgaggcaga agatcttggc gtgtactttt gttcccagag tacccacgta 360 cccccttaca cattcggagg agggaccaag ttggagatca aaggaggggg gggcagtggg 420 gggggcggtt ccggaggggg aggaagccaa gtacaactgc aacaaagcgg acccgagctc 480 gtcaaacctg gtgctagcgt caaaatttcc tgtaagacca gcggctacgc ttttagcttt 540 tcttggatga actgggtgaa atggggccg gggcagggcc tggagtggat aggacgaatt 600 tatcccggga atgggggatac aaattacaat ggtaaattca aggggaaagc caactgact 660 gcagataaaa gctccaacac ggcatacatg cagctcagct ccctcacttc tgtggattct 720 gctgtttact tttgtgccag gtgggtttac tctggtaata actatgccgt ggattactgg 780 gggcaaggca cctctgttac agtgagttcc accacaacac ctgctccaag gccccccaca 840 cccgctccaa ctatagccag ccaaccattg agcctcagac ctgaagcttg caggcccgca 900 gcaggaggcg ccgtccatac gcgaggcctg gacttcgcgt gtgatattta tatttgggcc 960 cctttggccg gaacatgtgg ggtgttgctt ctctcccttg tgatcactct gtattgtaag 1020 cgcgggagaa agaagctcct gtacatcttc aagcagcctt ttatgcgacc tgtgcaaacc 1080 actcaggaag aagatgggtg ttcatgccgc ttccccgagg aggaagaagg agggtgtgaa 1140 ctgagggtga aattttctag aagcgccgat gctcccgcat atcagcaggg tcagaatcag 1200 ctctacaatg aattgaatct cggcaggcga gaagtacg atgttctgga caagacgg 1260 ggcagggatc ccgagatggg gggaaagccc cggagaaaaa atcctcagga ggggttgtac 1320 aatgagctgc agaaggacaa gatggctgaa gcctatagcg agatcggaat gaaaggcgaa 1380 agacgcagag gcaaggggca tgacggtctg taccagggtc tctctacagc caccaaggac 1440 acttatgatg cgttgcatat gcaagccttg ccaccccgct aa 1482 <210> 35 <211> 1458 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti-CD79A-02_HL CAR Polynucleotide Construct <400> 35 atggcgcttc ctgtcactgc cctgcttctg cccctcgctc ttctgctgca cgccgctcgc 60 cccgaagttc agctgttgga aagcggaggc ggcctggttc aacccggtgg cagtctgagg 120 ctgtcctgcg ccgcctccgg tcttacattc acatcttacg ctatgaattg ggtgcgccaa 180 gcccccggca agggcctgga gtgggttagt ggtatcagcg ggtccggagg agggacttac 240 tacgccgact ctgtaaaggg acggttcact atctcccggg acaactccaa aaacacactt 300 tacctgcaga tgaactcctt gcgagccgaa gacacagccg tgtactactg tgctaagctt 360 ggaggctgga gtgattattg gggtcagggg accttggtga cagtgtcttc cgggggtggg 420 ggatccggtg gcggtggatc aggagggggt ggctcccaga gcgctctcac acagccggcc 480 agtgtgtcag gatcacctgg acagtccatt acgatctctt gcactggaac aagctctgac 540 tttggctcct ataatctcgt gtcctggtat cagcagcacc ccgggaaggc tcccaagctt 600 atgatttacg agggctcaaa acgcccttca ggtgtatcca accgattcag cggaagcaaa 660 tccggcaata ccgcaagtct caccataagt ggcctgcagg cagaagacga agccgactac 720 tattgttgtt cttatgccgg cggctcaaca tgggtgttcg gcgggggcac caagctgact 780 gtattgacca caacacctgc tccaaggccc cccacacccg ctccaactat agccagccaa 840 ccattgagcc tcagacctga agcttgcagg cccgcagcag gaggcgccgt ccatacgcga 900 ggcctggact tcgcgtgtga tatttatatt tgggcccctt tggccggaac atgtggggtg 960 ttgcttctct cccttgtgat cactctgtat tgtaagcgcg ggagaaagaa gctcctgtac 1020 atcttcaagc agccttttat gcgacctgtg caaaccactc aggaagaaga tgggtgttca 1080 tgccgcttcc ccgaggagga agaaggaggg tgtgaactga gggtgaaatt ttctagaagc 1140 gccgatgctc ccgcatatca gcagggtcag aatcagctct acaatgaatt gaatctcggc 1200 aggcgagaag agtacgatgt tctggacaag agacggggca gggatcccga gatgggggga 1260 aagccccgga gaaaaaatcc tcaggagggg ttgtacaatg agctgcagaa ggacaagatg 1320 gctgaagcct atagcgagat cggaatgaaa ggcgaaagac gcagaggcaa ggggcatgac 1380 ggtctgtacc agggtctctc tacagccacc aaggacactt atgatgcgtt gcatatgcaa 1440 gccttgccac cccgctaa 1458 <210> 36 <211> 1458 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Laboratory Preparation - Anti - CD79A - 02_LH CAR Polynucleotide Construct <400> 36 atggctctgc ctgtaactgc tctgctgctt ccccttgctc tgctcctgca tgccgcccgg 60 ccgcaaagcg ccctgactca gccagcttcc gtgtctggct ctccggggca gtccatcacc 120 atctcatgca caggaacctc cagcgacttc ggatcatata acctggtttc atggtatcag 180 cagcatcctg ggaaggctcc gaagttgatg atctacgagg gaagcaagag gcccagtggg 240 gtcagtaacc gcttctctgg aagtaagtca ggcaataccg ctagtctgac catctccgga 300 ttgcaagctg aggacgaagc tgattactac tgctgttcat atgcagggg ttctacttgg 360 gtgttcggcg gtggaaccaa gcttaccgtt ctgggaggcg gaggctccgg gggaggcgga 420 tcaggcggag gtggatccga agtgcagctg cttgagtcag gagggggatt ggtccagccg 480 gggggatcat tgaggctgtc ttgtgccgca agtggactca cttttaccag ttatgcaatg 540 aactgggtgc ggcaggcacc cgggaaggga ctggaatggg tgagtggcat atctggcagc 600 ggggggcggaa cttactacgc tgattctgtc aagggaaggt ttactatctc acgagacaac 660 agcaaaaaca ccctctacct gcagatgaac tcactcagag cagaggatac tgcagtctac 720 tattgtgcta agctgggtgg ttggagcgac tactggggac aagggacgtt ggtgaccgtg 780 tcttcaacca caacacctgc