CD79A chimeric antigen receptor

Abstract

To provide improved compositions for adoptive cell therapies for cancers expressing CD79A.SOLUTION: Provided herein is a chimeric antigen receptor (CAR) comprising an extracellular domain that comprises: a) an anti-CD79A antibody or antigen binding fragment thereof that binds one or more epitopes of human CD79A polypeptide, the antibody comprising a variable light chain sequence provided with CDRL1, CDRL2, and CDRL3 of specific sequences respectively and / or a variable heavy chain sequence provided with CDRH1, CDRH2, and CDRH3 of specific sequences respectively; and b) a transmembrane domain; c) one or more intracellular co-stimulatory signaling domain; and / or d) a primary signaling domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 685,078, filed June 14, 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Description The sequence listing associated with this application has been provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named BLBD_100_01WO_ST25.txt. The text file is 64kB in size, was created on June 14, 2019, and was submitted electronically via EFS-Web concurrently with the filing of the specification.

[0003] The present invention relates to improved compositions and methods for treating cancer. More particularly, the present invention relates to improved anti-CD79A chimeric antigen receptors (CARs), genetically modified immune effector cells, and the use of these compositions to effectively treat cancers that express CD79A. [Background technology]

[0004] Cancer is a serious health problem worldwide. Based on rates calculated between 2008 and 2010, 40.76% of men and women born today will be diagnosed with some form of cancer at some point in their lifetime. The cancer incidence rate between the ages of 50 and 69 is estimated to be 15.30% for women and 20.37% for men. In the United States, the number of currently alive men and women who have had cancer as of January 1, 2010, was approximately 13,027,914, including 6,078,974 men and 6,948,940 women. In 2013, an estimated 1,660,290 men and women (854,790 men and 805,500 women) in the United States were diagnosed with cancer at any site, and 580,350 men and women died from cancer. See Howlader et al. 2013.

[0005] Malignant transformation of B cells leads to cancer, including, but not limited to, lymphomas such as multiple myeloma and non-Hodgkin's lymphoma. The majority of patients suffer from B-cell malignancies, such as non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), resulting in significantly increased cancer mortality. B-cell malignancies respond differently to various forms of treatment. Conventional treatments for B-cell malignancies, including chemotherapy and radiation therapy, have limited efficacy due to adverse side effects. Immunotherapy using therapeutic antibodies, such as anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD52, anti-CD80, and anti-HLA-DR, has also met with limited success. This is due to poor pharmacokinetic profiles, rapid clearance of antibodies by serum proteases and glomerular filtration, insufficient tumor penetration, and insufficient expression levels of target antigens in cancer cells. Attempts to use genetically engineered cells expressing chimeric antigen receptors (CARs) have met with limited success. Furthermore, the therapeutic efficacy of the specific antigen-binding domain used in a particular CAR can be unpredictable. If the antigen-binding domain binds too strongly, the CAR-T cells may induce massive cytokine release, potentially triggering a potentially fatal immune response known as a "cytokine storm." If the antigen-binding domain binds too weakly, the CAR-T cells may not be sufficiently effective in eliminating cancer cells. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides improved vectors for generating adoptive cellular therapy agents and methods of using same. More specifically, the present invention provides anti-CD79ACAR molecules and their use in treating, preventing, or ameliorating cancers that express CD79A.

[0007] In various embodiments, a chimeric antigen receptor (CAR) is provided, comprising: a) an extracellular domain comprising an anti-CD79A antibody or antigen-binding fragment thereof that binds 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 having a CDRL1 sequence through a CDRL3 sequence set forth in SEQ ID NO:1-3, SEQ ID NO:9-11, or SEQ ID NO:17-19, and / or a variable heavy chain sequence having a CDRH1 sequence through a CDRH3 sequence set forth in SEQ ID NO:4-6, SEQ ID NO:12-14, or SEQ ID NO:20-22; b) a transmembrane domain; c) one or more intracellular costimulatory signaling domains; and / or d) a primary signaling domain.

[0008] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment that binds a 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), an Fv, a single-chain Fv protein ("scFv"), a bis-scFv (bis-scFv), an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0009] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment that binds a human CD79A polypeptide is an scFv.

[0010] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs set forth in any one of SEQ ID NOs:1-3 and / or one or more heavy chain CDRs set forth in any one of SEQ ID NOs:4-6.

[0011] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs set forth in any one of SEQ ID NOs:9-11 and / or one or more heavy chain CDRs set forth in any one of SEQ ID NOs:12-14.

[0012] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs set forth in any one of SEQ ID NOs: 17-19 and / or one or more heavy chain CDRs set forth in any one of SEQ ID NOs: 20-22.

[0013] In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:23, and / or a variable heavy chain sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:16, or SEQ ID NO:24.

[0014] In certain embodiments, an 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 NO:7, SEQ ID NO:15, or SEQ ID NO: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 NO:8, SEQ ID NO:16, or SEQ ID NO:24.

[0015] In a further embodiment, the anti-CD79A antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:7 and / or the variable heavy chain sequence set forth in SEQ ID NO:8.

[0016] In a further embodiment, the anti-CD79A antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:15 and / or the variable heavy chain sequence set forth in SEQ ID NO:16.

[0017] In a further embodiment, the anti-CD79A antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:23 and / or the variable heavy chain sequence set forth in SEQ ID NO:24.

[0018] In further embodiments, the transmembrane domain is derived from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1.

[0019] In another embodiment, the transmembrane domain is derived from a polypeptide selected from the group consisting of CD8α, CD28, CD4, CD45, PD1, and CD152.

[0020] In one embodiment, the transmembrane domain is from CD8α.

[0021] In further embodiments, the one or more costimulatory signaling domains are derived from a costimulatory molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

[0022] In certain embodiments, the one or more costimulatory signaling domains are derived from a costimulatory molecule selected from the group consisting of CD28, CD134, and CD137.

[0023] In certain embodiments, the one or more costimulatory signaling domains are derived from CD137.

[0024] 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.

[0025] In certain embodiments, the primary signaling domain is isolated from CD3ζ.

[0026] In another embodiment, the CAR further comprises a hinge region polypeptide.

[0027] In certain embodiments, the hinge region polypeptide comprises the hinge region of CD8α.

[0028] In another embodiment, the CAR further comprises a spacer region.

[0029] In a further embodiment, the CAR further comprises a signal peptide.

[0030] In certain embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide.

[0031] In certain embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NO:25 to SEQ ID NO:30.

[0032] In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:25.

[0033] In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:26.

[0034] In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:27.

[0035] In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:28.

[0036] In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:29.

[0037] In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:30.

[0038] In various embodiments, a polypeptide is provided that comprises the amino acid sequence of a CAR discussed herein.

[0039] In various embodiments, a polynucleotide encoding a CAR discussed herein is provided.

[0040] In certain embodiments, a polynucleotide encoding a CAR discussed herein comprises the sequence set forth in any one of SEQ ID NO:31 to SEQ ID NO:36.

[0041] In various embodiments, a vector is provided that comprises a polynucleotide encoding a CAR discussed herein.

[0042] In certain embodiments, the vector is an expression vector.

[0043] In certain embodiments, the vector is an episomal vector.

[0044] In a further embodiment, the vector is a viral vector.

[0045] In a further embodiment, the vector is a retroviral vector.

[0046] In certain embodiments, the vector is a lentiviral vector.

[0047] 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), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0048] In certain embodiments, the vector comprises a left (5') retroviral LTR, a psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral transport element, a promoter operably linked to the aforementioned polynucleotide, and a right (3') retroviral LTR.

[0049] In a further embodiment, the vector further comprises a heterologous polyadenylation sequence.

[0050] In certain embodiments, the vector further comprises a Hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE).

[0051] In a further embodiment, the promoter of the 5'LTR is replaced with a heterologous promoter.

[0052] In further embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40) promoter.

[0053] In certain embodiments, the 5'LTR or 3'LTR is a lentiviral LTR.

[0054] In certain embodiments, the 3'LTR comprises one or more modifications.

[0055] In certain embodiments, the 3'LTR comprises one or more deletions.

[0056] In certain embodiments, the 3'LTR is a self-inactivating (SIN) LTR.

[0057] In certain embodiments, the polyadenylation sequence is the bovine growth hormone polyadenylation or the signal rabbit beta globin polyadenylation sequence.

[0058] In a further embodiment, the polynucleotide comprises an optimized Kozak sequence.

[0059] In further embodiments, the promoter operably linked to the polynucleotide is selected from the group consisting of a cytomegalovirus immediate early gene promoter (CMV), an elongation factor 1 alpha promoter (EF1-α), a phosphoglycerate kinase 1 promoter (PGK), a ubiquitin C promoter (UBQ-C), a cytomegalovirus enhancer / chicken beta-actin promoter (CAG), a polyoma enhancer / herpes simplex thymidine kinase promoter (MC1), a beta-actin promoter (β-ACT), a simian virus 40 promoter (SV40), and a dl587rev primer binding site substitution (MND) promoter of a myeloproliferative sarcoma virus enhancer with deleted negative regulatory regions.

[0060] In various embodiments, immune effector cells are provided that comprise a vector encoding a CAR discussed herein.

[0061] In certain embodiments, the immune effector cells are selected from the group consisting of T lymphocytes, natural killer (NK) cells, and NKT cells.

[0062] In one embodiment, immune effector cells are transduced with a vector discussed herein and activated and stimulated in the presence of an inhibitor of the PI3K pathway, resulting in preserved proliferation of the transduced immune effector cells compared to proliferation of transduced immune effector cells activated and stimulated in the absence of the inhibitor of the PI3K pathway.

[0063] In certain embodiments, immune effector cells activated and stimulated in the presence of an inhibitor of the PI3K pathway 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 following markers: CD62L, CD127, CD197, and CD38, compared to immune effector cells activated and stimulated in the absence of an inhibitor of the PI3K pathway.

[0064] In certain embodiments, immune effector cells activated and stimulated in the presence of an inhibitor of the PI3K pathway 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 following markers: CD62L, CD127, CD27, and CD8, compared to immune effector cells activated and stimulated in the absence of an inhibitor of the PI3K pathway.

[0065] In one embodiment, the PI3K inhibitor is ZSTK474.

[0066] In various embodiments, compositions are provided that include the immune effector cells discussed herein and a physiologically acceptable excipient.

[0067] In various embodiments, provided is a method of generating a population of immune effector cells comprising a CAR as discussed herein, the method comprising introducing a vector encoding a CAR as discussed herein into the population of immune effector cells.

[0068] In certain embodiments, the method further comprises contacting immune effector cells with an antibody that binds CD3 and an antibody that binds CD28 to stimulate the cells and induce proliferation of the cells, thereby expanding the immune effector cell population.

[0069] In certain embodiments, immune effector cells are stimulated and induced to proliferate prior to introducing the vector.

[0070] In a further embodiment, the immune effector cell population comprises T cells.

[0071] In one embodiment, the immune effector cell population comprises NK cells.

[0072] In certain embodiments, the cells are activated and stimulated in the presence of an inhibitor of the PI3K pathway, which results in preserved proliferation of the transduced immune effector cells compared to proliferation of immune effector cells activated and stimulated in the absence of the inhibitor of the PI3K pathway.

[0073] In one embodiment, immune effector cells activated and stimulated in the presence of an inhibitor of the PI3K pathway 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 following markers: CD62L, CD127, CD197, and CD38, compared to immune effector cells activated and stimulated in the absence of an inhibitor of the PI3K pathway.

[0074] In certain embodiments, immune effector cells activated and stimulated in the presence of an inhibitor of the PI3K pathway 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 following markers: CD62L, CD127, CD27, and CD8, compared to immune effector cells activated and stimulated in the absence of an inhibitor of the PI3K pathway.

[0075] In one embodiment, the PI3K inhibitor is ZSTK474.

[0076] In various embodiments, methods are provided for increasing the cytotoxicity of CD79A-expressing cancer cells in a subject, the methods comprising administering to the subject a composition as discussed herein in an amount sufficient to increase the cytotoxicity of the CD79A-expressing cancer cells compared to the cytotoxicity of the CD79A-expressing cancer cells prior to administration.

[0077] In various embodiments, methods are provided for reducing the number of CD79A-expressing cancer cells in a subject, the methods comprising administering to the subject a composition as discussed herein, in an amount sufficient to reduce the number of CD79A-expressing cancer cells compared to the number of CD79A-expressing cancer cells prior to administration.

[0078] In various embodiments, there is provided a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition discussed herein.

[0079] In certain embodiments, the cancer is a liquid cancer.

[0080] In certain embodiments, the cancer is a hematological malignancy.

[0081] 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).

[0082] In certain 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).

[0083] 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 myelogenous leukemia (CML).

[0084] In certain embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0085] In certain embodiments, the cancer is multiple myeloma (MM) selected from the group consisting of overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0086] In various embodiments, methods are provided for ameliorating one or more symptoms associated with a CD79A-expressing cancer in a subject, the methods comprising administering to the subject a composition as discussed herein, in an amount sufficient to ameliorate at least one symptom associated with the CD79A-expressing cancer cells.

[0087] In certain embodiments, the one or more symptoms that are improved are selected from the group consisting of weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain, bone or joint pain, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination. The present invention provides, for example, the following items. (Item 1) a) an extracellular domain comprising an anti-CD79A antibody or antigen-binding fragment thereof that binds 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 having a CDRL1 sequence to CDRL3 sequence set forth in SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:9 to SEQ ID NO:11, or SEQ ID NO:17 to SEQ ID NO:19, and a variable heavy chain sequence having a CDRH1 sequence to CDRH3 sequence set forth in SEQ ID NO:4 to SEQ ID NO:6, SEQ ID NO:12 to SEQ ID NO:14, or SEQ ID NO:20 to SEQ ID NO:22; and b) a transmembrane domain; c) one or more intracellular costimulatory signaling domains; and d) a primary signaling domain; and A chimeric antigen receptor (CAR) comprising: (Item 2) 2. The CAR of item 1, wherein the anti-CD79A antibody or antigen-binding fragment that binds 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), an Fv, a single-chain Fv protein ("scFv"), a bis-scFv (bis-scFv), an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single-domain antibody (sdAb, nanobody). (Item 3) 3. The CAR of item 1 or item 2, wherein the anti-CD79A or antigen-binding fragment that binds the human CD79A polypeptide is an scFv. (Item 4) 4. The CAR of any one of items 1 to 3, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs set forth in any one of SEQ ID NOs: 1 to 3 and / or one or more heavy chain CDRs set forth in any one of SEQ ID NOs: 4 to 6. (Item 5) Item 4. The CAR of any one of Items 1 to 3, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs set forth in any one of SEQ ID NOs: 9 to 11 and / or one or more heavy chain CDRs set forth in any one of SEQ ID NOs: 12 to 14. (Item 6) Item 4. The CAR of any one of Items 1 to 3, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises one or more light chain CDRs set forth in any one of SEQ ID NOs: 17 to 19 and / or one or more heavy chain CDRs set forth in any one of SEQ ID NOs: 20 to 22. (Item 7) 6. The CAR of any one of items 1 to 5, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in any one of SEQ ID NO: 7, SEQ ID NO: 15, or SEQ ID NO: 23, and / or a variable heavy chain sequence set forth in any one of SEQ ID NO: 8, SEQ ID NO: 16, or SEQ ID NO: 24. (Item 8) 8. The CAR of any one of items 1 to 7, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in SEQ ID NO: 7 and / or a variable heavy chain sequence set forth in SEQ ID NO: 8. (Item 9) 8. The CAR of any one of items 1 to 7, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in SEQ ID NO: 15 and / or a variable heavy chain sequence set forth in SEQ ID NO: 16. (Item 10) 8. The CAR of any one of items 1 to 7, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in SEQ ID NO: 23 and / or a variable heavy chain sequence set forth in SEQ ID NO: 24. (Item 11) 11. The CAR of any one of paragraphs 1 to 10, wherein the transmembrane domain is isolated from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. (Item 12) 12. The CAR of any one of items 1 to 11, wherein the transmembrane domain is isolated from a polypeptide selected from the group consisting of CD8α, CD28, CD4, CD45, PD1, and CD152. (Item 13) 13. The CAR of any one of items 1 to 12, wherein the transmembrane domain is isolated from CD8α. (Item 14) 14. The CAR of any one of paragraphs 1 to 13, wherein the one or more costimulatory signaling domains are isolated from a costimulatory molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. (Item 15) 15. The CAR of any one of paragraphs 1 to 14, wherein the one or more costimulatory signaling domains are isolated from a costimulatory molecule selected from the group consisting of CD28, CD134, and CD137. (Item 16) 16. The CAR of any one of paragraphs 1 to 15, wherein the one or more costimulatory signaling domains are isolated from CD137. (Item 17) 17. The CAR of any one of paragraphs 1 to 16, wherein 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. (Item 18) 18. The CAR of any one of items 1 to 17, wherein the primary signaling domain is isolated from CD3ζ. (Item 19) 19. The CAR of any one of items 1 to 18, further comprising a hinge region polypeptide. (Item 20) 20. The CAR of item 19, wherein the hinge region polypeptide comprises the hinge region of CD8α. (Item 21) 21. The CAR of any one of items 1 to 20, further comprising a spacer region. (Item 22) 23. The CAR of any one of items 1 to 21, further comprising a signal peptide. 23. The CAR of item 22, wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. (Item 24) 24. The CAR according to any one of items 1 to 23, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 25 to 30. (Item 25) 25. The CAR of any one of items 1 to 24, comprising the amino acid sequence set forth in SEQ ID NO: 25. (Item 26) 25. The CAR of any one of items 1 to 24, comprising the amino acid sequence set forth in SEQ ID NO: 26. (Item 27) 25. The CAR of any one of items 1 to 24, comprising the amino acid sequence set forth in SEQ ID NO: 27. (Item 28) 25. The CAR of any one of items 1 to 24, comprising the amino acid sequence set forth in SEQ ID NO: 28. (Item 29) 25. The CAR of any one of items 1 to 24, comprising the amino acid sequence set forth in SEQ ID NO: 29. (Item 30) 25. The CAR of any one of items 1 to 24, comprising the amino acid sequence set forth in SEQ ID NO: 30. (Item 31) A polypeptide comprising the amino acid sequence of the CAR of any one of items 1 to 30. (Item 32) 32. A polynucleotide encoding the CAR of any one of items 1 to 31. (Item 33) A polynucleotide comprising any one of the sequences set forth in SEQ ID NO:31 to SEQ ID NO:36. (Item 34) A vector comprising the polynucleotide of Item 32 or 33. (Item 35) 35. The vector according to item 34, wherein the vector is an expression vector. (Item 36) Item 36. The vector of item 34 or item 35, wherein the vector is an episomal vector. (Item 37) 37. The vector of any one of items 34 to 36, wherein the vector is a viral vector. (Item 38) 38. The vector of any one of items 34 to 37, wherein the vector is a retroviral vector. (Item 39) 39. The vector of any one of items 34 to 38, wherein the vector is a lentiviral vector. (Item 40) 40. The vector of any one of items 34 to 39, wherein the vector is a lentiviral vector selected from the group consisting essentially of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). (Item 41) 39. The vector of any one of items 34 to 39, comprising a left (5') retroviral LTR, a psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral transport element, a promoter operably linked to the polynucleotide of item 32 or item 33, and a right (3') retroviral LTR. (Item 42) 42. The vector of any one of items 34 to 41, further comprising a heterologous polyadenylation sequence. (Item 43) 42. The vector of any one of items 34 to 41, further comprising a Hepatitis B virus post-transcriptional regulatory element (HPRE) or a Woodchuck post-transcriptional regulatory element (WPRE). (Item 44) 44. The vector of any one of items 41 to 43, wherein the promoter of the 5'LTR is replaced with a heterologous promoter. (Item 45) 45. The vector of item 44, wherein the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40) promoter. (Item 46) 46. ​​The vector of any one of items 41 to 45, wherein the 5'LTR or 3'LTR is a lentiviral LTR. (Item 47) 47. The vector of any one of items 41 to 46, wherein the 3'LTR comprises one or more modifications. (Item 48) 48. The vector of any one of items 41 to 47, wherein the 3'LTR comprises one or more deletions. (Item 49) 49. The vector of any one of items 41 to 48, wherein the 3'LTR is a self-inactivating (SIN) LTR. (Item 50) 49. The vector of any one of Items 41 to 49, wherein the promoter operably linked to the polynucleotide of Item 32 or 33 is selected from the group consisting of a cytomegalovirus immediate early gene promoter (CMV), an elongation factor 1 alpha promoter (EF1-α), a phosphoglycerate kinase 1 promoter (PGK), a ubiquitin C promoter (UBQ-C), a cytomegalovirus enhancer / chicken beta-actin promoter (CAG), a polyoma enhancer / herpes simplex thymidine kinase promoter (MC1), a beta-actin promoter (β-ACT), a simian virus 40 promoter (SV40), and a dl587rev primer binding site-substituted (MND) U3 promoter of a myeloproliferative sarcoma virus enhancer with a deleted negative regulatory region. (Item 51) 51. An immune effector cell comprising the vector of any one of items 34 to 50. (Item 52) 52. The immune effector cell of item 51, wherein the immune effector cell is selected from the group consisting of T lymphocytes, natural killer (NK) cells, and NKT cells. (Item 53) 53. A composition comprising the immune effector cells of item 51 or 52 and a physiologically acceptable excipient. (Item 54) 50. A method of producing a population of immune effector cells comprising the CAR of any one of items 1 to 30, the method comprising introducing the polynucleotide of item 32 or item 33 or the vector of any one of items 34 to 50 into the population of immune effector cells. (Item 55) 55. The method of claim 54, further comprising contacting the immune effector cells with an antibody that binds CD3 and an antibody that binds CD28 to stimulate the cells and induce proliferation of the cells, thereby expanding the immune effector cell population. (Item 56) 55. The method of claim 54, wherein the immune effector cells comprise T cells, NK cells, and / or NKT cells. (Item 57) 57. The method of claim 56, wherein the immune effector cells comprise T cells. (Item 58) 54. A method for increasing the cytotoxicity of CD79A-expressing cancer cells in a subject, comprising administering to the subject the composition of claim 53 in an amount sufficient to increase the cytotoxicity of CD79A-expressing cancer cells compared to the cytotoxicity of the CD79A-expressing cancer cells prior to said administration. (Item 59) 54. A method for reducing the number of CD79A-expressing cancer cells in a subject, comprising administering to the subject the composition of claim 53 in an amount sufficient to reduce the number of CD79A-expressing cancer cells compared to the number of CD79A-expressing cancer cells before said administration. (Item 60) 54. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of claim 53. (Item 61) Item 61. The method of item 60, wherein the cancer is a liquid cancer. (Item 62) 62. The method of claim 60 or 61, wherein the cancer is a hematological malignancy. (Item 63) 63. The method of any one of items 60 to 62, wherein the cancer is non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML). (Item 64) 64. The method of item 63, wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL). (Item 65) 64. The method of item 63, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL). (Item 66) The method of any one of items 60 to 62, wherein the cancer is acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML). 63. The method of any one of items 60 to 62, wherein the cancer is multiple myeloma (MM) selected from the group consisting of overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma. (Item 68) 54. A method of ameliorating one or more symptoms associated with a CD79A-expressing cancer in a subject, comprising administering to the subject the composition of claim 53, wherein the composition is administered in an amount sufficient to ameliorate at least one symptom associated with CD79A-expressing cancer cells. (Item 69) 69. The method of item 68, wherein the one or more symptoms improved are selected from the group consisting of weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain, bone or joint pain, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination. [Brief explanation of the drawings]