tccaaggccc cccacacccg ctccaactat agccagccaa 840 ccattgagcc tcagacctga agcttgcagg cccgcagcag gaggcgccgt ccatacgcga 900 ggcctggact tcgcgtgtga tatttatatt tgggcccctt tggccggaac atgtggggtg 960 ttgcttctct cccttgtgat cactctgtat tgtaagcgcg ggagaaagaa gctcctgtac 1020 atcttcaagc agccttttat gcgacctgtg caaaccactc aggaagaaga tgggtgttca 1080 tgccgcttcc ccgaggagga agaaggaggg tgtgaactga gggtgaaatt ttctagaagc 1140 gccgatgctc ccgcatatca gcagggtcag aatcagctct acaatgaatt gaatctcggc 1200 aggcgagaag agtacgatgt tctggacaag agacggggca gggatcccga gatgggggga 1260 aagccccgga gaaaaaatcc tcaggagggg ttgtacaatg agctgcagaa ggacaagatg 1320 gctgaagcct atagcgagat cggaatgaaa ggcgaaagac gcagaggcaa ggggcatgac 1380 ggtctgtacc agggtctctc tacagccacc aaggacactt atgatgcgtt gcatatgcaa 1440 gccttgccac cccgctaa 1458 <210> 37 <211> 226 <212> PRT <213> Homo sapiens <400> 37 Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile Gln Asn Val 85 90 95 Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val Gln Glu Gly Asn 100 105 110 Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro 115 120 125 Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn Arg Ile 130 135 140 Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly 145 150 155 160 Thr Leu Leu Leu Phe Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu 165 170 175 Asp Ala Gly Asp Glu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190 Leu Asp Asp Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205 Thr Tyr Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215 220 Lys Pro 225 <210> 38 <211> 188 <212> PRT <213> Homo sapiens <400> 38 Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Glu Pro Pro Pro Arg Pro Phe Leu 85 90 95 Asp Met Gly Glu Gly Thr Lys Asn Arg Ile Ile Thr Ala Glu Gly Ile 100 105 110 Ile Leu Leu Phe Cys Ala Val Val Pro Gly Thr Leu Leu Leu Phe Arg 115 120 125 Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp Glu Tyr 130 135 140 Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser Met 145 150 155 160 Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr Gln Asp Val Gly 165 170 175 Ser Leu Asn Ile Gly Asp Val Gln Leu Glu Lys Pro 180 185 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequence <400> 39 Asp Gly Gly Gly Ser 1 5 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 40 Thr Gly Glu Lys Pro 1 5 <210> 41 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 41 Gly Gly Arg Arg 1 <210> 42 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 42 Gly Gly Gly Gly Ser 1 5 <210> 43 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 43 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp 1 5 10 <210> 44 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 44 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 45 Gly Gly Arg Arg Gly Gly Gly Ser 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 46 Leu Arg Gln Arg Asp Gly Glu Arg Pro 1 5 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 47 Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro 1 5 10 <210> 48 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 48 Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro 1 5 10 15 <210> 49 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Linker Sequences <400> 49 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Cleavage sequence by TEV protease <220> <221> SITE <222> (2)..(3) <223> Xaa is any amino acid <220> <221> SITE <222> (5) <223> Xaa is any amino acid <220> <221> SITE <222> (7) <223> Xaa = Gly or Ser <400> 50 Glu Xaa Xaa Tyr Xaa Gln Xaa 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Cleavage sequence by TEV protease <400> 51 Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> 52 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Cleavage sequence by TEV protease <400> 52 Glu Asn Leu Tyr Phe Gln Ser 1 5 <210> 53 <211> twenty two <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Self-cleaving peptide containing 2A site <400> 53 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 54 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Self-cleaving peptide containing 2A site <400> 54 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 55 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Self-cleaving peptide containing 2A site <400> 55 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 1 5 10 <210> 56 <211> ...