[0088] [Figure 1A]

[0033] Figures show CD79A expression in target cells, CAR expression, and anti-CD79ACAR-T cell antigen-dependent activity in the presence of CD79A-expressing target cells. A) Panel showing CD79A expression in Pfeiffer and Daudi cells, but not in K562 cells. B) Panel showing representative anti-CD79ACAR expression in T cells measured using flow cytometry. C) Panel showing IFNγ secretion of anti-CD79ACAR-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). [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 2A]

[0033] Figures show CAR expression, CD79A expression, and anti-CD79ACAR-T cell antigen-dependent activity in the presence of CD79A-expressing target cells. A) Panel showing CD79A receptor density on Daudi, NU-DUL-1, SU-DHL-2, and Pfeiffer cells. B) Panel showing representative anti-CD79ACAR expression on T cells measured using flow cytometry. C) Panel showing IFNγ secretion of anti-CD79ACAR-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+). [Figure 2B] Same as above. [Figure 2C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0089] A brief description of arrays SEQ ID NO: 1 to SEQ ID NO: 24 show 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 anti-CD79 ACARs discussed herein. SEQ ID NOs: 25 to 30 show the amino acid sequences of exemplary anti-CD79 ACARs. SEQ ID NO:31 to SEQ ID NO:36 show the nucleic acid sequences of exemplary anti-CD79 ACARs. SEQ ID NO: 37-38 show the amino acid sequences of exemplary human CD79A polypeptides. SEQ ID NO: 39 to SEQ ID NO: 49 show the amino acid sequences of various linkers. SEQ ID NO: 50 to SEQ ID NO: 74 show the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

[0090] A. Overview The present invention relates to improved compositions and methods for preventing or treating CD79A-expressing cancers or for preventing, treating, or ameliorating at least one symptom associated with CD79A-expressing cancers. In certain embodiments, the present invention relates to improved adoptive cell therapy of CD79A-expressing cancers using genetically modified immune effector cells. Genetic approaches hold promise as a means to enhance immune recognition and promote elimination of cancer cells. One promising strategy is to genetically modify immune effector cells to express chimeric antigen receptors (CARs), which redirect cytotoxicity against cancer cells.

[0091] Improved adoptive cell therapy compositions and methods contemplated herein provide genetically engineered immune effector cells that are readily propagated, exhibit long-term persistence in vivo, and exhibit antigen-dependent cellular cytotoxicity against cells expressing CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain, membrane-associated immunoglobulin-related protein (MB1, MB-1), surface IgM-related protein, and Ig alpha (IGA)). Examples of polynucleotide sequences encoding CD79A include, but are not limited to, NM_001783.3, NM_021601.3, ENST00000221972 (uc002orv.3), ENST00000597454 (uc060zdj.1), ENST00000444740 (uc002oru.4), Hs.631567, and AK223371. Examples of polypeptide sequences encoding CD79A include, but are not limited to, P11912-1, P11912-2, ENSP00000400605, ENSP00000468922, ENSP00000221972, NP_001774.1, and NP_067612.1.

[0092] CD79 consists of two proteins, CD79A and CD79B. CD79A is located on chromosome 19q13.2 and encodes a 226-amino acid glycoprotein of approximately 47 kDa. The exact molecular weight varies depending on the degree of glycosylation. CD79B is located on chromosome 17q23 and encodes a 229-amino acid glycoprotein of approximately 37 kDa. Both CD79A and CD79B share an exon-intron structure and contain a single Ig domain of the Ig superfamily (SF) (111-residue C-type for CD79A and 129-residue V-type for CD79B). Each contains a highly conserved transmembrane domain and a 61-amino acid cytoplasmic tail (CD79A) or a 48-amino acid cytoplasmic tail (CD79B) that show significant evolutionary amino acid conservation. CD79A and CD79B are expressed by early B cell precursors. CD79A / B heterodimers are found on the surface of early B-cell precursors in the absence of mu heavy chains, although neither protein is essential for the precursor's commitment to the B-cell lineage. Later in development, CD79A and CD79B are coexpressed on the surface of B cells with Ig of all isotypes as part of the mature BCR complex. The CD79 protein is B-lineage specific and is expressed throughout B-lymphopoiesis. CD79A and CD79B can be used as markers to identify B-cell neoplasms, such as diffuse large B-cell lymphoma (DLBCL), most precursor B-cell acute leukemias, and B-cell lymphomas, as well as some myelomas.

[0093] In various embodiments, CARs comprising anti-CD79A antibody sequences are highly effective, grow robustly in vivo, recognize CD79A-expressing cancer cells, and exhibit cytotoxic activity against CD79A-expressing cancer cells.

[0094] In one embodiment, a CAR is provided that includes an anti-CD79A antibody or antigen-binding fragment, a transmembrane domain, and one or more intracellular signaling domains.

[0095] In one embodiment, the immune effector cells are genetically modified to express a CAR. T cells that express a CAR are referred to herein as CAR-T cells or CAR-modified T cells.

[0096] In various embodiments, the genetically modified immune effector cells are administered to a subject with CD79A-expressing cancer cells, including, but not limited to, subjects with liquid tumors and hematologic tumors. In one embodiment, anti-CD79A CAR-T cells are administered to a subject with DLBCL.

[0097] Techniques for recombinant (i.e., modified) DNA, peptide, and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification, and related techniques and procedures can generally be performed as described in various general and more specialized works in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, cited and discussed throughout this specification, 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, revised July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985), Current Protocols in Immunology (edited by John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY), Real-Time PCR: Current Technology and Applications, Edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK, Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992), Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991), Oligonucleotide Synthesis (N. Gait, Ed., 1984), Nucleic Acid The Hybridization (B. Hames & S. Higgins, Eds., 1985), Transcription and Translation (B. Hames & S. Higgins, Eds., 1984), Animal Cell Culture (R. Freshney, Ed., 1986), Perbal, A Practical Guide to Molecular Cloning (1984), Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH), PCR Protocols (Methods in Molecular Biology) (Park, Ed., 3rd Edition, 2010 Humana Press), Immobilized Cells And Enzymes (IRL Press, 1986), Specialized Book Methods In Enzymology (Academic Press, Inc., N.Y.), Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory), Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987), Handbook Of Experimental Immunology, Volumes I-IV (DM Weir andCC Blackwell, eds., 1986), Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988), Current Protocols in Immunology (QE Coligan, AM Kruisbeek, See journal articles such as DH Margulies, EM Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0098] B. Definition Before setting forth the present disclosure in further detail, it is believed to be helpful to an understanding thereof to set forth definitions of certain terms used herein.

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of the compositions, methods, and materials are described herein. In this disclosure, the following terms are defined below. Additional definitions are provided throughout this disclosure.

[0100] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one or one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0101] When an alternative conjunction (eg, "or") is used, it should be understood to mean one, both, or any combination of the alternatives.

[0102] The term "and / or" should be understood to mean one or both of the alternatives.

[0103] The term "about" or "approximately," as used herein, means that an amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length varies by about 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the referenced amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of about ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the referenced amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length.

[0104] In one embodiment, a range (eg, 1 to 5, about 1 to 5, or about 1 to about 5) refers to each number encompassed by the range. For example, in a non-limiting and merely exemplary embodiment, the range "1 to 5" is equivalent to 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0105] The term "approximately," as used herein, refers to an amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the referenced amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length that produces approximately the same effect, e.g., the same physiological effect, as the referenced amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length.

[0106] As used herein, unless expressly stated otherwise, "comprises" and "comprises" are used interchangeably. The words "composed of" and "comprising" will be understood to mean the inclusion of the stated step or element or steps or elements, but not the exclusion of any other step or element or steps or elements. "Consisting of" means including and limited to everything that follows the phrase. Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means that any elements listed following the phrase are present. and is limited to other elements that do not inhibit or impart to the listed elements the activity or effect detailed in this disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are necessary or essential. indicates the absence of any other components that substantially affect the activity or action of the listed components.

[0107] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "another embodiment," or "a further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that affirmatively reciting a feature in one embodiment constitutes a basis for excluding that feature in a particular embodiment.

[0108] C. Chimeric Antigen Receptor In various embodiments, engineered receptors are provided that redirect the cytotoxicity of immune effector cells against CD79A-expressing cancer cells. These engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine the specificity of an antibody against a desired antigen (e.g., CD79A) with a T cell receptor activating intracellular domain to produce a chimeric protein that exhibits specific anti-CD79A cellular immune activity. The term "chimeric," as used herein, indicates that the protein is composed of separate protein or DNA segments derived from different sources.

[0109] In particular embodiments, the CAR comprises an extracellular domain (also referred to as a binding domain or antigen-specific binding domain) that binds to CD79A, a transmembrane domain, and an intracellular signaling domain. When the anti-CD79A antigen-binding domain of the CAR binds to CD79A on the surface of the target cell, the CAR clusters and delivers an activating stimulus to the CAR-containing cell. A key feature of CARs is their ability to redirect the specificity of immune effector cells, thereby inducing proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of target antigen-expressing cells regardless of major histocompatibility (MHC) status, thereby leveraging the cell-specific targeting capabilities of monoclonal antibodies, lytic ligands, or cell-specific co-receptors.

[0110] In various embodiments, the CAR comprises an extracellular binding domain comprising a CD79A-specific binding domain, a transmembrane domain, and one or more intracellular costimulatory signaling domains and / or primary signaling domains.

[0111] In certain embodiments, the CAR comprises an extracellular binding domain comprising an anti-CD79A antibody or antigen-binding fragment thereof, one or more hinge or spacer domains, a transmembrane domain, and one or more intracellular costimulatory signaling domains and / or primary signaling domains.

[0112] 1. Binding Domain In certain embodiments, the CAR comprises an extracellular binding domain comprising an anti-CD79A antibody or antigen-binding fragment thereof that specifically binds to a human CD79A polypeptide expressed on a target cell, e.g., a cancer cell. The terms "binding domain," "extracellular domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably herein and refer to a CAR that can specifically bind to a target antigen of interest, e.g., CD79A. The binding domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0113] The terms "specific binding affinity," or "specifically binds," or "specifically bound," or "specific binding," or "specifically targets," as used herein, indicate that an anti-CD79A antibody or antigen-binding fragment thereof (or a CAR comprising the same) binds to CD79A with a binding affinity higher than background binding. The binding domain (or a CAR comprising the binding domain, or a fusion protein comprising the binding domain) binds to CD79A with an affinity or K a (i.e., the equilibrium binding constant for a particular binding interaction, with units of 1 / M) is, for example, about 10 5 M -1 Greater than or about 10 5 M -1 In certain embodiments, a binding domain (or fusion protein thereof) "specifically binds" to a CD79A polypeptide if it binds or associates with CD79A with a value equal to K a is 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 A "high affinity" binding domain (or single-chain fusion protein thereof) binds to the target with a K greater than or equal to a is 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 higher.

[0114] Alternatively, affinity can be expressed as the equilibrium dissociation constant (K) of a particular binding interaction, which has units M. d ) (e.g., 10 -5 M~10 -13 The affinity of the binding domain polypeptides and CAR proteins of the present disclosure can be readily measured using conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay) or using labeled ligands or the Biacore (New Jersey, USA) assay. Binding association or displacement assays using surface plasmon resonance devices such as the Biacore T100 (Piscataway, AL) or optical biosensor technology such as the EPIC system from Corning or the EnSpire from Perkin Elmer (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660, and U.S. Pat. Nos. 5,283,173 and 5, 468,614, or equivalent).

[0115] 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.

[0116] In certain embodiments, the extracellular binding domain of the CAR comprises an antibody or antigen-binding fragment thereof. An "antibody" refers to a polypeptide binding agent comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant (e.g., one recognized by an immune cell). An "isolated antibody or antigen-binding fragment thereof" is one that has been identified, separated, and / or recovered from a component of its natural environment.

[0117] "Antigen (Ag)" means a compound, composition, or substance capable of stimulating the production of antibodies or a T-cell response in an animal, including, for example, compositions (such as those containing cancer-specific proteins) that are injected or absorbed into an animal. Antigens react with specific humoral or cellular immune products, including those induced by heterologous antigens, such as those of the present disclosure. In certain embodiments, the target antigen is an epitope of a CD79A polypeptide.

[0118] "Epitope" or "antigenic determinant" refers to a region of an antigen to which a binding agent binds. An epitope can be composed of contiguous amino acids or non-contiguous amino acids that are closely adjacent due to tertiary folding of a protein. Epitopes composed of contiguous amino acids typically retain their structure when exposed to denaturing solvents, whereas epitopes composed of tertiary folding typically lose their structure when treated with denaturing solvents. Epitopes often include at least three, more commonly at least five, about nine, or about eight to ten amino acids in a unique conformation.

[0119] Antibodies include antigen-binding fragments thereof, such as camelid Ig, IgNAR, Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins ("scFv"), bis-scFv (bis-scFv), (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and single-domain antibodies (sdAbs, nanobodies), as well as the portion of a full-length antibody that is involved in antigen binding. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997. I want to be.

[0120] A complete antibody, as understood by those skilled in the art and described elsewhere herein, comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies have a "Y" structure. The stem of the Y consists of the interconnected second and third constant regions of the two heavy chains (and a fourth constant region for IgE and IgM), with disulfide bonds (interchain) formed in the hinge region. The heavy chains λ, α, and δ have a constant region composed of three tandem Ig domains and a hinge region that allows flexibility, while the heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The second constant region is called the "CH2 domain," and the third constant region is called the "CH3 domain." Each arm of the Y contains the variable region and first constant region of a single heavy chain 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.

[0121] The light and heavy chain variable regions contain a "framework" region flanked by three hypervariable regions (also called "complementarity-determining regions" or "CDRs"). The CDRs can be sequenced using conventional methods, for example, using the sequences of 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)) (Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991), or using the structure of Chothia et al. Lesk, AM, J Mol. Biol., 196(4): 901-917 (1987), Chothia, C. et al, Nature, 342: 877-883 (1989)), characterized, or identified.

[0122] Specific examples of criteria for identifying light chain CDRs are as follows: CDR-L1 begins at approximately residue 24, begins with Cys, is about 10 to 17 residues long, and is followed by Trp (usually Trp-Tyr-Gln, but can also be Trp-Leu-Gln, Trp-Phe-Gln, or Trp-Tyr-Leu). CDR-L2 begins approximately 16 residues after the end of CDR-L1, is usually preceded by Ile-Tyr, but can also be preceded by Val-Tyr, Ile-Lys, or Ile-Phe, and is 7 residues long. CDR-L3 begins approximately 33 residues after the end of CDR-L2, begins with Cys, is 7 to 11 residues long, and is followed by Phe-Gly-XXX-Gly (SEQ ID NO: 76) (XXX is any amino acid).

[0123] Specific examples of criteria for identifying heavy chain CDRs are as follows: CDR-H1 begins at approximately residue 26, begins with Cys-XXX-XXX-XXX (SEQ ID NO: 77), is followed by Trp (usually Trp-Val, but may also be Trp-Ile or Trp-Ala), and is about 10 to 12 residues long. CDR-H2 begins approximately 15 residues after the end of CDR-H1, and is usually preceded by Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 78) or several variants, is followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala, and is 16 to 19 residues long. CDR-H3 begins approximately 33 residues after the end of CDR-H2, begins with Cys-XXX-XXX (usually Cys-Ala-Arg), and is 3 to 25 residues long, followed by Trp-Gly-XXX-Gly (SEQ ID NO: 79). In one embodiment, the light chain and heavy chain CDRs are identified using the method of Kabat.

[0124] In one embodiment, the light chain CDRs and the heavy chain CDR2 and CDR3 are isolated using the method of Kabat. The heavy chain CDR1 is identified using the AbM method, which is equivalent to the Kabat and Clothia methods. See, for example, Whitelegg N & Rees AR, Protein Eng. 2000 Dec;13(12):819-24, and Methods Mol Biol. 2004;248:51-91. Programs are also publicly available, such as AbYsis (www.bioinf.org.uk / abysis / ). There is.

[0125] The sequences of the framework regions of various light and heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, consisting of the combined framework regions of the constituent light and heavy chains of the antibody, positions and aligns the CDRs in three-dimensional space. CDRs are primarily responsible for binding to an antigen epitope. The CDRs of each chain are typically designated CDR1, CDR2, and CDR3, are numbered consecutively starting from the N-terminus, and are generally identified by the chain in which the particular CDR resides. Thus, the CDRs in the variable domain of an antibody heavy chain are designated CDRH1, CDRH2, and CDRH3, while the CDRs in the variable domain of an antibody light chain are designated CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different binding sites for various antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are designated specificity-determining residues (SDRs). Specific examples of light chain CDRs suitable for constructing anti-CD79 ACARs contemplated in certain embodiments include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 1 to 3, 9 to 11, and 17 to 19. Specific examples of heavy chain CDRs suitable for constructing anti-CD79 ACARs contemplated in certain embodiments include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 4 to 6, 12 to 14, and 20 to 22.

[0126] "V L " or "VL" refers to the variable region of an immunoglobulin light chain, and includes the variable region of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragments described herein. Specific examples of light chain variable regions suitable for constructing anti-CD79 ACARs contemplated in certain embodiments include, but are not limited to, the light chain variable region sequences set forth in SEQ ID NO:7, SEQ ID NO:15, and SEQ ID NO:23.

[0127] "V H" or "VH" refers to the variable region of an immunoglobulin heavy chain, and includes the variable region of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragments described herein. Specific examples of heavy chain variable regions suitable for constructing anti-CD79 ACARs contemplated in certain embodiments include, but are not limited to, the heavy chain variable region sequences set forth in SEQ ID NO:8, SEQ ID NO:16, and SEQ ID NO:24.

[0128] A "monoclonal antibody" is an antibody obtained from a single clone of B lymphocytes or from a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies can be produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0129] A "chimeric antibody" is one that has framework residues from one species, such as human, and CDRs (typically the portions that provide antigen binding) from another species, such as mouse. In certain preferred embodiments, the CAR comprises the antigen-specific binding domain of a chimeric antibody or antigen-binding fragment thereof.

[0130] 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-derived phage display library with known human constant domain sequences, as described above. Alternatively, human monoclonal antibodies can be produced by hybridoma technology. Human myeloma and mouse-human heteromyeloma cell lines suitable for producing human monoclonal antibodies are described, for example, by Kozbor J. Immunol., 133: 3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147: 86 (1991). Furthermore, transgenic animals (e.g., mice) can be used to produce the entire repertoire of human antibodies without producing endogenous immunoglobulins. 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 be used to produce a full repertoire of human antibodies. Thus, it is possible to obtain human antibodies derived from non-human (e.g., rodent) antibodies that have similar affinities and specificities to the original non-human antibodies (see International Application PCT93 / 06213, published April 1, 1993). Unlike conventional CDR-grafting methods for humanizing non-human antibodies, this method produces fully human antibodies that have no FR or CDR residues of non-human origin.

[0131] In one embodiment, a CAR comprises a "humanized" antibody. A humanized antibody is an immunoglobulin comprising a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if present, the constant regions should be substantially identical to those of human immunoglobulins, i.e., at least about 85% to 90%, e.g., about 95% or more identical. Thus, all parts of a humanized immunoglobulin (possibly excluding the CDRs) are substantially identical to the corresponding parts of a native human immunoglobulin sequence. Humanized antibodies or other monoclonal antibodies may contain additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (see, eg, US Pat. No. 5,585,089).

[0132] In certain embodiments, anti-CD79A antibodies or antigen-binding fragments thereof include, but are not limited to, camelid Igs (Camelidae antibodies (VHHs)), IgNARs, Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2s), trispecific Fab trimers (Fab3s), Fvs, single-chain Fv proteins ("scFvs"), bis-scFvs (bis-scFvs), (scFv)2s, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFvs"), and single domain antibodies (sdAbs, nanobodies).

[0133] As used herein, "camelid Ig" or "camelid VHH" refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte, et al, FASEB J., 21: 3490-3498 (2007)). "Heavy chain antibody" or "camelid antibody" refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al, J. Immunol. Methods 231:25-38 (1999); WO 94 / 04678; WO 94 / 25591; U.S. Pat. No. 6,005,079).

[0134] "Immunoglobulin neoantigen receptor" or "IgNAR" refers to a class of antibodies derived from the shark immune repertoire, consisting of a homodimer of one variable neoantigen receptor (VNAR) domain and five constant neoantigen receptor (CNAR) domains. IgNARs are examples of the smallest known immunoglobulin-based protein scaffolds, with high stability and efficient binding properties. The high stability of IgNARs is thought to result from (i) a base Ig scaffold with a significantly higher number of charged and affinity-surface-exposed residues compared to the VH and VL domains of conventional antibodies found in murine antibodies, and (ii) stabilizing structural features of the complementarity-determining region (CDR) loops, such as intraloop disulfide bridges and intraloop hydrogen-bonding patterns.

[0135] 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, named for its high crystallizability. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0136] An "Fv" is the minimum antibody fragment that contains the entire antigen-binding site. In one embodiment, two-chain Fvs consist of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. Single-chain Fv (scFv) species comprise one heavy- and one light-chain variable domain covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that of a two-chain Fv. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (i.e., half of an Fv with only three antigen-specific HVRs) is capable of recognizing and binding antigen, albeit with lower affinity than the entire binding site.

[0137] Fab fragments contain heavy and light chain variable domains, as well as the light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH, as used herein, represents Fab' in which the cysteine ​​residues in the constant domains bear free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known. A bispecific Fab dimer (Fab2) contains two Fab' fragments, each of which binds a different antigen. A trispecific Fab trimer (Fab3) contains three Fab' fragments, each of which binds a different antigen.

[0138] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with complementary domains in another chain, forming two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are described in further detail, for example, in EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., PNAS USA 90: 6444-6448 (1993). Bodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0139] "Single domain antibody," or "sdAb," or "nanobody" refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL domain) (Holt, L., et al, Trends in Biotechnology, 21(11): 484-490). .

[0140] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, with the two domains organized in a single polypeptide chain and in either orientation (e.g., VL-VH or VH-VL). Typically, scFv polypeptides further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form a structure suitable for antigen binding. For a review of scFvs, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0141] In a preferred embodiment, the anti-CD79A antigen-binding fragment is an scFv. In a specific embodiment, the scFv is a mouse scFv, a human scFv, or a humanized scFv. Single-chain antibodies can be cloned from the V region genes of hybridomas specific to the desired target. The production of such hybridomas is routine. Techniques that can be used to clone the variable heavy chain (VH) and variable light chain (VL) are described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837.

[0142] In various embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising the CDRL1 through CDRL3 sequences set forth in SEQ ID NO:1-3, SEQ ID NO:9-11, or SEQ ID NO:17-19, and / or a variable heavy chain sequence comprising the CDRH1 through CDRH3 sequences set forth in SEQ ID NO:4-6, or SEQ ID NO:12-14. In some embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:15, or SEQ ID NO:23, and / or a variable heavy chain sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:16, or SEQ ID NO:24.

[0143] In a specific embodiment, the antigen-specific binding domain is an scFv that binds to a human CD79A polypeptide.

[0144] An example of a CD79A-specific binding domain is an immunoglobulin variable region specific for CD79A that includes at least one human framework region. By "human framework region" is meant a wild-type (i.e., native) framework region of a human immunoglobulin variable region, or a modified framework region in which 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 framework region of a human immunoglobulin variable region have been deleted or substituted (e.g., substituted with one or more amino acid residues from a non-human immunoglobulin framework region at the corresponding positions), or a modified framework region in which less than about 50% (e.g., less than 45%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%) of the amino acids in the framework region of a non-human immunoglobulin variable region have been deleted or substituted (e.g., deleted or substituted at exposed residue positions and / or substituted with one or more amino acid residues from a human immunoglobulin framework region at the corresponding positions), resulting in a framework region that, in one embodiment, has reduced immunogenicity.

[0145] In certain embodiments, the human framework regions are wild-type framework regions of a human immunoglobulin variable region. In other certain embodiments, the human framework regions are modified framework regions of a human immunoglobulin variable region having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid deletions or substitutions. In other embodiments, the human framework regions are modified framework regions of a non-human immunoglobulin variable region having one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid deletions or substitutions.

[0146] In certain embodiments, a CD79A-specific binding domain comprises at least 1, 2, 3, 4, 5, 6, 7, or 8 human framework regions (FRs) selected from 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.

[0147] Human FRs that can be included in a CD79A-specific binding domain also include variants of the exemplary FRs provided herein, in which one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids of the exemplary FRs have been substituted or deleted.

[0148] 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.

[0149] Furthermore, the CD79A-specific binding domains provided herein comprise one, two, three, four, five, or six CDRs. These CDRs may be non-human or modified non-human CDRs selected from light chain CDRL1, CDRL2, and CDRL3, and heavy chain CDRH1, CDRH2, and CDRH3. 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.

[0150] In certain embodiments, the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence comprising CDRL1 through CDRL3 sequences that have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acids set forth in SEQ ID NO:1-3, SEQ ID NO:9-11, or SEQ ID NO:17-19, and / or a variable heavy chain sequence comprising CDRH1 through CDRH3 sequences that have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acids set forth in SEQ ID NO:4-6, SEQ ID NO:12-14, or SEQ ID NO:20-22.

[0151] In one embodiment, the CD79A-specific binding domain comprises a light chain CDR sequence set forth in SEQ ID NO:1-3, SEQ ID NO:9-11, or SEQ ID NO:17-19. In a specific 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 the light chain CDR sequence set forth in SEQ ID NO:1-3, SEQ ID NO:9-11, or SEQ ID NO:17-19.

[0152] In one embodiment, the CD79A-specific binding domain comprises a heavy chain CDR sequence set forth in SEQ ID NO:4-6, SEQ ID NO:12-14, or SEQ ID NO:20-22. In certain embodiments, 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 the heavy chain CDR sequence set forth in SEQ ID NO:4-6, SEQ ID NO:12-14, or SEQ ID NO:20-22.

[0153] In certain embodiments, the anti-idiotype 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 NO:7, SEQ ID NO:15, or SEQ ID NO: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 NO:8, SEQ ID NO:16, or SEQ ID NO:24.

[0154] 2. Linker In certain embodiments, the anti-CD79 ACAR comprises linker residues between various domains, with the linker being added, for example, to achieve proper spacing and conformation of the molecule. In certain embodiments, the linker is a variable region linking sequence. A "variable region linking sequence" is an amino acid sequence that links the VH domain and the VL domain and provides a spacer function suitable for the interaction of the two sub-binding domains. The resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In certain embodiments, the CAR comprises one, two, three, four, five, or more linkers. In certain embodiments, the linker length 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 amino acid length therebetween. In certain embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0155] A specific example of the linker is a glycine polymer (G) n , glycine-serine polymer (G 1-5 S 1-5 ) n(where n is an integer of at least 1, 2, 3, 4, or 5), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Because glycine and glycine-serine polymers are relatively amorphous in structure, they can function as intermediate tethers between domains of fusion proteins, such as the CARs described herein. Glycine has access to significantly more ΦΨ space than alanine and is much less constrained than residues with long side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). The skilled artisan will recognize that the design of CARs in certain embodiments may include fully or partially flexible linkers, and thus, linkers may comprise a flexible linker and one or more moieties that confer less flexibility to achieve the desired CAR structure.

[0156] Other examples of 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., PNAS 5525-5530 (1997)), GGRR (SEQ ID NO: 41) (Pomerantz et al. 1995 , supra), (GGGGS) n (wherein n is 1, 2, 3, 4, or 5) (SEQ ID NO: 42) (Kim et al., PNAS 93, 1156-1160 (1996)), EGKSSGSGSESKVD( Examples of suitable flexible linkers include 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), LQRDGERP (SEQ ID NO: 46), LRQKDGGGSERP (SEQ ID NO: 47), and LRQKD(GGGS)2ERP (SEQ ID NO: 48). Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling DNA binding sites and their peptides (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994)), or using phage display methods. In embodiments, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 49) (Cooper et al., Blood, 101(4): 1637-1644 (2003)).

[0157] 3. Spacer domain In certain embodiments, the binding domain of the anti-CD79 ACAR is followed by one or more "spacer domains," which are regions that distance the antigen binding domain from the effector cell surface to allow proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may comprise the amino acid sequence of a natural or modified immunoglobulin hinge region.

[0158] In one embodiment, the spacer domain comprises the CH2 and CH3 of IgG1, IgG4, or IgD.

[0159] 4. Hinge domain The binding domain of an anti-CD79 ACAR is typically followed by one or more "hinge domains," which serve to distance the antigen-binding domain from the effector cell surface, allowing for proper cell-cell contact, antigen binding, and activation. Anti-CD79 ACARs typically contain one or more hinge domains between the binding domain and the transmembrane (TM) domain. The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a natural immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0160] A "modified hinge region" is defined as (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 naturally occurring hinge region at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids) with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions). "Hinge region" refers to a portion of a native immunoglobulin hinge region, or (c) a portion of a native immunoglobulin hinge region comprising the core hinge region (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, or at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acids in length). In certain embodiments, one or more cysteine ​​residues of the native immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). Alternatively, or in addition, a modified immunoglobulin hinge region may have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue (e.g., a serine residue).

[0161] Examples of hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins, such as CD8α and CD4, which may be the wild-type hinge region of the type I membrane protein or may be modified. In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge, and in another embodiment, the hinge domain comprises a CD8α hinge region.

[0162] 5. Transmembrane (TM) domain The "transmembrane domain" is the portion of the anti-CD79 ACAR that fuses the extracellular binding portion with the intracellular signaling domain, anchoring the CAR to the plasma membrane of an immune effector cell. The TM domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The TM domain can be derived from or include at least the transmembrane region of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In certain embodiments, the TM domain is synthetic and contains primarily hydrophobic residues such as leucine and valine.

[0163] In one embodiment, the CAR comprises a TM domain from PD1, CD152, CD28, or CD8α. In another embodiment, the CAR comprises a TM domain from PD1, CD152, CD28, or CD8α and a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, connecting the TM domain to the intracellular signaling domain of the CAR. Glycine-serine based linkers are particularly preferred linkers.

[0164] 6. Intracellular signaling domains In certain embodiments, the anti-CD79 ACAR comprises one or more intracellular signaling domains. "Intracellular signaling domain" refers to a portion of the CAR that transmits a message to the interior of an immune effector cell that the anti-CD79 ACAR is substantially bound to a human CD79A polypeptide, thereby eliciting effector cell function. Such functions include, for example, activation, cytokine production, proliferative activity, and cytotoxic activity, such as the release of cytotoxic factors toward target cells to which the CAR is bound, or other cellular responses elicited by antigen binding to the extracellular CAR domain.

[0165] The term "effector function" refers to a specific function of an immune effector cell. Effector function of T cells is thought to involve, for example, cytolytic or adjuvant activity, or cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and induces a cell to perform a specific function. While the entire intracellular signaling domain can be used, it is often not necessary to use the entire domain. When a truncated portion of the intracellular signaling domain is used, it can be used in place of the entire domain as long as the truncated portion transmits the effector function signal. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0166] It is known that the signal generated by TCR alone is insufficient to fully activate T cells, and a secondary signal or costimulatory signal is also required. Therefore, T cell activation is also said to be mediated by two different classes of intracellular signaling 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 antigen-independently to generate a secondary signal or costimulatory signal. In a preferred embodiment, the CAR comprises an intracellular signaling domain with one or more "costimulatory signaling domains" and a "primary signaling domain."

[0167] The primary signaling domain regulates the primary activation of the TCR complex, either stimulatory or inhibitory. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).

[0168] Specific examples of ITAMs comprising primary signaling domains useful in certain embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79A, CD79B, and CD66d. In certain preferred embodiments, an anti-CD79 ACAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domains may be linked in tandem to the carboxyl terminus of the transmembrane domain in any order.

[0169] In certain embodiments, the CAR comprises one or more costimulatory signaling domains that increase the potency and proliferation of T cells expressing the CAR receptor. The term "costimulatory signaling domain" or "costimulatory domain," as used herein, refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or an Fc receptor, that, upon binding to an antigen, provides a second signal necessary for effective activation and function of T lymphocytes. Specific examples of 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 costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and the primary signaling domain of CD3ζ.

[0170] In another embodiment, the CAR comprises the costimulatory signaling domains of CD28 and CD137 and the CD3ζ primary signaling domain.

[0171] In yet another embodiment, the CAR comprises the costimulatory signaling domains of CD28 and CD134 and the CD3ζ primary signaling domain.

[0172] In one embodiment, the CAR comprises the costimulatory signaling domains of CD137 and CD134 and the CD3ζ primary signaling domain.

[0173] In one embodiment, the CAR comprises a CD137 costimulatory signaling domain and a CD3ζ primary signaling domain.

[0174] In one embodiment, the CAR comprises a CD134 costimulatory signaling domain and a CD3ζ primary signaling domain.

[0175] In one embodiment, the CAR comprises a CD28 costimulatory signaling domain and a CD3ζ primary signaling domain.

[0176] In certain embodiments, the CAR comprises an anti-CD79A antibody or antigen-binding fragment thereof that specifically binds to a CD79A polypeptide expressed on cancer cells.

[0177] In one embodiment, the CAR comprises an anti-CD79AscFv binding a CD79A polypeptide, a transmembrane domain derived from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN1, and PD1, and a transmembrane domain derived from a polypeptide selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15, TLR16, TLR17, TLR18, TLR19, TLR20, TLR21, TLR22, TLR23, TLR24, TLR25, TLR26, TLR27, TLR28, TLR29, TLR30, TLR31, TLR32, TLR33, TLR34, TLR35, TLR36, TLR37, TLR38, TLR39, TLR40, TLR41, TLR42, TLR43, TLR44, TLR45, TLR46, TLR47, TLR48, TLR49, TLR50, TLR51, TLR52, TLR53, TLR54, TLR55, TLR56, TLR57, TLR58, TLR59, TLR59, TLR50, TLR510, TLR511, TLR52, TLR53, TLR54, TLR55, TLR56, TLR57, TLR and one or more intracellular costimulatory signaling domains derived from a costimulatory molecule selected from the group consisting of 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 selected from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79A, CD79B, and CD66d.

[0178] In one embodiment, the CAR comprises an anti-CD79AscFv binding 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 the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN1, and PD1; and a transmembrane domain derived from a polypeptide 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 selected from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79A, CD79B, and CD66d.

[0179] In one embodiment, the CAR comprises an anti-CD79AscFv binding 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 (TM) domain derived from a polypeptide selected from the group consisting of the alpha or beta chain of the T cell receptor, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN1, and PD1; and a transmembrane (TM) domain, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 The antibody comprises a short oligo- or polypeptide linker of 6, 7, 8, 9, or 10 amino acids; one or more intracellular costimulatory signaling domains derived from a costimulatory molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70; and a primary signaling domain selected from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79A, CD79B, and CD66d.

[0180] In certain embodiments, the CAR comprises an anti-CD79AscFv that binds 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 costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0181] In certain embodiments, the CAR comprises an anti-CD79AscFv that binds 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 costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0182] In certain embodiments, the CAR comprises an anti-CD79AscFv that binds 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 costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0183] The CARs contemplated in certain embodiments are engineered to provide improved proliferation, long-term persistence, and cytotoxicity in T cells expressing the CAR compared to unmodified T cells or T cells modified to express other CARs.

[0184] D. Polypeptides Various polypeptides, fusion polypeptides, and polypeptide variants are contemplated herein, including, but not limited to, CAR polypeptides and fragments thereof. In preferred embodiments, a polypeptide comprising one or more CARs is provided. In a specific embodiment, the CAR is an anti-CD79ACAR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 25 to 30.

[0185] The terms "polypeptide," "peptide," and "protein," unless otherwise specified, are used interchangeably and according to their conventional meaning, i.e., to refer to a sequence of amino acids. A polypeptide is not limited to a particular length and can include, for example, a full-length polypeptide or a polypeptide fragment. A polypeptide can also include one or more post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and other natural and non-natural modifications known in the art. In various embodiments, a CAR polypeptide includes a signal sequence (or leader sequence) at the N-terminus of the protein, which directs protein import during or after translation. Specific examples of suitable signal sequences useful for the CARs discussed in certain embodiments include, but are not limited to, an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha polypeptide. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides discussed herein include CARs of the present disclosure or sequences of the CARs discussed herein in which one or more amino acids have been deleted, added, and / or substituted.

[0186] "Isolated polypeptide" and like expressions, as used herein, refer to a peptide or polypeptide molecule that has been synthesized in vitro, isolated, and / or purified from its cellular environment and from association with other components of a cell. That is, the polypeptide is not significantly associated with in vivo materials. In certain embodiments, an isolated polypeptide is a synthetic polypeptide, a semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0187] Polypeptides include "polypeptide variants." Polypeptide variants can differ from naturally occurring polypeptides by possessing one or more substitutions, deletions, additions, and / or insertions. Such variants can be natural or synthetically produced, and can be created, for example, by modifying one or more of the above-described polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of a CAR by introducing one or more substitutions, deletions, additions, and / or insertions into the binding domain, hinge domain, TM domain, costimulatory signaling domain, or primary signaling domain of the CAR polypeptide. In certain embodiments, the polypeptide comprises a polypeptide having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of the reference sequences discussed herein, and often the variant retains at least one biological activity of the reference sequence. In certain embodiments, the biological activity is binding affinity. In certain embodiments, the biological activity is cytolytic activity.

[0188] Polypeptides include "polypeptide fragments." A polypeptide fragment refers to a polypeptide, which may be monomeric or multimeric, having an amino-terminal deletion, a carboxyl-terminal deletion, and / or internal deletions or substitutions of a naturally occurring or recombinantly produced polypeptide. Specific examples of biologically active polypeptide fragments include antibody fragments. The terms "biologically active fragment" or "minimal biologically active fragment," as used herein, refer to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of the naturally occurring polypeptide. In preferred embodiments, biological activity refers to binding affinity to an idiotype. In certain embodiments, a polypeptide fragment may include an amino acid chain at least 5 to about 500 amino acids in length. In certain embodiments, the length of the fragment 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 fragments of antibodies. In the case of anti-CD79A antibodies, useful fragments include, but are not limited to, CDR regions, CDR3 regions of the heavy or light chain, heavy or light chain variable regions, portions of antibody chains or variable regions comprising two CDRs, and the like.

[0189] The polypeptides may be fused in-frame or conjugated to linkers or other sequences to facilitate polypeptide synthesis, purification, or identification (e.g., poly-His, etc.), or to enhance binding of the polypeptide to a solid support.