Claims

1. A chimeric antigen receptor (CAR), comprising: an extracellular domain, wherein the CAR comprises: a) an anti-CD79A antibody or an antigen-binding fragment thereof that binds to one or more epitopes of a human CD79A polypeptide, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising a CDRL1-CDRL3 sequence set forth in SEQ ID NOs: 1-3, and a variable heavy chain sequence comprising a CDRH1-CDRH3 sequence set forth in SEQ ID NOs: 4-6; b) transmembrane domain from CD8α; c) an intracellular co-stimulatory signaling domain from CD137; and d) the primary signaling domain from CD3ζ, The variable heavy chain of the anti-CD79A antibody or antigen-binding fragment thereof is located at the C-terminus of the variable light chain.

2. The CAR according to claim 1, wherein the anti-CD79A antibody or antigen-binding fragment that binds to the human CD79A polypeptide is selected from the group consisting of: Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein ("scFv"), dual scFv and (scFv)2.

3. The CAR of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment that binds to the human CD79A polypeptide is selected from the group consisting of: miniantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and disulfide-stabilized Fv proteins ("dsFv").

4. The CAR of claim 1, wherein the anti-CD79A antibody or antigen-binding fragment that binds to the human CD79A polypeptide is a scFv.

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

8.

6. The CAR of claim 1, further comprising a hinge region polypeptide.

7. The CAR of claim 6, wherein the hinge region polypeptide comprises the hinge region of CD8α.

8. The CAR of claim 1, further comprising a spacer region.

9. The CAR according to claim 1, further comprising a signal peptide.

10. The CAR of claim 9, wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor α signal polypeptide.

11. The CAR of claim 1, comprising the amino acid sequence as set forth in SEQ ID NO:

26.

12. A polypeptide comprising the amino acid sequence of the CAR according to any one of claims 1 to 11.

13. A polynucleotide encoding the CAR according to any one of claims 1 to 11.

14. A polynucleotide comprising the sequence as set forth in SEQ ID NO:

32.

15. A vector comprising the polynucleotide according to claim 13 or 14. The vector according to claim 15 , wherein the vector is an expression vector. The vector according to claim 15 , wherein the vector is an episomal vector.

18. The vector according to claim 15, wherein the vector is a viral vector.

19. The vector according to claim 15, wherein the vector is a retroviral vector.

20. The vector according to claim 15, wherein the vector is a lentiviral vector.

21. The vector of claim 15, wherein the vector is a lentiviral vector selected from the group consisting of human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2); Vesna-Medievirus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

22. The vector of claim 15, comprising a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element; a promoter operably linked to a polynucleotide of claim 13 or 14; and a right (3') retroviral LTR.

23. The vector of claim 15, further comprising a heterologous polyadenylation sequence.

24. The vector of claim 15, further comprising a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE).

25. The vector according to claim 22, wherein the promoter of the 5'LTR is replaced with a heterologous promoter.

26. The vector of claim 25, wherein the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40) promoter.

27. The vector of claim 22, wherein the 5'LTR or 3'LTR is a lentiviral LTR.

28. The vector of claim 22, wherein the 3'LTR comprises one or more modifications.

29. The vector of claim 22, wherein the 3'LTR comprises one or more deletions.

30. The vector of claim 22, wherein the 3' LTR is a self-inactivating (SIN) LTR.

31. The vector of claim 22, wherein the promoter operably linked to the polynucleotide of claim 13 or 14 is selected from the group consisting of: cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF1-α), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer / chicken β-actin promoter (CAG), polyoma enhancer / herpes simplex thymidine kinase promoter (MC1), β-actin promoter (β-ACT), simian virus 40 promoter (SV40) and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site replaced (MND) U3 promoter.

32. An immune effector cell comprising the vector according to any one of claims 15 to 31.

33. The immune effector cell of claim 32, wherein the immune effector cell is selected from the group consisting of: T lymphocytes, natural killer (NK) cells, and NKT cells.

34. A composition comprising the immune effector cells according to claim 32 or 33 and a physiologically acceptable excipient.

35. A method for producing an immune effector cell colony comprising a CAR according to any one of claims 1 to 11, comprising introducing a polynucleotide according to claim 13 or 14, or a vector according to any one of claims 15 to 31 into the immune effector cell colony.

36. The method of claim 35, further comprising stimulating immune effector cells by contacting the cells with an antibody that binds CD3 and an antibody that binds CD28 and inducing proliferation of the cells; thereby producing an expanded population of immune effector cells.

37. The method of claim 35, wherein the immune effector cells comprise T cells, NK cells and / or NKT cells.

38. The method of claim 37, wherein the immune effector cells comprise T cells.

39. Use of the composition of claim 34 in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the cancer is Burkitt's lymphoma or diffuse large B-cell lymphoma.

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