[0190] As noted above, in certain embodiments, polypeptides can be modified in various ways, including amino acid substitutions, deletions, truncations, and insertions. Such manipulation methods are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutating DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al. (1987, Methods in Enzymol, 154: 367-382), U.S. Patent No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. For guidance on appropriate amino acid substitutions that do not affect the biological activity of a protein of interest, see the model of Dayhoff et al. ((1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0191] In certain embodiments, polypeptide variants include one or more conservative substitutions. A "conservative substitution" is one in which one amino acid is substituted for another amino acid with similar properties, but which one skilled in the art of peptide chemistry would predict will not substantially alter the secondary structure and hydropathic properties of the polypeptide. Modifications can also be made to the polynucleotide and polypeptide structures discussed in certain embodiments to further obtain functional molecules that encode variant or derivative polypeptides with desired properties. If it is desired to alter the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, one skilled in the art can, for example, change one or more of the codons in the encoding DNA sequence, e.g., according to Table 1. [Table 1]

[0192] Methods for determining amino acid residues that can be substituted, inserted, or deleted without losing biological activity can be found using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, MacVector, or Vector NTI software. The amino acid changes in the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids with similar side chains. Natural amino acids are typically divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. Suitable conservative substitutions of amino acids in peptides or proteins are known to those of skill in the art and can be made without altering the biological activity of the resulting molecule. It is generally recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0193] As outlined above, amino acids can be substituted on the basis of the relative similarity of the amino acid side-chain substituents, for example, hydrophobicity, hydrophilicity, charge, size, and the like.

[0194] Polypeptide variants further include glycosylated forms, conjugates with other molecules, and covalent conjugates with non-interacting chemical moieties (e.g., pegylated molecules). Covalent variants can be prepared by attaching functional groups to groups within the amino acid chain or to groups at the N- or C-terminal residues, as known in the art. Variants also include allelic variants, species variants, and muteins. Truncation or deletion of regions that do not affect the functional activity of the protein are also variants.

[0195] In one embodiment where expression of two or more polypeptides is required, the polynucleotide sequences encoding them may be separated by an IRES sequence as discussed elsewhere herein, hi another embodiment, the two or more polypeptides may be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences.

[0196] Polypeptides discussed in certain embodiments include fusion polypeptides. In preferred embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides, such as CARs, are provided. Fusion polypeptides and fusion proteins refer to polypeptides comprising at least two, three, four, five, six, seven, eight, nine, ten, or more polypeptide segments. Fusion polypeptides are typically linked C-terminus to N-terminus, but can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of a fusion protein can be in any order or a specific order. Fusion polypeptides or proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired transcriptional activity of the fusion polypeptide is maintained. Fusion polypeptides can be produced by chemical synthesis or by chemically linking two moieties, or in many cases, can be produced using other standard methods. The ligated DNA sequence comprising the fusion polypeptide is operably linked to appropriate transcriptional or translational control elements as discussed elsewhere herein.

[0197] In one embodiment, the fusion partner contains a sequence (expression enhancer) that aids in more efficient protein expression than the original recombinant protein. The other fusion partner can be selected to increase the solubility of the protein, to target the protein to a desired intracellular compartment, or to facilitate transport of the fusion protein across the cell membrane.

[0198] The fusion polypeptide may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. The polypeptide cleavage site can also be included in any linker peptide sequence. Examples of polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites, etc.), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004, Traffic, 5(8); 616-26).

[0199] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include the following: These include, but are not limited to, potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. In one embodiment, TEV (Tobacco Etch Virus) protease cleavage sites are preferred due to their high cleavage stringency, examples of which include EXXYXQ(G / S) (SEQ ID NO: 50), 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 between Q and S).

[0200] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0201] Specific examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0202] In one embodiment, the viral 2A peptide is selected from the group consisting of a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0203] Examples of 2A sites are shown in Table 2. [Table 2]

[0204] In a preferred embodiment, the polypeptide comprises an anti-CD79ACAR polypeptide.

[0205] E. Polynucleotides In preferred embodiments, polynucleotides encoding one or more CAR polypeptides are provided. The terms "polynucleotide" or "nucleic acid," as used herein, refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded, and can be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. A polynucleotide refers to a polymeric form of nucleotides, which may be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide, that is 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 10,000, or at least 15,000 nucleotides in length, or any length in between. It will be readily understood that "intermediate lengths" in this context means any length between the specified values, e.g., 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, a polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence.

[0206] Specific examples of polynucleotides include, but are not limited to, SEQ ID NOs: 31 to 36, and polynucleotides encoding SEQ ID NOs: 1 to 30.

[0207] "Isolated polynucleotide," as used herein, refers to a polynucleotide that has been purified from sequences that naturally flank it, e.g., a DNA fragment that has been removed from the sequences that normally flank it. In certain embodiments, "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that are not found in nature and are produced artificially. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0208] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide discussed herein. In certain embodiments, the mRNA comprises a cap, i.e., one or more nucleotides, and a poly(A) tail.

[0209] In certain embodiments, a polynucleotide may be codon-optimized. The term "codon optimization," as used herein, refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors that affect codon optimization include, but are not limited to, (i) differences in codon bias between two or more organisms or genes, or between comprehensively compiled bias tables; (ii) differences in codon bias within an organism, gene, or gene; These include one or more of: differences in the degree of codon bias within a gene group; (iii) systematic differences in codons including context; (iv) differences in codons based on decoding tRNA; (v) differences in codons based on GC% overall or at a position in a triplet; (vi) differences in similarity to a reference sequence, e.g., a natural sequence; (vii) differences in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence required for designing a codon substitution set; (x) systematic differences in the codon set for each amino acid; and / or (xi) isolation and elimination of false translation start sites.

[0210] The terms "polynucleotide variant" and "variant," and similar expressions, as used herein, refer to a polynucleotide that exhibits substantial sequence identity to a reference polynucleotide sequence, or that hybridizes to a reference sequence under stringent conditions, as defined below. These terms include polynucleotides in which one or more nucleotides have been added or deleted, or have been substituted with different nucleotides, compared to the reference polynucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, provided that the modified polynucleotide retains the biological function or activity of the reference polynucleotide.

[0211] Polynucleotide variants include polynucleotide fragments that encode biologically active polypeptide fragments or variants. The term "polynucleotide fragment," as used herein, refers to a polynucleotide fragment at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1100, 1111, 112, 113, 114, 115, 116, 117, 118 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides. A polynucleotide fragment refers to a polynucleotide that encodes a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or internal deletions or substitutions of one or more amino acids of a naturally occurring or recombinantly produced polypeptide.

[0212] The phrase "sequence identity," as used herein, including examples such as "50% identical sequences," refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, calculating the number of positions in both sequences with identical nucleic acid groups (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Also 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; often, the polypeptide variant retains at least one biological activity of the reference sequence.

[0213] Terms used to describe the sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison region," "sequence identity," "percent sequence identity," and "substantial identity." The length of a "reference sequence" is at least 12, but often 15-18 and often at least 25, monomeric units, including nucleotides and amino acid residues. Because two polynucleotides may each contain (1) sequence similarity between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) sequence differences between the two polynucleotides, sequence comparison between two (or more) polynucleotides is performed by comparing "comparison regions" of the two polynucleotide sequences to identify and compare local regions of sequence similarity. A "comparison region" refers to a conceptual segment of at least six contiguous positions, typically about 50 to about 100, more typically about 100 to about 150 contiguous positions. After optimal alignment of the two sequences, the sequence is compared to a reference sequence over the same number of contiguous positions. For optimal alignment of two sequences, the comparison region may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (a sequence that does not contain additions or deletions). Optimal alignment of sequences for aligning comparison regions can be achieved by computerized algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package release 7.0, manufactured by Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA) or by analysis and optimal alignment (i.e., the one that results in the highest percentage of homology in the comparison region) using any of a variety of selected methods. See also, for example, the BLAST family of programs disclosed in Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis is provided in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0214] Terms describing the orientation of a polynucleotide include 5' (typically the end of a polynucleotide with a free phosphate group) and 3' (typically the end of a polynucleotide with a free hydroxyl (OH) group). Polynucleotide sequences may be annotated in the 5' to 3' direction or the 3' to 5' direction. For DNA and mRNA, the 5' to 3' strand is referred to as the "sense," "plus," or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) (except for thymine (T) in DNA, which becomes uracil (U) in RNA). For DNA and mRNA, the complementary 3' to 5' strand is the strand that is transcribed by RNA polymerase and is referred to as the "template," "antisense," "minus," or "non-coding" strand. The term "reverse orientation" as used herein means a 5' to 3' sequence written in a 3' to 5' direction or a 3' to 5' sequence written in a 5' to 3' direction.

[0215] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complement of the DNA sequence 5'AGTCATG3' is 3'TCAGTAC5'. This latter sequence is often written as a reverse complement, 5'CATGACT3', with the 5' end on the left and the 3' end on the right. A sequence identical to this reverse complement is said to be a palindromic sequence. Complementarity can be "partial" if only a portion of the nucleic acid bases are matched according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids.

[0216] Those skilled in the art will also understand that, due to the degeneracy of the genetic code, there are numerous nucleotide sequences that encode fragments of the polypeptides described herein or their variants. Some of these polynucleotides may have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage, e.g., polynucleotides optimized for human and / or primate codon preferences, are specifically contemplated in certain embodiments. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein can also be used. Alleles are endogenous genes that have been modified by one or more mutations, e.g., nucleotide deletions, additions, and / or substitutions.

[0217] The term "nucleic acid cassette" or "expression cassette," as used herein, refers to a genetic sequence within a vector capable of expressing RNA and subsequently expressing a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. A vector may contain one, two, three, four, five, six, seven, eight, nine, ten, or more nucleic acid cassettes. The nucleic acid cassettes are positioned and ordered within the vector so that the nucleic acid within the cassette is transcribed into RNA, translated into a protein or polypeptide, if necessary, and subjected to the appropriate post-translational modifications required for activity in the transformed cell, and then transported to the appropriate region of biological activity by targeting to the appropriate intracellular region or secretion to the extracellular region. The cassette preferably has 3' and 5' ends suitable for immediate insertion into a vector, e.g., has a restriction endonuclease site at each end. In a preferred embodiment, the nucleic acid cassette contains the sequence of a chimeric antigen receptor used to increase the cytotoxicity of cancer cells expressing CD79A. The cassette can be removed or inserted as a single unit into a plasmid or viral vector.

[0218] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide encoding a polypeptide, polypeptide variant, or fusion polypeptide. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In certain embodiments, a polynucleotide of interest encodes a polypeptide that confers a therapeutic effect in the treatment or prevention of a disease or disorder. Polynucleotides of interest, and polypeptides encoded therefrom, include polynucleotides encoding wild-type polypeptides and functional variants and fragments thereof. In certain embodiments, functional variants have at least 80%, at least 90%, at least 95%, or at least 99% identity to a corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, functional variants or fragments have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the corresponding wild-type polypeptide.

[0219] The polynucleotides discussed herein, regardless of the length of their coding sequences, may be combined with other DNA sequences, including promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP sites, FRT sites, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, and the like, as disclosed elsewhere herein or known in the art. Thus, overall lengths may vary widely. Thus, polynucleotide fragments of almost any length may be used in certain embodiments, with the overall length preferably being limited by ease of preparation and use in a given recombinant DNA protocol.

[0220] Polynucleotides can be prepared, manipulated, and / or expressed using any of a variety of well-established techniques known and applicable in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0221] Specific 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.

[0222] Further 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.

[0223] Specific examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40).

[0224] Specific examples of expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, and lentiviral vectors for expression in mammalian cells. pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for mediated gene transfer and expression. In certain embodiments, the coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors to express the polypeptides in mammalian cells.

[0225] In certain embodiments, the vector is an episomal vector, or a vector that remains extrachromosomal. The term "episomal," as used herein, refers to a vector that can replicate without integrating into host chromosomal DNA and without being gradually lost from dividing host cells, i.e., an extrachromosomally or episomally replicating vector.

[0226] "Regulatory elements" or "regulatory sequences" contained in expression vectors are located in untranslated regions of the vector and interact with host cell proteins to effect transcription and translation, such as origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno or Kozak sequences), introns, polyadenylation sequences, and 5' and 3' untranslated regions. Such elements may vary in their strength and specificity. Depending on the vector system and host employed, any number of suitable transcription and translation elements may be used, such as ubiquitous promoters and inducible promoters.

[0227] In certain embodiments, vectors include, but are not limited to, expression vectors and viral vectors, and include exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous" regulatory sequence is a sequence that is naturally linked to a given gene in the genome. An "exogenous" regulatory sequence is a sequence that is placed in proximity to a gene by genetic engineering (i.e., molecular biological techniques) such that transcription of the gene is driven by the linked enhancer / promoter. A "heterologous" regulatory sequence is an exogenous sequence that originates from a species different from the cell being genetically engineered.

[0228] The term "promoter," as used herein, refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, a promoter operable in a mammalian cell includes an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated, and / or another sequence, a CNCAAT region (where N can be any nucleotide), approximately 70 to 80 bases upstream from the transcription start site.

[0229] The term "enhancer" refers to a DNA segment containing sequences that can enhance transcription and, in some instances, function independently of its orientation relative to other regulatory sequences. Enhancers can act cooperatively or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment containing sequences that can confer both promoter and enhancer functions.

[0230] The term "operably linked" refers to a juxtaposition or relationship permitting the described components to function in their intended manner. In one embodiment, the term refers to the functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, such that the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0231] The term "constitutive expression control sequence," as used herein, refers to a promoter, enhancer, or promoter / enhancer that permits continuous or continuous transcription of an operably linked sequence. A constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a wide variety of cell or tissue types, or a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in a limited variety of cell or tissue types.

[0232] Examples of ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the H5 promoter from vaccinia virus, the P7.5 promoter, and the P11 promoter. , elongation factor 1 alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase 1 (PGK) promoter, the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, the β-actin promoter, and the dl587rev primer binding site substitution (MND) U3 promoter of the myeloproliferative sarcoma virus enhancer with deleted negative regulatory regions (Haas et al. Journal of Virology. 2003;77(17):9439-9450).

[0233] In one embodiment, the vector comprises the MNDU3 promoter.

[0234] In one embodiment, the vector comprises the EF1a promoter with the first intron of the human EF1a gene.

[0235] In one embodiment, the vector comprises the EF1a promoter lacking the first intron of the human EF1a gene.

[0236] In certain embodiments, it may be desirable to express the polynucleotide comprising the CAR from a T cell-specific promoter.

[0237] "Conditional expression," as used herein, may refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, and expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell-type- or tissue-specific expression. In certain embodiments, conditional expression of a polynucleotide of interest is used, e.g., to control expression by exposing a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide of interest or that increases or decreases expression of a polynucleotide encoded by the polynucleotide of interest.

[0238] Specific examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as the promoters of the genes encoding the glucocorticoid receptor or estrogen receptor (inducible by treatment with the corresponding hormone), the metallothionein promoter (inducible by treatment with various heavy metals), the MX-1 promoter (inducible by interferon), the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumic acid-inducible gene switch (WO 2002 / 088346), and tetracycline-dependent regulatory system.

[0239] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, a vector includes at least one (typically two) site for recombination mediated by a site-specific recombinase. The term "recombinase" or "site-specific recombinase," as used herein, includes excision or integration proteins, enzymes, cofactors, or related proteins involved in recombination reactions at one or more (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.) recombination sites, which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequence or a fragment thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in certain embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0240] A vector contains one or more recombination sites for any of a variety of site-specific recombinases. It should be understood that a target site for a site-specific recombinase is a portion of a vector (e.g., a retroviral or lentiviral vector) other than any site required for integration. The terms "recombination sequence," "recombination site," or "site-specific recombination site," as used herein, refer to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0241] For example, one recombination site for Cre recombinase is the loxP, a 34-base pair sequence containing two 13-base pair inverted repeats (which act as recombinase binding sites) flanked by an 8-base pair core sequence (Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994) (see Figure 1). Other examples of 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).

[0242] 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), FR T(RE) (Senecoff et al., 1988).

[0243] Other examples of recognition sequences include the attB, attP, attL, and attR sequences recognized by the recombinase enzyme λ integrase, e.g., phi-c31. The phi-c31 SSR mediates recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). attB and attP are the names given to the attachment sites for phage integrase in the bacterial and phage genomes, respectively, containing imperfect inverted repeats thought to be bound by the φC31 homodimer (Groth et al., 2000). The product sites attL and attR are essentially inactive for further φC31-mediated recombination (Belteki et al., 2003), rendering the reaction irreversible. For the induction of insertion, attB It has been shown that inserting DNA into a genomic attP site is easier than inserting an attP site into a genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy involves inserting a desired DNA into a genomic attB site by homologous recombination. An attP-containing "docking site" is placed at the locus, which is then inserted in combination with another attB-containing sequence.

[0244] "Internal ribosome entry site" or "IRES," as used herein, refers to an element that directs entry of an internal ribosome at the start codon (e.g., ATG) of a cistron (protein coding region), thereby resulting in cap-independent translation of a gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83, and See Jackson and Kaminski. 1995. RNA 1(10):985-1000. In some embodiments, the vector comprises one or more polynucleotides of interest encoding one or more polypeptides. In certain embodiments, to efficiently translate each of the multiple polypeptides, the polynucleotide sequences can be separated by one or more IRES sequences or by a polynucleotide sequence encoding a self-cleaving polypeptide. In one embodiment, the IRES used in the polynucleotides discussed herein is the EMCV IRES.

[0245] The term "Kozak sequence," as used herein, refers to a short nucleotide sequence that strongly promotes the initial binding of mRNA to the small ribosomal subunit and increases translation. The consensus 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 Res. 15(20):8125-48). In certain embodiments, the vector comprises a conjugated nucleotide sequence. It includes a polynucleotide having a sensor Kozak sequence and encoding a desired polypeptide, such as a CAR.

[0246] Elements that induce efficient termination and polyadenylation of heterologous nucleic acid transcription increase heterologous gene expression. Transcription termination signals are generally located downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' of a polynucleotide encoding an expressed polypeptide. As used herein, the terms "polyA site" and "polyA sequence" refer to a DNA sequence that induces termination and polyadenylation of nascent RNA transcription by RNA polymerase II. Polyadenylation sequences can increase mRNA stability by adding a polyA tail to the 3' end of a coding sequence, thereby contributing to improved translation efficiency. Cleavage and polyadenylation are induced by the poly(A) sequence of the RNA. The core poly(A) sequence of mammalian pre-mRNAs contains two recognition elements flanking the cleavage-polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is located 20 to 50 nucleotides upstream of a highly variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements, with the addition of up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is an SV40 poly(A) sequence, bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA), variants thereof, or other suitable heterologous or endogenous poly(A) sequences known in the art.

[0247] In certain embodiments, the polynucleotide or cells harboring the polynucleotide utilize a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain aspects, the suicide gene is not immunogenic to the host harboring the polynucleotide or cells. Specific examples of suicide genes that can be used are caspase 9, caspase 8, or cytosine deaminase. Caspase 9 can be activated using a specific chemical inducer of dimerization (CID).

[0248] In certain embodiments, one or more polynucleotides encoding an anti-CD79 ACAR are introduced into cells (e.g., immune effector cells) using a non-viral or viral vector.

[0249] The term "vector" is used herein to mean a nucleic acid molecule capable of introducing or transporting another nucleic acid molecule. The nucleic acid to be introduced is typically linked, e.g., inserted, into the vector nucleic acid molecule. The vector may contain a sequence that induces autonomous replication in a cell or may contain a sequence that allows integration into host cell DNA. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides discussed herein to T cells. In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding an anti-CD79 ACAR.

[0250] Examples of non-viral vectors include, but are not limited to, mRNA, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0251] Specific examples of non-viral delivery of polynucleotides contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistic bombardment, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid-nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated delivery, gene guns, and heat shock.

[0252] Specific examples of polynucleotide delivery systems suitable for use in certain embodiments contemplated in certain embodiments include, but are not limited to, those provided by Amaxa Biosystems, Inc., Maxcyte, Inc., BTX Molecular Delivery Systems, Inc., and Copernicus Therapeutics, Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for transfection are described in the literature, e.g., Liu et al. (2003) Gene Therapy. 10:180-187, and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Delivery of non-biological nano-cell systems derived from bacteria is also contemplated in certain embodiments.

[0253] In various embodiments, the polynucleotide is an mRNA that is introduced into a cell to transiently express a desired polypeptide. "Transient," as used herein, refers to expression of an unintroduced transgene for a period of hours, days, or weeks, which is shorter than the period of expression of the polynucleotide when introduced into the genome or inserted into an appropriate plasmid replicon in the cell.

[0254] In certain embodiments, the mRNA encoding the polypeptide is in vitro transcribed mRNA. The term "in vitro transcribed RNA" as used herein refers to RNA, preferably mRNA synthesized in vitro. In vitro transcribed RNA is usually produced from an in vitro transcription vector. The in vitro transcription vector comprises a template used to produce in vitro transcribed RNA.

[0255] In certain embodiments, the mRNA may further comprise a 5' cap or modified 5' cap and / or poly(A) sequence. A 5' cap (also referred to as an RNA cap, RNA 7-methylguanosine cap, or RNAm7G cap), as used herein, is a modified guanine nucleotide added to the "head" or 5' end of a eukaryotic messenger RNA shortly after transcription initiation. The 5' cap is linked to the first transcribed nucleotide and includes a terminal group that is recognized by the ribosome and provides protection from ribonucleases. The capping moiety can be modified to modulate mRNA functionality, such as translational stability or efficiency. In certain embodiments, the mRNA comprises a poly(A) sequence consisting of about 50 to about 5,000 adenines. In one embodiment, the mRNA comprises a poly(A) sequence of about 100 to about 1,000 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 catalytically modified to modulate mRNA functionality, such as localization, translational stability, or translational efficiency.

[0256] Viral vectors containing polynucleotides contemplated in certain embodiments can be delivered in vivo by administration to an individual patient, generally by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection) or local administration, as described below. Alternatively, vectors can be delivered ex vivo to cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy, etc.) or to cells such as universal donor hematopoietic stem cells, which can then be reimplanted into the patient.

[0257] In one embodiment, a viral vector containing a polynucleotide encoding an anti-CD79ACAR is directly administered to an organism to transduce cells in vivo. Alternatively, naked DNA can be administered. Administration is by any route commonly used to maximize contact of molecules with blood or tissue cells, including, but not limited to, injection, infusion, topical administration, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art. While more than one route may be used to administer a particular composition, one particular route will often result in a more immediate and efficient response than another route.

[0258] Specific examples of viral vector systems suitable for use in certain embodiments discussed herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0259] In various embodiments, one or more polynucleotides encoding an anti-CD79 ACAR are incorporated into immune effector cells, e.g., T cells, by transduction with a recombinant adeno-associated virus (rAAV) containing the one or more polynucleotides.

[0260] AAV is a small (approximately 26 nm) and primarily episomal, replication-deficient, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of the host cell. Recombinant AAV (rAAV) typically consists of at least a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are approximately 145 bp in length. In a specific embodiment, rAAV comprises ITRs and a capsid sequence isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0261] In some embodiments, chimeric rAAV is used, in which the ITR sequence is isolated from one AAV serotype and the capsid sequence is isolated from a different AAV serotype. For example, a rAAV with an ITR sequence from AAV2 and a capsid sequence from AAV6 is referred to as AAV2 / AAV6. In certain embodiments, a rAAV vector can comprise an ITR from AAV2 and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, a rAAV comprises an ITR sequence from AAV2 and a capsid sequence from AAV6. In a preferred embodiment, a rAAV comprises an ITR sequence from AAV2 and a capsid sequence from AAV2.

[0262] In certain embodiments, engineering and selection methods can be applied to the AAV capsid to make it more likely to transduce cells of interest.

[0263] The construction, production, and purification of rAAV vectors are disclosed in 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 by reference herein in its entirety.

[0264] In various embodiments, one or more polynucleotides encoding an anti-CD79 ACAR are incorporated into immune effector cells by transducing the cells with a retrovirus (e.g., a lentivirus) containing the one or more polynucleotides.

[0265] The term "retrovirus," as used herein, refers to an RNA virus that reverse-transcribes genomic RNA into a linear, double-stranded DNA copy, which then covalently integrates the genomic DNA into the host genome. Specific examples of retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0266] The term "lentivirus," as used herein, refers to a group (or genus) of complex retroviruses. Specific examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus (including HIV types 1 and 2)), Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0267] In various embodiments, the lentiviral vectors discussed herein include one or more LTRs and one or more or all of the following accompanying elements: cPPT / FLAP, Psi(Ψ) packaging signal, transport element, poly(A), and optionally, a WPRE or HPRE, an insulator sequence, a selectable marker, and a cell suicide gene, as described anywhere herein.

[0268] In certain embodiments, the lentivirus vector discussed herein can be an integrating lentivirus, a non-integrating lentivirus, or an integration-defective lentivirus.The term " integration-defective lentivirus " or " IDLV " as used herein refers to the lentivirus that has an integrase that is unable to integrate viral genome into the genome of host cell.Integration-defective viral vectors are described in International Publication No. 2006 / 010834, and this patent application is incorporated herein in its entirety by reference.

[0269] Examples of mutations in the HIV-1 pol gene that are suitable for reducing integrase activity include, but are not limited to, H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A , E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, and K264H.

[0270] In one embodiment, the HIV-1 integrase defective pol gene includes the mutations D64V, D116I, D116A, E152G, or E152A, or the mutations D64V, D116I, and E152G, or the mutations D64V, D116A, and E152A.

[0271] In one embodiment, the HIV-1 integrase defective pol gene comprises a D64V mutation.

[0272] The term "long terminal repeat (LTR)" refers to the domain of base pairs located at the end of retroviral DNA which, in its native sequence configuration, is a direct repeat and contains the U3, R, and U5 regions.

[0273] The term "FLAP element" or "cPPT / FLAP," as used herein, refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus (e.g., 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, a lentiviral vector includes a FLAP element with one or more mutations in the cPPT element and / or the CTS element. In yet another embodiment, a lentiviral vector includes a cPPT element or a CTS element. In yet another embodiment, a lentiviral vector does not include a cPPT element or a CTS element.

[0274] The term "packaging signal" or "packaging sequence," as used herein, refers to the psi [Ψ] sequence in a retroviral genome that is required for the insertion of viral RNA into the viral capsid or viral particle (see, e.g., Clever et al. al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109).

[0275] The term "transport element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA transport elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65: 1053, and Cullen et al., 1991. Cell 58: 423) and the hepatitis B virus post-transcriptional regulatory element (HPRE).

[0276] In certain embodiments, the expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, high-efficiency polyadenylation sites, and optionally transcription termination signals into viral vectors.Various posttranscriptional regulatory elements can increase the expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE, Zufferey et al., 1999, J. Virol., 73:2886), the hepatitis B virus posttranscriptional regulatory element (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864), and similar elements (Liu et al., 1995, Genes Dev., 9:1766).

[0277] Lentiviral vectors preferably contain several safety enhancements as a result of modifications to the LTR. "Self-inactivating" (SIN) vectors refer to replication-deficient vectors, for example, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., deleted or replaced) to prevent viral transcription after the first viral replication cycle. Further safety enhancements can be achieved 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 promoters from the viruses 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).

[0278] The term "pseudotype" or "pseudotyping," as used herein, refers to a virus having a viral envelope protein replaced with a protein from another virus that has favorable properties. For example, HIV can be pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope protein, allowing HIV to infect a broad range of cells. Generally, the HIV envelope protein (the protein encoded by the env gene) inhibits the virus from infecting CD4 + This is because it targets the presenting cells.

[0279] In certain embodiments, lentiviral vectors are produced by known methods, e.g., Kutner et al., BMC Biotechnol. 2009;9:10. doi: 10.1186 / 1472-6750-9-10, Kutner et al. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038 / nprot.2009.22 Please refer to.

[0280] According to certain embodiments discussed herein, most or all of the viral vector backbone sequences are derived from a lentivirus (e.g., HIV-1). However, it should be understood that various sources of retroviral and / or lentiviral sequences can be used, and that multiple substitutions and modifications can be combined within the lentiviral sequences without impairing the ability of the transfer vector to perform the functions described herein. Additionally, various lentiviral vectors are known in the art, and are described in detail in Naldini et al., (1996a, 1996b, and 1998), Zufferey et al., (1997), Dull et al., 1998 See U.S. Patent Nos. 6,013,516 and 5,994,136. Many of these vectors can be adapted to produce the viral vectors or transfer plasmids discussed herein.

[0281] In various embodiments, one or more polynucleotides encoding an anti-CD79 ACAR are incorporated into immune effector cells by transduction with an adenovirus containing the one or more polynucleotides.

[0282] Adenovirus-based vectors can produce extremely high transduction efficiencies in many cell types and do not require cell division. High titers and high levels of expression have been obtained using these vectors. The vectors can be produced in large quantities using a relatively simple system. Most adenovirus vectors have been engineered so that a transgene replaces the AdE1a, AdE1b, and / or AdE3 genes, and the replication-deficient vector is then propagated in human 293 cells, which provide the deleted gene function in trans. Adenovirus (Ad) vectors can transduce multiple tissues in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney, and muscle. Ad vectors typically have high carrying capacity.

[0283] A unique helper cell line called 293 can be used to produce and propagate current replication-deficient adenoviral vectors. This cell line was transformed from human embryonic kidney cells with Ad5 DNA fragments and constitutively expresses E1 proteins (Graham et al., 1977). Because the E3 region is not essential for the adenoviral genome (Jones & Shenk, 1978), current adenoviral vectors utilize 293 cells to carry foreign DNA in the E1 region, the D3 region, or both (Graham & Prevec, 1991). Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Methods of administration to various tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). An example of the use of Ad vectors in clinical trials is polynucleotide therapy for anti-tumor immunization by intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0284] In various embodiments, one or more polynucleotides encoding an anti-CD79 ACAR are incorporated into cells by transducing immune effector cells with a herpes simplex virus (e.g., HSV-1, HSV-2) containing the one or more polynucleotides.

[0285] Mature HSV virions are composed of an enveloped icosahedral capsid with a viral genome consisting of a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV-based viral vector is deficient in 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 have deletions that eliminate one or more intermediate-early, early, or late HSV genes, preventing replication. For example, HSV vectors may be deficient in an immediate-early gene selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors are their ability to enter a latent stage, which can result in long-term DNA expression, and their large viral DNA genome, which can incorporate exogenous DNA inserts up to 25 kb. HSV-based vectors are described, for example, in U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, each of which is incorporated herein by reference in its entirety.

[0286] F. Genetically Modified Cells In various embodiments, cells are provided that have been genetically modified to express the CAR discussed herein, and are used to treat cancer. As used herein, the term "genetic manipulation" or "genetic modification" refers to the addition of additional genetic material, in the form of DNA or RNA, to the total genetic material of 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 of a cell to restore, repair, or modify the expression of a gene or to express a therapeutic polypeptide, such as a CAR.

[0287] In certain embodiments, the anti-CD79ACARs discussed herein are introduced into and expressed in immune effector cells to redirect their specificity to a target antigen of interest, e.g., a CD79A polypeptide. An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC). Examples of immune effector cells discussed herein are T lymphocytes, including, but not limited to, cytotoxic T cells (CTLs, CD8 + T cells), TIL, and helper T cells (HTL, CD4 + In certain embodiments, the cells comprise αβ T cells. In certain embodiments, the cells comprise γδ T cells. In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0288] Immune effector cells may be autologous / autonomous ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic). "Autologous," as used herein, means cells derived from the same subject. "Allogeneic," as used herein, means cells of the same type that are genetically different from the cells of a control. "Syngeneic," as used herein, means cells from a different subject that are genetically identical to the cells of a control. "Xenogeneic," as used herein, means cells of a different type than the cells of a control. In a preferred embodiment, the cells are allogeneic.

[0289] Examples of immune effector cells for use with the anti-CD79 ACARs contemplated in certain embodiments include T lymphocytes. The terms "T cell" or "T lymphocyte" are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, e.g., T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells may be helper T cells (HT1, CD4+ T cells)CD4 + T cells, cytotoxic T cells (CTL, CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The T cells may be T cells, or any other T cell subset. Other examples of T cells suitable for use in certain embodiments include naive T cells (TN), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF).

[0290] As will be appreciated by those skilled in the art, other cells can also be used as immune effector cells in conjunction with the anti-CD79 ACARs discussed herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, which can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include CD34 cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood. + The cell population includes precursors of immune effector cells, such as hematopoietic stem cells (HSCs), which differentiate into mature immune effector cells upon administration to a subject or can be induced in vitro to differentiate into mature immune effector cells. Immune effector cells engineered to contain a CD79A-specific CAR may be referred to as "CD79A-specific redirected immune effector cells" as used herein.

[0291] The term "CD34 + "CD34" as used herein refers to a cell that expresses the CD34 protein on its cell surface. "CD34" as used herein refers to a cell surface glycoprotein (e.g., a sialomucin protein) that generally acts as an intercellular adhesion factor and is involved in T cell entry into lymph nodes. CD34 +The cell population includes hematopoietic stem cells (HSCs), which upon administration to a patient differentiate into all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0292] Methods for producing immune effector cells expressing an anti-CD79 ACAR discussed herein are described in certain embodiments. In one embodiment, the method includes transfecting or transducing immune effector cells isolated from an individual to cause the immune effector cells to express one or more anti-CD79 ACARs discussed herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. These cells can be directly readministered to the individual. In a further embodiment, the immune effector cells are first activated and stimulated, expanded in vitro, and then genetically modified to express an anti-CD79 ACAR. In this regard, the immune effector cells can be cultured before and / or after being genetically modified (i.e., transduced or transfected to express an anti-CD79 ACAR discussed herein).

[0293] In certain embodiments, a source of the cells is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein, hi certain embodiments, the CAR-modified immune effector cells comprise T cells.

[0294] In certain embodiments, PBMCs may be directly genetically modified to express an anti-CD79 ACAR using the methods discussed herein. In certain embodiments, PBMCs are isolated followed by further isolation of T lymphocytes. Also, in certain embodiments, cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion.

[0295] Immune effector cells, such as T cells, can be isolated using known methods and then genetically modified, or the immune effector cells can be activated and expanded (or differentiated, in the case of precursors) in vitro before being genetically modified. In certain embodiments, immune effector cells, such as T cells, are genetically modified with a chimeric antigen receptor (e.g., transduced with a viral vector comprising a nucleic acid encoding an anti-CD79 ACAR) as discussed herein, followed by in vitro activation and expansion. In various embodiments, methods are used in accordance with, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,5 T cells may be activated and expanded before or after being genetically modified to express a CAR using methods described in U.S. Patent Application Publication Nos. 66, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0296] In one embodiment, CD34 + The cells are transduced with a nucleic acid construct as discussed herein. In certain embodiments, the transduced CD34 + The cells are administered to a subject, typically the subject from whom the isolated cells were obtained, and then differentiated in vivo into mature immune effector cells. + Cells may be stimulated in vitro with one or more of the following cytokines, Flt-3 ligand (FLT3), stem cell factor (SCF), megakaryocyte differentiation factor (TPO), IL-3, and IL-6, according to previously described methods, before exposure to a CAR or after being genetically modified with a CAR as described herein (Asheuer et al., 2004; Imren, et al., 2004).

[0297] In certain embodiments, the modified immune effector cell population for cancer treatment comprises the CAR discussed herein. For example, the modified immune effector cell population is prepared from peripheral blood mononuclear cells (PBMCs) obtained from patients (autologous donors) diagnosed with B-cell malignancies as described herein. PBMCs are CD4 + , CD8 + , or CD4 + and CD8 + These cells form a heterogeneous population of T lymphocytes that can become

[0298] PBMCs may also contain other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying a coding sequence for a CAR discussed in certain embodiments is introduced into a human donor T cell population, NK cell population, or NKT cell population. In certain embodiments, transduced T cells carrying the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells, which can then be further expanded to expand the number of CAR protein-expressing T cells, and cell activation can be enhanced using anti-CD3 and / or anti-CD28 antibodies and IL-2, or any method known in the art described elsewhere herein. T cells expressing the CAR protein T cells are cryopreserved using standard procedures for storage and / or preparation for use in human subjects. In one embodiment, the in vitro transduction, culture, and / or expansion of T cells is performed without the use of non-human animal-derived products, such as fetal bovine serum and fetal bovine serum. Because a heterogeneous population of PBMCs is genetically modified, the resulting transduced cells are a heterogeneous population of engineered cells containing the anti-CD79ACAR discussed herein.

[0299] In further embodiments, a mixture of several (e.g., one, two, three, four, five, or more) different expression vectors can be used to genetically modify a donor population of immune effector cells, each vector encoding a different chimeric antigen receptor protein as discussed herein. The resulting modified immune effector cells express two or more different CAR proteins, forming a mixed population of modified cells.

[0300] GT cell production method In various embodiments, the genetically modified T cells are expanded by contacting them with an agent that stimulates a CD3TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.

[0301] In certain embodiments, PBMCs or isolated T cells are contacted with stimulatory and costimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies bound to beads or other surfaces, in medium containing appropriate cytokines, such as IL-2, IL-7, and / or IL-15.

[0302] In certain embodiments, PBMCs or isolated T cells are contacted with stimulatory and costimulatory agents, such as soluble anti-CD3 and anti-CD28 antibodies, or antibodies bound to beads or other surfaces, in medium containing appropriate cytokines, such as IL-2, IL-7, and / or IL-15, and / or one or more agents that modulate the PI3K / Akt / mTOR cell signaling pathway.

[0303] In preferred embodiments, T cells produced by the methods discussed herein provide improved adoptive immunotherapy compositions. While not wishing to be bound by any particular theory, it is believed that T cell compositions produced by the methods of certain embodiments discussed herein have superior properties, such as increased survival, a tendency to remain relatively undifferentiated, and persistence in vivo. In one embodiment, a method for producing T cells includes contacting T cells with one or more agents that modulate the PI3K cell signaling pathway. In one embodiment, a method for producing T cells includes contacting T cells with one or more agents that modulate the PI3K / Akt / mTOR cell signaling pathway. In various embodiments, T cells can be obtained from any source and contacted with the agents during the activation and / or expansion phases of the production process. The resulting T cell composition is enriched for developmentally potent T cells that are capable of expanding and expressing one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, CD38, and CD8. In one embodiment, the cell population comprising T cells treated with one or more PI3K inhibitors includes CD8 T cells that co-express one or more, or all, of the following biomarkers: CD62L, CD127, CD197, and CD38. + It contains a large number of T cells.

[0304] In one embodiment, the cell population comprising T cells treated with one or more PI3K inhibitors includes CD8 T cells that co-express one or more, or all, of the following biomarkers: CD62L, CD127, CD27, and CD8. + It contains a large number of T cells.

[0305] In one embodiment, modified T cells are produced that have retained levels of proliferation and reduced differentiation. In a specific embodiment, the T cells are produced by stimulating the T cells to activate and proliferate in the presence of one or more stimulatory signals and an agent that is an inhibitor of the PI3K cell signaling pathway.

[0306] The T cells can then be engineered to express the anti-CD79 ACAR. In one embodiment, the T cells are engineered by transducing them with a viral vector comprising the anti-CD79 ACAR discussed herein. In a specific embodiment, the T cells are engineered prior to stimulation and activation in the presence of an inhibitor of the PI3K cell signaling pathway. In another embodiment, the T cells are engineered after stimulation and activation in the presence of an inhibitor of the PI3K cell signaling pathway. In a specific embodiment, the T cells are engineered 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.

[0307] After activation, the T cells are expanded in culture. The T cells can be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more expansion cycles for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, at least 2 weeks, at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.

[0308] In various embodiments, the T cell compositions are produced in the presence of one or more inhibitors of the PI3K / Akt / mTOR cell signaling pathway. The inhibitor may target one or more activities in the pathway, or a single activity. Without wishing to be bound by any particular theory, it is believed that treating or contacting T cells with one or more inhibitors of the PI3K pathway during the stimulation, activation, and / or expansion phases of the production process preferentially results in the expansion of immature T cells, resulting in a superior therapeutic T cell composition.

[0309] In certain embodiments, methods are provided for increasing the proliferation of T cells expressing an engineered T cell receptor. Such methods can include, for example, harvesting a source of T cells from a subject, stimulating and activating the T cells in the presence of one or more inhibitors of the PI3K pathway, modifying the T cells to express an anti-CD79 ACAR, and expanding the T cells in culture.

[0310] In certain embodiments, methods are contemplated for producing a population of T cells enriched for expression of one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, CD38, and CD8. In one embodiment, immature T cells include one or more, or all, of the biomarkers CD62L, CD127, CD197, and CD38.

[0311] In one embodiment, immature T cells include one or more, or all of the biomarkers CD62L, CD127, CD27, and CD8.

[0312] In one embodiment, immature T cells are provided that do not express CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3. As discussed elsewhere herein, the expression levels of immature T cell biomarkers correlate with the expression levels of biomarkers of more differentiated T cell or immune effector cell populations.

[0313] In one embodiment, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell manufacturing methods discussed herein. PBMCs are CD4 + , CD8 + , or CD4 + and CD8 +These cells form a heterogeneous population of T lymphocytes that can be transformed into T cells, and may include monocytes, B cells, NK cells, and other mononuclear cells such as NKT cells. An expression vector containing a polynucleotide encoding an engineered TCR or CAR as contemplated in certain embodiments is introduced into a human donor T cell, NK cell, or NKT cell population. In certain embodiments, the transduced T cells harboring the expression vector can be sorted using flow cytometry to isolate CD3-positive T cells, which can then be further expanded to expand the number of engineered T cells, and cell activation can be enhanced with anti-CD3 and / or anti-CD28 antibodies, as well as IL-2, IL-7, and / or IL-15.

[0314] The manufacturing methods discussed herein can also include cryopreservation of engineered T cells for storage and / or preparation for use in human subjects. In one embodiment, a method for preserving immune effector cells expressing a genetically engineered mouse CAR protein, a human CAR protein, or a humanized CAR protein that targets CD79A-expressing cells includes cryopreserving the immune effector cells so that they remain viable upon thawing. A fraction of the immune effector cells expressing the CAR protein can be cryopreserved by methods known in the art to provide a continuous source of cells for future treatment of patients with CD79A-expressing cancer cells. T cells are cryopreserved so that they remain viable upon thawing. If necessary, cryopreserved transformed immune effector cells can be thawed, grown, and expanded to expand. "Cryopreservation," as used herein, refers to the preservation of cells, for example, by cooling (typically) to a subzero temperature of 77K, i.e., -196°C (the boiling point of liquid nitrogen). Cryoprotectants are often used at subzero temperatures to prevent damage to preserved cells caused by freezing at low temperatures or by warming to room temperature. The cryoprotectant and optimal cooling rate can protect cells from damage. Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). A preferred cooling rate is 1 °C / min to 3 °C / min. After at least 2 hours, the T cells will reach -80°C and can be placed directly into, for example, liquid nitrogen (-196°C) in a long-term cryogenic storage container for long-term storage.

[0315] 1.T cells The production of improved CAR-T cell compositions is described in specific embodiments. T cells used for CAR-T cell production can be autologous / autonomous ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic). In preferred embodiments, T cells are obtained from a mammalian subject. In more preferred embodiments, T cells are obtained from a primate subject. In most preferred embodiments, T cells are obtained from a human subject.

[0316] 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 an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any of a variety of techniques known to those of skill in the art, such as sedimentation techniques, e.g., FICOLL™ separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place them in an appropriate buffer or medium for subsequent processing. Cells can be washed with PBS or other appropriate solutions containing little or no calcium, magnesium, or divalent cations. As will be appreciated by those skilled in the art, washing steps can be performed by methods known in the art, such as using a semi-automated flow-through centrifuge. Examples include the Cobe 2991 cell processing device and the Baxter CytoMate. After washing, the cells can be resuspended in a variety of biocompatible buffers or other saline solutions, with or without buffers. In certain embodiments, undesirable components in the apheresis sample can be removed directly in the medium in which the cells are resuspended.

[0317] In certain embodiments, a population of cells comprising T cells, e.g., PBMCs, is used in the manufacturing methods discussed herein. In other embodiments, an isolated or purified population of T cells is used in the manufacturing methods discussed herein. For example, centrifugation on a PERCOLL™ gradient Cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes using isolation. In certain embodiments, after PBMC isolation, cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after activation, expansion, and / or genetic modification.

[0318] In certain embodiments, a population of cells comprising T cells, e.g., PBMCs, is used in the manufacturing methods discussed herein. In other embodiments, an isolated or purified population of T cells is used in the manufacturing methods discussed herein. For example, centrifugation on a PERCOLL™ gradient Cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes using isolation. In certain embodiments, after PBMC isolation, cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after activation, expansion, and / or genetic modification.

[0319] In certain embodiments, immune effector cell populations are produced from PBMCs that have been genetically modified to express a CAR using the methods discussed herein, but have not been subjected to either positive or negative selection. In certain embodiments, after isolating the PBMCs, T lymphocytes are further isolated. Also, in certain embodiments, cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion.

[0320] In certain embodiments, specific subpopulations of T cells expressing one or more of the following markers CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques. In one embodiment, specific subpopulations of T cells expressing one or more markers selected from the group consisting of: i) CD62L, CCR7, CD28, CD27, CD122, CD127, and CD197; ii) CD62L, CD127, CD197, and CD38; or iii) CD62L, CD127, CD27, and CD8 are further isolated by positive or negative selection techniques. In various embodiments, the T cell compositions produced do not express, or substantially do not express, one or more of the following markers CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0321] 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 without a PI3K inhibitor. + Contains T cells.

[0322] In one embodiment, the expression of one or more 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 with a PI3K inhibitor. +Contains T cells.

[0323] In one embodiment, the manufacturing methods discussed herein expand the number of CAR-T cells that contain one or more markers of naive T cells or developmentally potent T cells. Without wishing to be bound by any particular theory, the inventors believe that treating a cell population comprising T cells with one or more PI3K inhibitors increases the expansion of developmentally potent T cells, resulting in a more robust and effective adoptive CAR-T cell immunotherapy compared to current CAR-T cell therapies.

[0324] Specific examples of markers of increased naive or developmentally potent T cells in T cells produced using the methods contemplated in certain embodiments include, but are not limited to, i) CD62L, CD127, CD197, and CD38, or ii) CD62L, CD127, CD27, and CD8. In certain embodiments, naive T cells do not express, or substantially do not express, one or more of the following markers: CD57, CD244, CD160, PD-1, BTLA, CD45RA, CTLA4, TIM3, and LAG3.

[0325] With respect to T cells, the T cell populations obtained by the various expansion methods discussed herein may have a variety of specific phenotypic characteristics depending on the context of use. In various embodiments, the expanded T cell populations include one or more of the following phenotypic markers: CD62L, CD27, CD127, CD197, CD38, CD8, and HLA-DR.

[0326] 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, CD8 T cells are characterized by expression of naive T cell phenotypic markers, including CD62L, CD127, CD197, and CD38. + Proliferates T lymphocytes.

[0327] 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 naive T cell phenotypic markers includes CD8, characterized by expression of naive T cell phenotypic markers including CD62L, CD127, CD27, and CD8. + Proliferates T lymphocytes.

[0328] In certain 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 are negative for granzyme B. In certain embodiments, central memory T cells are CD45RO + T cells, CD62L + T cells, CD8 + T cells.

[0329] In certain embodiments, naive CD4 + characterized by the expression of phenotypic markers of CD4 cells and negative for the expression of CD45RA and / or CD45RO, + In one embodiment, CD4 + The cells are CD62L-containing central memory CD4 + In one embodiment, the cells are characterized by the expression of phenotypic markers of CD45RO and are CD45RO positive. + The cells are CD62L positive and CD45RO negative.

[0330] In certain embodiments, T cells are isolated from an individual, activated and stimulated, expanded in vitro, and then genetically modified to express an anti-CD79 ACAR. In this regard, the T cells can be cultured before and / or after being genetically modified (i.e., transduced or transfected to express an anti-CD79 ACAR as discussed herein).

[0331] 2. Activation and proliferation To obtain a sufficient therapeutic dose of a T cell composition, the T cells often undergo one or more cycles of stimulation, activation, and / or expansion. Generally, T cells can be activated and expanded using methods described in the following documents, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041, each of which is incorporated herein by reference in its entirety. T cells engineered to express an anti-CD79 ACAR can be activated and / or expanded before and / or after engineering the T cells. Additionally, the T cells may 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 produced by the methods discussed herein may be activated and expanded for one, two, three, four, five, or more cycles, each of which may include one or more agents that modulate the PI3K / Akt / mTOR cell signaling pathway.

[0332] Artificial antigen-presenting cells (aAPCs) express functional human CD8+ cells without the need for exogenous cytokines, unlike natural APCs. + The ex vivo growth and long-term proliferation of T cells is sustained. In certain embodiments, PBMCs or isolated T cells are contacted with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in medium containing appropriate cytokines, such as IL-2, IL-7, and / or IL-15.

[0333] In other embodiments, artificial APCs (aAPCs) have been generated by engineering K562, U937, 721.221, T2, and C1R cells to induce stable expression and secretion of various costimulatory molecules and cytokines. In specific embodiments, K32 or U32 aAPCs are used to induce the presentation of one or more antibody-based stimulatory molecules on the aAPC cell surface. T cell populations can be expanded with aAPCs expressing various costimulatory molecules, including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and / or CD80 or CD86. aAPCs can expand genetically modified T cells and express CD8 + This provides an effective platform for maintaining CD28 expression on T cells. The aAPCs described in WO 03 / 057171 and U.S. Patent Application Publication No. 2003 / 0147869 are incorporated herein by reference in their entireties.

[0334] In one embodiment, a costimulatory ligand is presented on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a desired T cell response, in addition to the primary signal provided by, for example, engagement of the TCR / CD3 complex. Suitable costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3.

[0335] In certain embodiments, the costimulatory ligand comprises an antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule presented 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.

[0336] Suitable costimulatory ligands further include target antigens, which may be in soluble form or expressed on an APC or aAPC that bind the modified TCR or CAR expressed on the modified T cell.

[0337] In various embodiments, the methods of producing T cells discussed herein include activating a cell population comprising T cells and expanding the T cell population. T cells can be activated by providing a primary stimulatory signal via the TCR / CD3 complex or via stimulation of the CD2 surface protein on the T cells, and by providing a secondary costimulatory signal via an accessory molecule, such as CD28.

[0338] The TCR / CD3 complex can be stimulated by contacting the T cells with a suitable CD3-binding agent, such as a CD3 ligand or an anti-CD3 monoclonal antibody. Specific examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1.

[0339] In another embodiment, a CD2 binding agent can be used to provide a primary stimulatory signal to T cells. Specific examples of CD2 binding agents include, but are not limited to, CD2 ligands and anti-CD2 antibodies, such as the T11.3 antibody in combination with the T11.1 antibody or the T11.3 antibody in combination with the T11.2 antibody (Meuer, SC et al. (1984) Cell 36:897-906), and the 9.6 antibody (which recognizes the same epitope as TI 1.1) in combination with the 9-1 antibody (Yang, SY et al. (1986) J. Immunol. 137:1097-1100). Other antibodies that bind to the same epitope as any of the above-mentioned antibodies can also be used. Additional antibodies or antibody combinations can be prepared and identified by standard methods as described elsewhere herein.

[0340] Induction of a T cell response requires a secondary costimulatory signal in addition to the primary stimulatory signal provided via the TCR / CD3 complex or via CD2. In certain embodiments, a CD28-binding agent can be used to provide the costimulatory signal. Specific examples of CD28-binding agents include, but are not limited to, natural CD28 ligands, e.g., natural ligands of CD28 (e.g., members of the B7 family of proteins, such as B7-1 (CD80) and B7-2 (CD86)), and anti-CD28 monoclonal antibodies or fragments thereof capable of cross-linking the CD28 molecule, such as monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10).

[0341] In one embodiment, a molecule that provides a primary stimulatory signal, eg, a molecule that stimulates via the TCR / CD3 complex or via CD2, and a costimulatory molecule are associated on the same surface.

[0342] In certain embodiments, the binding agents that provide stimulatory and costimulatory signals are localized on the cell surface, which can be achieved by transfecting or transducing the cells with a nucleic acid encoding the binding agent in a form suitable for expression at the cell surface, or by binding the binding agent to the cell surface.

[0343] In another embodiment, a molecule that provides a primary stimulatory signal, e.g., a molecule that stimulates via the TCR / CD3 complex or via CD2, and a costimulatory molecule are presented on the antigen-presenting cell.

[0344] In one embodiment, the molecule that provides the primary stimulatory signal, for example, a molecule that stimulates via the TCR / CD3 complex or via CD2, and the costimulatory molecule are provided on separate surfaces.

[0345] In certain embodiments, one of the binding agents that provides the stimulatory signal and the costimulatory signal is soluble (provided in solution), while the other agent is provided on one or more surfaces.

[0346] In certain embodiments, the binding agents that provide the stimulatory signal and the costimulatory signal are both provided in dissolved form (provided in solution).

[0347] In various embodiments, the methods of producing T cells discussed herein include activating T cells with anti-CD3 and anti-CD28 antibodies.

[0348] In certain embodiments, the T cell compositions produced by the methods contemplated include T cells that have been activated and / or expanded in the presence of one or more agents that inhibit the PI3K cell signaling pathway. T cells that are modified to express an anti-CD79 ACAR can be activated and expanded before and / or after modifying the T cells. In certain embodiments, a population of T cells is activated and modified to express an anti-CD79 ACAR and then cultured for expansion.

[0349] In one embodiment, the T cells produced by the methods discussed herein express markers indicative of high proliferative potential and self-renewal capacity, but do not express, or express substantially undetectable, markers of T cell differentiation, and can be repeatedly activated and expanded in a robust manner to provide improved therapeutic T cell compositions.

[0350] 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 without a PI3K inhibitor.

[0351] In one embodiment, a population of T cells characterized by expression of markers indicative of immature T cell activation and expansion 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, relative to a population of T cells activated and expanded without a PI3K inhibitor.

[0352] In one embodiment, expanding T cells activated by the methods discussed herein further comprises culturing the population of cells comprising the T cells for a period of time ranging from a few hours (about 3 hours) to about 7 to about 28 days, or any integer value of one hour therebetween. 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 to 3 days. Additionally, multiple cycles of stimulation / activation / expansion may be desirable to culture the T cells for 60 days or more.

[0353] In certain embodiments, suitable conditions for T cell culture include an appropriate medium (e.g., Minimal Essential Media, or RPMI Media 1640, or X-vivo 15 (Lonza)), and The medium contains one or more factors necessary for growth and viability, including, but not limited to, serum (e.g., fetal bovine serum 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 additive suitable for cell growth known to those of skill in the art.

[0354] Further specific examples of cell culture media include, but are not limited to, RPMI1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo15, and X-Vivo20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a typical set of hormones and / or cytokines in amounts sufficient to grow and proliferate T cells.

[0355] Specific examples of other additives for expanding T cells include, but are not limited to, detergents, piasmanate, pH buffers such as HEPES, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol.

[0356] Antibiotics such as penicillin and streptomycin are included only in the experimental medium, not in the cell culture medium to be infused into the subject. The target cells are maintained under the conditions necessary to maintain growth, e.g., the appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0357] 3. Drugs In various embodiments, methods for producing T cells are provided that expand undifferentiated T cells or developmentally potent T cells, comprising contacting T cells with an agent that modulates the PI3K pathway in the T cells. In various embodiments, methods for producing T cells are provided that expand undifferentiated T cells or developmentally potent T cells, comprising contacting T cells with an agent that modulates the PI3K / Akt / mTOR pathway in the T cells. The cells can be contacted before, during, or after activation and expansion. The T cell composition retains sufficient T cell potency to allow multiple cycles of expansion without substantially increasing differentiation.

[0358] The terms "modulate," "modulator," or "modulator," or similar terms, as used herein, refer to the ability of an agent to effect a change in a cell signaling pathway. A modulator can increase or decrease the amount, activity of a pathway component, or can increase or decrease a desired effect or output of a cell signaling pathway. In one embodiment, a modulator is an inhibitor. In another embodiment, a modulator is an activator.

[0359] The term "agent" refers to a chemical compound, a small molecule, e.g., a small organic molecule, a nucleic acid, a polypeptide, or a fragment, isoform, variant, analog, or derivative thereof, used to modulate the PI3K / Akt / mTOR pathway.

[0360] "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 0.5 kD. Small molecules may include nucleic acids, peptides, polypeptides, peptidomimetics, peptoids, carbohydrates, lipids, components thereof, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art, and any assay can be used to screen the libraries. Examples of methods for synthesizing molecular libraries are described in the following references (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et al., 1994).

[0361] "Analog" is intended to mean a small organic compound, nucleotide, protein, or polypeptide, or a compound having the same or equivalent activity or function as the compound having the desired activity, but which does not necessarily have the same or identical sequence or structure as the sequence or structure of the preferred embodiments.

[0362] "Derivative" refers to a compound, protein, or polypeptide containing the amino acid sequence of a parent protein or polypeptide that has been modified by the introduction of amino acid substitutions, deletions, or additions, or a nucleic acid or nucleotide that has been modified either by the introduction of nucleotide substitutions, deletions, additions, or mutations. A derivative nucleic acid, nucleotide, protein, or polypeptide has the same or similar function as the parent polypeptide.

[0363] In various embodiments, an agent that modulates the PI3K pathway activates a component of the PI3K pathway. "Activator" or "agonist" refers to an agent that promotes, increases, or induces the activity of one or more molecules in the PI3K / Akt / mTOR pathway, including, but not limited to, molecules that activate one or more activities of PI3K.

[0364] In various embodiments, an agent that modulates the PI3K pathway inhibits a component of the PI3K pathway. "Inhibitor" or "antagonist" refers to an agent that inhibits, decreases, or reduces the activity of one or more 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 certain embodiments, the inhibitor can inhibit molecular species with the same or substantially equivalent activity (pan-inhibitor) or can specifically inhibit the activation of a molecule (selective or specific inhibitor). Inhibition can be irreversible or reversible.

[0365] 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 can be calculated using any conventional technique known in the art. For example, IC50 can be calculated by measuring the activity of a particular enzyme at different concentrations of the inhibitor under consideration. The experimentally obtained enzyme activity values ​​are plotted against the inhibitor concentration used. The concentration of the inhibitor that exhibits 50% enzyme activity (compared to the activity without any inhibitor) is taken as the "IC50" value. Similarly, other inhibitory concentrations can be determined by appropriate activity measurements.

[0366] In various embodiments, T cells are contacted, treated, or cultured with one or more modulators of the PI3K / Akt / mTOR pathway 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.

[0367] In certain embodiments, T cells may be contacted, treated, or cultured with one or more modulators of the PI3K / Akt / mTOR pathway for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more expansion cycles for at least 12 hours, 18 hours, at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, at least 2 weeks, at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.

[0368] The phosphatidylinositol-3 kinase / Akt / mammalian target of rapamycin pathway acts as a conduit, linking growth factor signaling with cell proliferation, differentiation, metabolism, and survival. PI3Ks are a family of highly conserved intracellular lipid kinases. Class IA PI3Ks are activated by growth factor receptor tyrosine kinases (RTKs) either directly or through interaction with the insulin receptor substrate family of adaptor molecules. This activity results in the production of phosphatidylinositol-3,4,5-triphosphate (PIP3), a regulator of the serine / threonine kinase Akt. mTOR acts through the canonical PI3K pathway via two distinct complexes, each characterized by distinct binding partners that confer unique activities. mTORC1 (mTOR complexed with PRAS40, RAPTOR, and mLST8 / GbL) acts as a downstream effector of PI3K / Akt signaling, linking growth factor signals to protein translation, cell growth, proliferation, and survival. mTORC2 (mTOR complexed with RICTOR, mSIN1, PROTOR, and mLST8) acts as an upstream activator of Akt.

[0369] Upon growth factor receptor-mediated activation of PI3K, Akt is recruited to the membrane via interaction between its pleckstrin homology domain and PIP3, where it is phosphorylated at threonine 308 (Thr308) by constitutively active phosphoinositide-dependent protein kinase 1 (PDK1), exposing its activation loop. For maximal activation, Akt is further phosphorylated by mTORC2 at serine 473 (Ser473) in its C-terminal hydrophobic motif. DNA-PK and HSPs have also been shown to be important for regulating Akt activity. Akt activates mTORC1 through the inhibitory phosphorylation of TSC2, whereas TSC2, together with TSC1, negatively regulates mTORC1 by inhibiting Rheb GTPase, a positive regulator of mTORC1. mTORC1 has two well-defined substrates, p70S6K (hereafter referred to as S6K1) and 4E-BP1, which critically regulate protein synthesis, and is therefore a key downstream effector of PI3K, linking growth factor signaling to protein translation and cell proliferation.

[0370] a.PI3K inhibitors The term "PI3K inhibitor," as used herein, refers to a nucleic acid, peptide, compound, or small organic molecule that binds to PI3K and inhibits at least one activity of PI3K. PI3K proteins can be classified into three classes: class 1 PI3K, class 2 PI3K, and class 3 PI3K. Class 1 PI3K exists as a heterodimer consisting of one of four p110 catalytic subunits (p110α, p110β, p110δ, and p110γ) and one of two families of regulatory subunits. PI3K inhibitors are preferably directed to class 1 PI3K inhibitors. In one embodiment, the PI3K inhibitor exhibits the selectivity for one or more isoforms of Class 1 PI3K inhibitors (i.e., selectivity for p110α, p110β, p110δ, and p110γ, or selectivity for one or more of p110α, p110β, p110δ, and p110γ). In another aspect, the PI3K inhibitor does not exhibit isoform selectivity and is considered a "pan-PI3K inhibitor." In one embodiment, the PI3K inhibitor competes with ATP for binding to the PI3K catalytic domain.

[0371] In certain embodiments, a PI3K inhibitor can target, for example, PI3K and another protein in the PI3K / Akt / mTOR pathway. In certain embodiments, a PI3K inhibitor that targets both mTOR and PI3K may be referred to as an mTOR inhibitor or a PI3K inhibitor. A PI3K inhibitor that targets only PI3K may be referred to as a selective PI3K inhibitor. In one embodiment, a selective PI3K inhibitor may be understood to refer to a drug that exhibits a 50% inhibitory concentration against PI3K that is at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more lower than the IC50 value of the inhibitor against mTOR and / or other proteins in this pathway.

[0372] In certain embodiments, typical PI3K inhibitors inhibit PI3K with an IC50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, about 1 nM or less, 100 μM or less, 50 μM or less, 25 μM or less, 10 μM or less, or 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.

[0373] Specific examples of PI3K inhibitors suitable for use in the T cell production methods contemplated in certain embodiments include, but are not limited to, BKM120 (Class 1 PI3K inhibitor, Novartis), XL147 (Class 1 PI3K inhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline), and PX-866 (Class 1 PI3K inhibitor, p1 p110α, p110β, and p110γ isoforms (Oncothyreon) do.

[0374] Other specific examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145.

[0375] Additionally, specific examples of pan-PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

[0376] In a preferred embodiment, the PI3K inhibitor is ZSTK474.

[0377] b.Akt inhibitors The term "Akt inhibitor," as used herein, refers to a nucleic acid, peptide, compound, or small organic molecule that inhibits at least one activity of Akt. Akt inhibitors can be divided into several classes, such as lipid-based inhibitors (e.g., inhibitors that target the pleckstrin homology domain of Akt, preventing Akt from localizing to the plasma membrane), ATP-competitive inhibitors, and allosteric inhibitors. In one embodiment, an Akt inhibitor acts by binding to the Akt catalytic site. In certain embodiments, an Akt inhibitor acts by inhibiting phosphorylation of downstream Akt targets, such as mTOR. In another embodiment, the activity of Akt is inhibited by inhibiting input signals that activate Akt, for example, by inhibiting DNA-PK activation of Akt, PDK-1 activation of Akt, and / or mTORC2 activation of Akt.

[0378] Akt inhibitors can target all three Akt isoforms, Akt1, Akt2, and Akt3, but can also be isoform-selective and target only one or two Akt isoforms. In one embodiment, Akt inhibitors can target Akt and other proteins in the PI3K / Akt / mTOR pathway. Akt inhibitors that target only Akt may 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 against Akt that is at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more lower than the IC50 values ​​of the inhibitors against other proteins in this pathway.

[0379] In certain embodiments, typical Akt inhibitors inhibit Akt with an IC50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, about 1 nM or less, 100 μM or less, 50 μM or less, 25 μM or less, 10 μM or less, or 1 μM or less. In one embodiment, an Akt inhibitor 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.

[0380] Specific 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).

[0381] A non-limiting example of a selective Akt1 inhibitor is A-674563.

[0382] A non-limiting example of a selective Akt2 inhibitor is CCT128930.

[0383] In certain embodiments, the Akt inhibitor inhibits DNA-PK activation of Akt, PDK-1 activation of Akt, mTORC2 activation of Akt, or HSP activation of Akt.

[0384] Specific examples of DNA-PK inhibitors include, but are not limited to, NU7441, PI-103, NU7026, PIK-75, and PP-121.

[0385] c. mTOR inhibitors 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 of the mTOR protein, e.g., its serine / threonine protein kinase activity toward at least one of its substrates (e.g., p70S6 kinase 1, 4E-BP1, Akt / PKB, and eEF2). An mTOR inhibitor can directly bind to and inhibit mTORC1, mTORC2, or both mTORC1 and mTORC2.

[0386] Inhibition of mTORC1 and / or mTORC2 activity can be measured by a decrease in signal transduction of the PI3K / Akt / mTOR pathway. Various readouts can be used to reveal the reduction in the output of the signal transduction pathway. Non-limiting examples of some readouts include: (1) a decrease in the phosphorylation of Akt at residues (including but not limited to residues 5473 and T308); (2) a decrease in Akt activation, as evidenced by a decrease in the phosphorylation of Akt substrates (e.g., substrates including but not limited to, Fox01 / O3a T24 / 32, GSK3a / β, S21 / 9, and TSC2 T1462); (3) a decrease in the phosphorylation of mTOR downstream signaling molecules (e.g., ribosomal S6 S240 / 244, 70S6K T389, and 4EBP1 T37 / 46); and (4) inhibition of cancer cell proliferation.

[0387] In one embodiment, the mTOR inhibitor is an active site inhibitor. These mTOR inhibitors bind to the ATP-binding site (also known as the ATP-binding pocket) of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2. One class of active site inhibitors suitable for use in the T cell production methods discussed in certain embodiments are dual-specific inhibitors that target and directly inhibit both PI3K and mTOR. Dual-specific inhibitors bind to the ATP-binding sites of both mTOR and PI3K. Specific examples of such inhibitors include, but are not limited to, imidazoquinazolines, wortmannin, LY294002, PI-103 (Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765 (Exelixis), and NVP-BEZ235 (Novartis).

[0388] Another class of mTOR active site inhibitors suitable for use in the methods contemplated in certain embodiments selectively inhibits the activity of mTORC1 and mTORC2 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 do not bind to PI3K. Specific examples of such inhibitors include, but are not limited to, pyrazolopyrimidines, 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).

[0389] In one embodiment, a selective mTOR inhibitor refers to an agent that exhibits a median inhibitory concentration (IC50) against 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 of the inhibitor against one, two, three, or more type I PI3 kinases, or all type I PI3 kinases.

[0390] Another class of mTOR inhibitors is referred to herein as "rapalogs." As used herein, the term "rapalog" refers to compounds that specifically bind to the FRB domain (FKBP rapamycin-binding domain) of mTOR, are structurally similar to rapamycin, and retain mTOR inhibitory properties. The term rapalog excludes rapamycin. Rapalogs include esters, ethers, oximes, hydrazones, and hydroxylamines of rapamycin, as well as compounds in which functional groups of the rapamycin core structure have been modified, for example, by reduction or oxidation. Pharmaceutically acceptable salts of such compounds are also considered rapamycin derivatives. Specific examples of rapalogs suitable for use in the methods discussed in certain embodiments include, but are not limited to, temsirolimus (CC1779), everolimus (RAD001), deforolimus (AP23573), AZD8055 (AstraZeneca), and OSI-027 (OSI).

[0391] In one embodiment, the agent is the mTOR inhibitor rapamycin (sirolimus).

[0392] In certain embodiments, exemplary mTOR inhibitors inhibit mTORC1, mTORC2, or both mTORC1 and mTORC2 with an IC50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, about 1 nM or less, 100 μM or less, 50 μM or less, 25 μM or less, 10 μM or less, or 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.

[0393] In one embodiment, a typical mTOR inhibitor inhibits PI3K and mTORC1, PI3K and mTORC2, or both mTORC1 and mTORC2 and PI3K with an IC50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, about 1 nM or less, 100 μM or less, 50 μM or less, 25 μM or less, 10 μM or less, or 1 μM or less. In one aspect, an mTOR inhibitor inhibits PI3K and mTORC1, PI3K and mTORC2, or both mTORC1 and mTORC2 and 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.

[0394] Further specific examples of mTOR inhibitors suitable for use in certain embodiments include, but are not limited to, AZD8055, INK128, rapamycin, PF-04691502, and everolimus.

[0395] mTOR has been shown to exhibit robust and specific catalytic activity towards its physiological substrate proteins, p70 S6 ribosomal protein kinase I (p70S6K1) and eIF4E-binding protein 1 (4EBP1), as measured by Western blot with phospho-specific antibodies.

[0396] 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.

[0397] H. Compositions and Formulations The compositions discussed herein may comprise one or more CAR polypeptides, polynucleotides, vectors comprising them, genetically modified immune effector cells, etc., as described herein. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in a pharmaceutically or physiologically acceptable solution for administration to a cell or animal, either alone or in combination with one or more other therapies. Of course, if necessary, the composition can also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There are virtually no limitations on other components that can be included in the composition, provided that the additional agents do not adversely affect the composition's ability to perform its intended therapy.

[0398] As used herein, the phrase "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0399] A "pharmaceutically acceptable carrier, diluent, or excipient," as used herein, includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as applicable for human or veterinary use. Examples of 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, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter; waxes; animal and vegetable fats; paraffin; silicone; bentonite; silicic acid; zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and any other compatible substance used in pharmaceutical formulations.

[0400] In certain embodiments, the composition comprises an amount of a CAR-expressing immune effector cell as discussed herein. The term "amount," as used herein, means an "effective amount" or "amount effective" of genetically modified therapeutic cells, e.g., T cells, that provides a beneficial or desired prophylactic or therapeutic result (including a clinical result).

[0401] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease.

[0402] A "therapeutically effective amount" of genetically modified therapeutic cells may vary depending on factors such as the disease stage, age, sex, and weight of the individual, as well as the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is one in which the beneficial effects of the treatment outweigh any toxic or harmful effects of the virus or transduced therapeutic cells. The term "therapeutically effective amount" includes an amount effective to treat a subject (e.g., a patient). When determining a therapeutic amount, the exact amount of the composition to be administered can be determined by a physician, taking into account the individual's age, weight, tumor size, extent of infection or metastasis, and the patient's (subject's) condition. The pharmaceutical compositions comprising T cells described herein may be administered in an amount of 10 per kg of body weight. 2 ~10 pieces 10 preferably 10 per kg of body weight 5 ~10 pieces 6 The number of cells may vary depending on the intended use of the composition, depending on the cell type contained in the composition. For the uses described herein, the cells are typically in a volume of 1 liter or less, but may also be in a volume of 500 mL or less, or even 250 mL or less, or 100 mL or less. Thus, the desired cell density is generally 10 6 cells / mL, typically 10 7 greater than 10 cells / mL, often 10 8 Clinically meaningful numbers of immune cells are allocated to multiple infusions, but the number is limited to 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces or 10 12 In some embodiments, the total number of injected cells is cumulatively equal to or exceeds 10 cells, since all injected cells are redirected to a specific target antigen. 6 Pieces / kilogram (10 per patient) 6 ~10 pieces 11Relatively small numbers of cells may be administered, in the range of 100-1500 cells (800-1500 cells). The CAR-expressing cell composition can be administered multiple times at doses within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy. Optionally, the treatment may also involve administration of a mitogen (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to further induce an immune response.

[0403] Compositions comprising the activated and expanded cells described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In certain embodiments, compositions comprising the CAR-modified T cells discussed herein are used to treat cancer. The CAR-modified T cells can be administered alone or as a pharmaceutical composition in combination with other components, such as a carrier, diluent, excipient, and / or IL-2 or other cytokines or cells. In certain embodiments, the pharmaceutical composition comprises a quantity of genetically modified T cells together with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0404] Pharmaceutical compositions comprising a population of CAR-expressing immune effector cells, such as T cells, may include a buffer, such as neutral buffered saline or phosphate buffered saline, a carbohydrate, such as glucose, mannose, sucrose, or dextran, a protein, a polypeptide or amino acid, such as glycine, an antioxidant, a chelating agent, such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and a preservative. The compositions are preferably formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0405] Liquid pharmaceutical compositions, whether in solution, suspension, or other form, may contain one or more of the following: sterile diluents for injection, saline (preferably saline, Ringer's solution, isotonic saline), fixed oils (such as synthetic monoglycerides or diglycerides that can function as solvents or suspending media), polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; 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 osmolality such as sodium chloride or dextrose. Parenteral preparations may be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials. Pharmaceutical compositions for injection are preferably sterile.

[0406] In one embodiment, the T cell compositions discussed herein are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0407] Serum-free media offer several advantages over serum-containing media, including simple and well-defined compositions, low levels of contamination, absence of potential sources of infectious agents, and low cost. In various embodiments, serum-free media are animal-free and optionally protein-free. Optionally, the media may contain biopharmaceutical-acceptable recombinant proteins. "Animal-free" media refers to media whose compositions are derived from non-animal sources. In animal-free media, recombinant proteins are used in place of natural animal proteins, and nutrients are obtained from synthetic, plant, or microbial sources. "Protein-free" media, on the other hand, are defined as those that are substantially free of protein.

[0408] Specific examples of serum-free media used in certain compositions include, but are not limited to, QBSF-60 (Quality Biologicals), StemPro-34 (Life Technologies), and X-VIVO10.

[0409] In one preferred embodiment, the compositions comprising the immune effector cells discussed herein are formulated in a solution comprising PlasmaLyte A.

[0410] In another preferred embodiment, the compositions comprising immune effector cells discussed herein are formulated in a solution comprising a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative agent can be used to maintain high cell viability after thawing. Specific examples of cryopreservation media for use in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0411] In a more preferred embodiment, the compositions comprising immune effector cells discussed herein are formulated in a solution comprising 50:50 PlasmaLyte A and CryoStor CS10.

[0412] In certain embodiments, the composition comprises an effective amount of CAR-expressing immune effector cells, alone or in combination with one or more therapeutic agents. Thus, the CAR-expressing immune effector cell composition can be administered 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 administered in combination with antibiotics. Such therapeutic agents are accepted in the art as standard treatments for certain conditions (e.g., certain cancers) described herein. Examples of therapeutic agents contemplated in certain embodiments include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active and adjuvant agents.

[0413] In certain embodiments, the compositions comprising the CAR-expressing immune effector cells disclosed herein can be administered in combination with any number of chemotherapeutic agents, including alkylating agents such as thiotepa, cyclophosphamide (CYTOXAN™), and alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylameramines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine regimes; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofluvial, thiazolinone, thiazolinone; Nitrogen mustards such as phosphamide and uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine, and aclacinomycins, actinomycin, ausramycin, azaserine, bleomycins, cactinomycin, calicheamicin, and calabimycin. Antibiotics such as benzodiazepine, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, and the like, as well as antibiotics such as methotrexate and 5-fluorouracil (5-FU). antimetabolites, folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate, purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, anti-adrenal agents such as aminoglutethimide, mitotane, trilostane, and florinic acid Folic acid supplements such as aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, and edatla. edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, and PS K®, razoxane, sizofiran, spirogermanium, tenuazonic acid, triazicon, 2,2′,2″-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), cyclophosphamide, thiotepa, and taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ). Princeton) and doxetaxel (TAXOTERE®, Antony Rhone-Poulenc Rorer, France), chlorambucil, gemcitabine, and 6-thioglucan. anthraquinone, 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, the topoisomerase inhibitor RFS2000, difluoromethylomithine (DMFO), retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin), and ONTAK™ (denileukin diftitox). (e.g., esperamicins, capecitabine, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.) Antihormonal agents that function to regulate or inhibit hormone action on cancer are included in this definition, such as antiestrogens, such as tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston), and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0414] Various other therapeutic agents can be used in combination with the compositions described herein. In one embodiment, a composition comprising CAR-expressing immune effector cells is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (e.g., betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, etc.), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolic acid, etc.).

[0415] Examples of other NSAIDs are ibuprofen, naproxen, naproxen sodium, VIOXX® (rofecoxib) and Celebrex® (celecoxib). Examples of analgesics are selected from the group consisting of acetaminophen, oxycodone, propoxyphene hydrochloride, tramadol. Examples of glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Examples of biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL)®), adalimumab (HUMIRA®), and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Examples of biological response modifiers include monoclonal antibodies and recombinant molecules. Examples of DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[0416] Specific examples of therapeutic antibodies suitable for use in combination with the CAR-modified T cells contemplated in certain embodiments include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, and konatsu. daratumumab, crizotinib, daratumumab, durigotumab, dacetuzumab , dalotuzumab, durvalumab, elotuzumab (HuLuc63), gemtuzumab, ibrix Momab, indatuximab, inotuzumab, ipilimumab, lorvox Lorvotuzumab, lucatumumab, milatuzumab, moxetumomab (moxetumomab), nivolumab, ocaratuzumab, ofatumumab, These include mbrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0417] In certain embodiments, the compositions described herein are administered in combination with a cytokine. As used herein, "cytokine" refers to a general term for a protein released by one group of cells that acts as an intercellular mediator on another group of cells. Examples of such cytokines are lymphokines, monokines, and general polypeptide hormones. Among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; glycoprotein hormones such as parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor alpha and tumor necrosis factor beta, Müllerian inhibitory factor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factors such as NGF beta, platelet growth factors, and transferrins such as TGF alpha and TGF beta. These include transforming growth factors (TGF), insulin-like growth factor I and insulin-like growth factor II, erythropoietin (EPO), osteoinductive factors, interferons such as interferon alpha, interferon beta, and interferon gamma, 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-1 alpha, 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 factors such as TNF alpha or TNF beta, and other polypeptide factors including LIF and Kit ligand (KL). The term cytokine, as used herein, includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0418] In certain embodiments, the composition comprises CAR-T cells described herein that have been cultured in the presence of a PI3K inhibitor as discussed herein and that express one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, and HLA-DR, and may be further isolated by positive or negative selection techniques. In one embodiment, the composition comprises a specific subpopulation of T cells that express one or more 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, and may be further isolated 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.

[0419] In one embodiment, the 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, relative to a population of T cells activated and expanded without a PI3K inhibitor.

[0420] 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-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, relative to a population of T cells activated and expanded without a PI3K inhibitor.

[0421] 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 with a PI3K inhibitor.

[0422] I. Target Cells and Antigens In certain embodiments, genetically modified immune effector cells are provided that include a CAR having a binding domain that is redirected to a target cell (e.g., a cancer cell) and binds to CD79A on the target cell. The term "cancer," as used herein, generally refers to a class of diseases or conditions in which abnormal cells divide indefinitely and can invade nearby tissues.

[0423] The term "malignant," as used herein, refers to a cancer in which tumor cells exhibit one or more of the following: uncontrolled growth (i.e., dividing above normal limits), invasion (i.e., invading and destroying adjacent tissues), and metastasis (i.e., spreading to other parts of the body via the lymphatics or blood). The term "metastasizing," as used herein, means that the cancer spreads from one part of the body to another. Tumors formed by the spread cells are called "metastatic tumors" or "metastases." Metastatic tumors contain cells similar to those of the original (primary) tumor.

[0424] The terms "benign" or "non-malignant," as used herein, refer to a tumor that may grow in size but does not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0425] "Cancer cell" refers to an individual cell of a cancerous growth or cancerous tissue. Cancer cells include solid and liquid cancers. "Tumor" or "tumor cell" generally refers to a swelling or lesion formed by abnormal cell proliferation, which may be benign, precancerous, or malignant. Many cancers form tumors, but liquid cancers, such as leukemia, do not necessarily form tumors. With respect to such cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of the tumor.

[0426] The term "recurrence" refers to a diagnosis of cancer recurrence or symptoms and signs of cancer recurrence after a period of improvement or remission.

[0427] "Remission" is sometimes referred to as "clinical remission" and includes partial and complete remission. In a partial remission, some, but not all, signs and symptoms of cancer have disappeared. In a complete remission, all signs and symptoms of cancer have disappeared, although cancer may still be present in the body.

[0428] "Refractory" refers to a cancer that is resistant or non-responsive to therapy with a particular therapeutic agent. A cancer may be refractory from the beginning of treatment (i.e., non-responsive to initial exposure to the therapeutic agent) or may become refractory as a result of developing resistance to the therapeutic agent over the course of the initial treatment period or during subsequent treatment periods.

[0429] In one embodiment, the target cell expresses an antigen, eg, a target antigen that is substantially not found on the surface of other normal (desired) cells.

[0430] In one embodiment, the target cell is a hematopoietic cell, a lymphoid cell, or a myeloid cell.

[0431] In certain embodiments, the target cells are part of blood, lymphoid tissue, or bone marrow tissue.

[0432] In certain embodiments, the target cells are cancer cells or cancer stem cells that express CD79A.

[0433] In certain embodiments, the target cells are liquid or hematological cancer cells that express CD79A.

[0434] Specific examples of liquid or hematological cancers that may be prevented, treated, or ameliorated using the compositions contemplated in certain embodiments include, but are not limited to, leukemia, lymphoma, and multiple myeloma.

[0435] Specific examples of cells that can be targeted by the anti-CD79 ACARs contemplated in certain embodiments include, but are not limited to, cells of leukemias such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera.

[0436] Specific examples of cells that may be targeted by the compositions and methods contemplated in certain embodiments include cells of lymphomas such as, but not limited to, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, and non-Hodgkin's lymphoma, including B-cell non-Hodgkin's lymphomas such as, but not limited to, Burkitt's 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's lymphomas such as mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, and precursor T-lymphoblastic lymphoma.

[0437] Specific examples of cells that may be targeted by the compositions and methods contemplated in certain embodiments include, but are not limited to, overt multiple myeloma, smoldering multiple myeloma (MGUS), plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0438] In a preferred embodiment, the CD79A-expressing target cells are DLBCL cancer cells.

[0439] J. Treatment method The genetically modified immune effector cells discussed herein provide improved adoptive immunotherapy methods for use in the prevention, treatment, and amelioration of CD79A-expressing cancers, or for preventing, treating, or ameliorating at least one symptom associated with CD79A-expressing cancers.

[0440] Additionally, in various embodiments, the genetically modified immune effector cells discussed herein provide improved adoptive immunotherapy methods for use in increasing the cytotoxicity of CD79A-expressing cancer cells in a subject or for use in reducing the number of CD79A-expressing cancer cells in a subject.

[0441] In certain embodiments, primary immune effector cells are genetically modified with a CAR described herein, thereby redirecting the specificity of the primary immune effector cells to cells that express CD79A, e.g., cancer cells. In various embodiments, a viral vector is used to genetically modify the immune effector cells with a specific polynucleotide that encodes a CAR that includes an anti-CD79A antigen-binding domain that binds a CD79A polypeptide, a hinge domain, a transmembrane (TM) domain, a short oligo- or polypeptide linker connecting the TM domain to the intracellular signaling domain of the CAR, one or more intracellular costimulatory signaling domains, and a primary signaling domain.

[0442] In one embodiment, a type of cell therapy is provided in which T cells are genetically modified to express an anti-CD79A CAR that targets CD79A-expressing cancer cells, and the CAR-T cells are infused into a recipient in need. The infused cells can kill disease-causing cells in the recipient. Unlike antibody therapy, CAR-T cells can self-renew in vivo, resulting in long-term persistence that can sustain cancer therapy.

[0443] In one embodiment, the anti-CD79ACAR-T cells are capable of robust in vivo T cell expansion and can persist for extended periods of time. In another embodiment, the anti-CD79ACAR-T cells are converted into specific memory T cells or stem cell memory T cells that can be reactivated to prevent any further tumor formation or tumor growth.

[0444] In certain embodiments, compositions comprising immune effector cells equipped with a CAR as discussed herein are used to treat conditions associated with CD79A-expressing cancer cells or cancer stem cells. Specific examples of conditions that can be treated, prevented, or ameliorated using immune effector cells comprising a CAR are discussed in certain embodiments.

[0445] In certain embodiments, compositions comprising the CAR-modified T cells discussed herein are used to treat liquid or hematological cancers.

[0446] In certain embodiments, the liquid or hematological cancer is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.

[0447] In certain embodiments, the liquid or hematological cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma (SMLL), and leukemias including ... SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0448] In certain embodiments, the liquid or hematological cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

[0449] In a preferred embodiment, the liquid or hematological cancer is DLBCL.

[0450] In a preferred embodiment, the liquid or hematological cancer is relapsed / refractory DLBCL.

[0451] In certain embodiments, methods are provided that include administering a therapeutically effective amount of the anti-CD79A CAR-expressing immune effector cells discussed herein, or a composition comprising the cells, to a patient in need thereof, alone or in combination with one or more therapeutic agents. In certain embodiments, the cells are used to treat a patient at risk of developing a pathology associated with CD79A-expressing cancer cells. Thus, in certain embodiments, methods are discussed herein for treating, preventing, or ameliorating at least one symptom of cancer, comprising administering a therapeutically effective amount of the CAR-modified cells to a subject in need thereof.

[0452] The terms "individual" and "subject," as used herein, are often used interchangeably and refer to any animal exhibiting symptoms of a disease, disorder, or condition that can be treated with a gene therapy vector, cell-based therapy, and any of the methods described herein. In preferred embodiments, a subject includes any animal exhibiting symptoms of a cancer-related disease, disorder, or condition that can be treated with a gene therapy vector, cell-based therapy, and any of the methods described herein. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), livestock, and domestic animals or pets (e.g., cats or dogs). Non-human primates, and preferably human patients, are also included. Exemplary subjects include human patients who have, have been diagnosed with, or are at risk for having a CD79A-expressing cancer (e.g., DLBCL).

[0453] The term "patient," as used herein, means a subject diagnosed with a particular disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapies, and methods disclosed anywhere herein.

[0454] "Treatment" or "treating," as used herein, includes any beneficial or desired effect on the symptoms of a disease or condition or pathology, and may include minimally reducing one or more measurable markers of the disease or condition being treated. Treatment may optionally include alleviating the disease or condition or slowing the progression of the disease or condition. "Treatment" does not necessarily imply complete eradication or cure of the disease or condition or its associated symptoms.

[0455] The terms "prevent" and similar terms such as "prevented" and "preventing," as used herein, refer to a means of preventing, inhibiting, or reducing the likelihood of the onset or recurrence of a disease or condition. These terms also refer to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. "Prevention," and similar terms, as used herein, includes reducing the extent, impact, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0456] The phrase "ameliorating at least one symptom," as used herein, means alleviating one or more symptoms of the disease or condition for which the subject is being treated. In certain embodiments, the disease or condition being treated is cancer, wherein the one or more symptoms that are ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (due to abdominal organomegaly), bone or joint pain, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (due to kidney dysfunction).

[0457] "Enhancement," or "promotion," or "increase," or "proliferation" means that a composition discussed herein (e.g., a genetically modified T cell or vector encoding a CAR) can produce, induce, or cause an increase in the physiological response (i.e., a downstream effect) compared to the response produced by a vehicle or control molecule / composition. Measurable physiological responses can include increased T cell proliferation, increased activation, increased persistence, and / or increased cancer cell killing capacity, among others, as would be apparent from the understanding in the art and herein. An "increase" or "enhancement" amount is typically a "statistically significant" amount and can include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points between these numbers and above 1, including, for example, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, etc.) relative to the response produced by a vehicle or control composition.

[0458] "Reduction," or "lowering," or "reducing," or "reducing," or "mitigating," generally means that the compositions discussed herein can produce, induce, or cause a decrease in the physiological response (i.e., a downstream effect) compared to the response produced by a vehicle or control molecular composition. A "decrease" or "reduced" amount is typically a "statistically significant" amount and can include a decrease of 1 / 1.1, 1 / 1.2, 1 / 1.5, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 30, or more (e.g., 1 / 500, 1 / 1000) (the denominator value includes all integers and decimal points between and greater than 1, including, for example, 1.5, 1.6, 1.7, 1.8, etc.) relative to the response produced by a vehicle or control composition (reference response) or the response of a particular cell line.

[0459] "Maintain" or "sustain" or "maintained" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the property of the compositions discussed herein to produce, induce, or cause a similar response in a cell (i.e., a downstream effect) compared to the physiological response or response of a particular cell lineage produced by a vehicle or control molecule / composition. A similar response is one that is not substantially different or measurably different from the reference response.

[0460] In one embodiment, a method for treating cancer in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising the genetically modified immune effector cells discussed herein. The amount and frequency of administration will depend on factors such as the patient's condition, the type and severity of the patient's disease, and the appropriate dosage can also be determined through clinical trials.

[0461] In one embodiment, the composition administered to a subject comprises at least 0.1 x 10 immune effector cells, e.g., T cells. 5 cells, at least 0.5 x 10 5 cells, at least 1 x 10 5 cells, at least 5 x 10 5 cells, at least 1 x 10 6 cells, at least 0.5 x 10 7 cells, at least 1 x 10 7 cells, at least 0.5 x 10 8 cells, at least 1 x 10 8 cells, at least 0.5 x 10 9 cells, at least 1 x 10 9 cells, at least 2 x 10 9 cells, at least 3 x 10 9 cells, at least 4 x 10 9 cells, at least 5 x 10 9 cells, or at least 1 x 10 10 Each cell is an individual cell.

[0462] In certain embodiments, about 1×10 7 T cells ~ approximately 1 x 10 9 T cells, approximately 2 x 10 7 T cells ~ approx. 0.9 x 10 9 T cells, approximately 3 x 10 7 T cells ~ approx. 0.8 x 10 9 T cells, approximately 4 x 10 7 T cells ~ approx. 0.7 x 10 9 T cells, approximately 5 x 10 7 T cells ~ approx. 0.6 x 10 9 T cells, or approximately 5 x 10 7 T cells ~ approximately 0.5 x 10 9 T cells are administered to the subject.

[0463] In one embodiment, the composition administered to a subject comprises immune effector cells, e.g., T cells, in an amount of at least 0.1 x 10 per kg of body weight. 4 cells, at least 0.5 x 10 per kg body weight 4 cells, at least 1 x 10 per kg body weight 4 cells, at least 5 x 10 per kg body weight 4 cells, at least 1 x 10 per kg body weight 5 cells, at least 0.5 x 10 per kg body weight 6 cells, at least 1 x 10 per kg body weight 6 cells, at least 0.5 x 10 per kg body weight 7 cells, at least 1 x 10 per kg body weight 7 cells, at least 0.5 x 10 per kg body weight 8 cells, at least 1 x 10 per kg body weight 8 cells, at least 2 x 10 per kg body weight 8 cells, at least 3 x 10 per kg body weight 8 cells, at least 4 x 10 per kg body weight 8 cells, at least 5 x 10 per kg body weight 8 cells, or at least 1 x 10 cells per kg of body weight 9 Each cell is an individual cell.

[0464] In certain embodiments, about 1 x 10 per kg of body weight 6 T cells ~ approximately 1 x 10 per kg of body weight 8 T cells, approximately 2 x 10 per kg of body weight 6 T cells ~ approximately 0.9 x 10 per kg of body weight 8 T cells, approximately 3 x 10 per kg of body weight 6 T cells ~ approximately 0.8 x 10 per kg of body weight 8 T cells, approximately 4 x 10 per kg of body weight 6 T cells ~ approximately 0.7 x 10 per kg of body weight 8 T cells, approximately 5 x 10 per kg of body weight 6 T cells ~ approximately 0.6 x 10 per kg of body weight 8 T cells, approximately 5 x 10 per kg of body weight 6 T cells ~ approximately 0.5 x 10 per kg of body weight 8 T cells are administered to the subject.

[0465] Those skilled in the art will appreciate that multiple administrations of the compositions discussed herein may be necessary to provide the desired treatment. For example, the composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.

[0466] In certain embodiments, it may be preferable to administer activated immune effector cells to a subject, followed by a second blood draw (or apheresis), activation of the immune effector cells obtained therefrom, and reinfusion of the activated and expanded immune effector cells back into the patient. This process can be performed multiple times, every few weeks. In certain embodiments, immune effector cells obtained from a blood draw of 10 cc to 400 cc can be activated. In certain embodiments, immune effector cells obtained from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or 400 cc or more are activated. Without being bound by theory, this multiple blood draw / multiple reinfusion protocol can be used to select for specific populations of immune effector cells.

[0467] The compositions discussed herein can be administered by any conventional method, including, for example, aerosol inhalation, injection, oral ingestion, infusion, injection, or implantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "administering parenterally" refer to modes of administration, typically by injection, other than enteral administration and topical administration, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In one embodiment, the compositions discussed herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0468] In one embodiment, an effective amount of the composition is administered to a subject in need thereof to increase the subject's cellular immune response against a B-cell-related pathology. The immune response may include cellular immune responses mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses, which can kill infected cells. A humoral immune response, primarily mediated by helper T cells, which can activate B cells and thereby produce antibodies, may also be induced. Various techniques can be used to analyze the type of immune response induced by the composition, which are described in detail in the art, for example, in *Current Protocols in Immunology*, edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0469] In the case of T cell-mediated killing, binding of a CAR to a ligand initiates CAR signaling to the T cell, which in turn activates various T cell signaling pathways that induce the T cell to produce or release proteins that can induce apoptosis of the target cell by a variety of mechanisms, including (but not limited to) the transfer of intracellular cytotoxic granules from the T cell to the target cell, the secretion of inflammatory cytokines by the T cell that can induce target cell death directly (or indirectly via the recruitment of other death effector cells), and the upregulation of death receptor ligands (e.g., FasL) on the T cell surface that induce apoptosis of the target cell after binding to a cognate death receptor (e.g., Fas) on the target cell.

[0470] In one embodiment, a method of treating a subject diagnosed with a CD79A-expressing cancer is provided, comprising harvesting immune effector cells from the subject diagnosed with a CD79A-expressing cancer, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a CAR as discussed herein, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0471] In certain embodiments, provided is a method of stimulating an immune effector cell-mediated immunomodulatory response against a target cell population in a subject, the method comprising administering to the subject a population of immune effector cells that express a nucleic acid construct encoding a CAR molecule.

[0472] Methods of administering the cell compositions contemplated in certain embodiments include any method effective for reintroducing ex vivo genetically modified immune effector cells that directly express a CAR in the subject, or reintroducing genetically modified precursors of the immune effector cells that differentiate into mature immune effector cells that express a CAR when introduced into the subject. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid construct discussed herein and returning the transduced cells to the subject.

[0473] 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, and issued patent was specifically and individually indicated to be incorporated by reference.

[0474] While the above embodiments have been described in detail, with illustrations and examples provided for clarity of understanding, it will be readily apparent to those skilled in the art, in light of the teachings discussed herein, that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. The following examples are offered by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield substantially similar results. [Example]

[0475] Example 1 Anti-CD79ACAR constructs A CAR containing a humanized anti-CD79AscFv antibody was designed to contain an MND promoter operably linked to the anti-CD79AscFv, a hinge domain and transmembrane domain from CD8α, a CD137 costimulatory domain, followed by the intracellular signaling domain of the CD3ζ chain. The anti-CD79ACAR contains a CD8α signal peptide (SP) sequence suitable for surface expression on immune effector cells. Table 3 shows the unique characteristics, GenBank reference numbers, source names, and citations of various nucleotide segments of the anti-CD79ACAR lentiviral vector. Exemplary CD79ACAR polypeptide sequences are set forth in SEQ ID NOs: 25 to 30, and exemplary CD79ACAR polynucleotide sequences are set forth in SEQ ID NOs: 31 to 36. [Table 3-1] [Table 3-2]

[0476] Example 2 Evaluation of human anti-CD79ACAR-T cells Chimeric antigen receptors (CARs) specific for CD79A (e.g., SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 30) were evaluated for CAR expression and biological activity on CD79A-expressing cells.

[0477] Target antigen expression In one experiment, K562 cells, Pfeiffer cells, and Daudi target cells were examined for CD79A expression using an anti-CD79A antibody. The cells were incubated with the CD79A antibody, and expression was assessed by flow cytometry. CD79A expression was undetectable in K562 cells, moderately expressed in Pfeiffer cells, and highly expressed in Daudi cells. See Figure 1A.

[0478] In another experiment, CD79A expression was measured in Daudi, NU-DUL-1, SU-DHL-2, and Pfeiffer target cells using an anti-CD79A antibody. These cells were incubated with the CD79A antibody, and expression was assessed using a flow cytometer. Similar to the previous results, CD79A expression was highest in Daudi cells, followed by NU-DUL-1, SU-DHL-2, and Pfeiffer cells, in that order (see Figure 2A).

[0479] Anti-CD79ACAR expression CAR-T cells were produced using a system that is readily scalable to large-scale clinical manufacturing processes. Briefly, peripheral blood mononuclear cells (PBMCs) were incubated with IL-2 (CellGenix The cells were cultured in medium containing a lentivirus encoding anti-CD79ACAR (manufactured by Miltenyi Biotec) and antibodies specific for CD3 and CD28 (manufactured by Miltenyi Biotec). One day after the initiation of culture, lentivirus encoding anti-CD79ACAR was added. Anti-CD79ACAR-T cells were maintained in logarithmic phase by adding fresh medium containing IL-2 for the entire 10 days of culture. At the end of the culture, the expression of anti-CD79ACAR-T cells was examined using flow cytometry. In one experiment, primary human T cells engineered with lentivirus expressing anti-CD79ACAR were stained with goat anti-mouse (GaM) conjugated to biotin and detected with PE-conjugated streptavidin. See Figure 1B. In a separate experiment, anti-CD79ACAR expression on T cells was assessed using goat anti-mouse (GaM) staining, and binding of soluble CD79A antigen to anti-CD79ACAR-T cells was assessed by staining with phycoerythrin (PE)-labeled CD79A extracellular domain-Fc fusion protein (see Figure 2B). These reagents specifically identified T cells that expressed anti-CD79ACAR.

[0480] Antigen-dependent anti-CD79ACAR-T cell activity In one experiment, the biological activity of anti-CD79ACAR-T cells against CD79A-positive (Pfeiffer and Daudi) and CD79A-negative (K562) cell lines was assessed using an interferon-gamma (IFNγ) release assay. Anti-CD79ACAR-T cells were co-cultured in the absence of target cells or with K562 cells (CD79A-), Pfeiffer cells (low CD79A expression), and Daudi cells (high CD79A expression) for 24 hours. Anti-CD79ACAR-T cells released IFNγ only in the presence of CD79A-positive cell lines. See Figure 1C.

[0481] In a separate experiment, the biological activity of anti-CD79ACAR-T cells against CD79A-positive (Huh7.CD79A, Daudi, NU-DUL-1, SU-DHL-2, and Pfeiffer) and CD79A-negative (Huh7) cell lines was assessed using an interferon gamma (IFNγ) release assay. Anti-CD79ACAR-T cells were cocultured in the absence of target cells (T cells alone) or with Huh7 (CD79A-), Huh7.CD79A (CD79A+), Daudi (CD79A+), NU-DUL-1 (CD79A+), SU-DHL-2 (CD79A+), or Pfeiffer (CD79A+) cells. See Figure 2C.

[0482] In the following claims, the terms used generally should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but rather to include all possible embodiments, including the full scope of equivalents to which the claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. A chimeric antigen receptor (CAR), a) an extracellular domain comprising an anti-CD79A antibody or antigen-binding fragment thereof that binds 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 having CDRL1 through CDRL3 sequences set forth in SEQ ID NOs: 1-3, and a variable heavy chain sequence having CDRH1 through CDRH3 sequences set forth in SEQ ID NOs: 4-6; b) a transmembrane domain; and c) one or more intracellular costimulatory signaling domains; and d) a primary signaling domain; and wherein the variable heavy chain of the anti-CD79A antibody or antigen-binding fragment thereof is positioned C-terminal to the variable light chain; and A chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence having at least 90% amino acid identity to the amino acid sequence set forth in SEQ ID NO:

26.

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

3. 3. The CAR of claim 1 or claim 2, wherein the anti-CD79A antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in SEQ ID NO: 7 and a variable heavy chain sequence set forth in SEQ ID NO:

8.

4. The CAR according to any one of claims 1 to 3, wherein the transmembrane domain is isolated from CD8α.

5. The CAR according to any one of claims 1 to 4, wherein the intracellular costimulatory signaling domain is isolated from CD137.

6. The CAR of any one of claims 1 to 5, wherein the primary signaling domain is isolated from CD3ζ.

7. The CAR according to any one of claims 1 to 6, further comprising a hinge region polypeptide.

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

9. The CAR according to any one of claims 1 to 8, further comprising a signal peptide.

10. a) an anti-CD79A scFv that binds 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 having CDRL1 through CDRL3 sequences set forth in SEQ ID NOs: 1-3 and a variable heavy chain sequence having CDRH1 through CDRH3 sequences set forth in SEQ ID NOs: 4-6; b) an isolated transmembrane domain from CD8α; and c) an isolated intracellular costimulatory signaling domain from CD137; and d) the isolated primary signaling domain from CD3ζ; The CAR according to any one of claims 1 to 9, comprising:

11. a) an anti-CD79A scFv that binds 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 having CDRL1 through CDRL3 sequences set forth in SEQ ID NOs: 1-3 and a variable heavy chain sequence having CDRH1 through CDRH3 sequences set forth in SEQ ID NOs: 4-6; b) a CD8α hinge region polypeptide; and c) an isolated transmembrane domain from CD8α; and d) an isolated intracellular costimulatory signaling domain from CD137; and e) an isolated primary signaling domain from CD3ζ; and The CAR according to any one of claims 1 to 10, comprising:

12. The CAR according to any one of claims 1 to 11, comprising the amino acid sequence set forth in SEQ ID NO:

26.

13. A polynucleotide encoding the CAR according to any one of claims 1 to 12, or a polynucleotide comprising the sequence set forth in SEQ ID NO:

32.

14. A vector comprising the polynucleotide of claim 13.

15. The vector of claim 14 , wherein the vector is a lentiviral vector.

16. An immune effector cell comprising the CAR according to any one of claims 1 to 12, the polynucleotide according to claim 13, or the vector according to claim 14 or 15.

17. 17. The immune effector cell of claim 16, wherein the immune effector cell is selected from the group consisting of a T lymphocyte, a natural killer (NK) cell, and an NKT cell.

18. 18. A composition comprising the immune effector cells of claim 16 or claim 17 and a physiologically acceptable excipient.

19. 20. The composition of claim 18 for use in treating cancer in a subject.

20. 20. The composition of claim 19, wherein the cancer is a hematological malignancy.

21. 21. The composition of claim 19 or claim 20, wherein the cancer is non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

22. 22. The composition of claim 21, wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL).

23. 22. The composition of claim 21, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL).

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