BR112017001498B1 - ANTI-BCMA CHIMERIC ANTIGEN RECEPTOR (CAR), POLYNUCLEOTIDE THAT ENCODS SAID CAR, VECTOR, COMPOSITION AND USE THEREOF TO TREAT A B CELL RELATED CONDITION - Google Patents

Abstract

BCMA CHIMERIC ANTIGEN RECEPTORS. The invention provides improved compositions for adoptive T cell therapies for B cell related conditions.

Description

CROSS REFERENCE TO RELATED ORDERS

[001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/028,664, filed July 24, 2014, U.S. Provisional Application No. 62/044,103, filed August 29, 2014, and Order U.S. Provisional No. 62/152,575, filed April 24, 2015, each of which is incorporated herein by reference in its entirety.

DECLARATION REGARDING SEQUENCE LISTING

[002] The Sequence Listing associated with this application is provided in text format rather than a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is BLBD_037_03WO_ST25.txt. The text file is 124 KB, was created on July 23, 2015, and is being submitted electronically through EFS-Web, concurrent with the presentation of the specification.

FUNDAMENTALS Technical Field

[003] The present invention relates to improved compositions and methods for treating conditions related to B cells. More particularly, the invention relates to improved chimeric antigen receptors (CARs) comprising humanized anti-BCMA antibodies or B cell-binding fragment. antigens thereof, immune effector cells genetically modified to express these CARs, and use of these compositions to effectively treat B cell-related conditions.

Description of Related Technique

[004] Several significant diseases involve B lymphocytes, that is, B cells. Abnormal B cell physiology can also lead to the development of autoimmune diseases including, but not limited to, systemic lupus erythematosus (SLE). Malignant transformation of B cells leads to cancers including, but not limited to, lymphomas, e.g., multiple myeloma and non-Hodgkin's lymphoma.

[005] The vast majority of patients having B-cell malignancies, including non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), are significant contributors to cancer mortality. The response of B cell malignancies to various forms of treatment is varied. Traditional methods of treating B-cell malignancies, including chemotherapy and radiation therapy, have limited utility due to toxic side effects. Immunotherapy with anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD52, anti-CD80, and anti-HLA-DR therapeutic antibodies has provided limited success, due in part to poor pharmacokinetic profiles, rapid antibody clearance by serum proteases and filtration in the glomerulus, and limited penetration into the tumor site and target antigen expression levels in cancer cells. Attempts to use genetically modified cells expressing chimeric antigen receptors (CARs) have also had limited success due to poor in vivo expansion of CAR T cells, rapid disappearance of the cells after infusion, and disappointing clinical activity.

BRIEF SUMMARY

[006] The invention generally provides improved vectors for generating T cell therapies and methods of using them. More particularly, the invention provides humanized anti-BCMA CAR molecules and their uses in treating, preventing or ameliorating B cell-related conditions.

[007] Without wishing to be bound by any particular theory, the inventors discovered that certain humanized anti-BCMA CARs evoke antigen-independent (“tonic”) cytokine release, a characteristic that would make humanized anti-BCMA CARs unsuitable for use in therapeutics. Surprisingly, the inventors have discovered that tonic cytokine release by certain T cells from humanized anti-BCMA CARs can be made antigen-dependent by altering one or more characteristics of at least the transmembrane domain of the humanized anti-BCMA CARs.

[008] In various embodiments, a chimeric antigen receptor (CAR) is provided comprising: an extracellular domain comprising a humanized anti-BCMA antibody (B cell maturation antigen) or antigen-binding fragment that binds to one or more epitopes of a human BCMA polypeptide; a transmembrane domain, one or more intracellular costimulatory signaling domains and a primary signaling domain.

[009] In particular embodiments, the humanized anti-BCMA antibody or antigen-binding fragment that binds to the human BCMA polypeptide is selected from the group consisting of: a Camel Ig, NAR Ig, Fab fragments, Fab fragments ', F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv antibody (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, Fv protein disulfide stabilized (“dsFv”) and single domain antibody (sdAb, Nanobody).

[010] In additional embodiments, the humanized anti-BCMA antibody or antigen-binding fragment that binds to the human BCMA polypeptide is an scFv.

[011] In some embodiments, the humanized anti-BCMA antibody or antigen-binding fragment thereof comprises one or more CDRs as set forth in any of SEQ ID NOs: 1 to 3.

[012] In particular embodiments, the humanized anti-BCMA antibody or antigen-binding fragment thereof comprises one or more CDRs as set forth in any of SEQ ID NOs: 4 to 6.

[013] In certain embodiments, the humanized anti-BCMA antibody or antigen-binding fragment thereof comprises a variable light chain sequence as set forth in any of SEQ ID NOs: 7 to 9.

[014] In particular embodiments, the variable light chain sequence comprises the CDR sequences presented in SEQ ID NOs: 1 to 3.

[015] In other embodiments, the humanized anti-BCMA antibody or antigen-binding fragment thereof comprises a variable heavy chain sequence as set forth in any of SEQ ID NOs: 10 to 14.

[016] In additional embodiments, the variable heavy chain sequence comprises the CDR sequences shown in SEQ ID NOs: 4 to 6.

[017] In other embodiments, the transmembrane domain is a polypeptide selected from the group consisting of: alpha, beta or zeta chain of the T cell receptor, CD3ε, CD3Z, CD4, CD5, CD8α, CD9, CD 16 , CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154 and PD1.

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

[019] In some embodiments, the transmembrane domain is PD1.

[020] In some embodiments, the transmembrane domain is from CD152.

[021] In certain embodiments, the transmembrane domain is CD8α.

[022] In particular embodiments, one or more costimulatory signaling domains are from a costimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM ), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70.

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

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

[025] In additional embodiments, one or more costimulatory signaling domains is from CD28.

[026] In particular embodiments, one or more costimulatory signaling domains is from CD134.

[027] In other embodiments, one or more costimulatory signaling domains is from CD137.

[028] In certain embodiments, a CAR comprises a hinge region polypeptide.

[029] In other embodiments, the hinge region is a polypeptide selected from the group consisting of: CD8α, PD1 and CD152.

[030] In other embodiments, the hinge region polypeptide comprises a PD1 hinge region

[031] In other embodiments, the hinge region polypeptide comprises a CD152 hinge region.

[032] In other embodiments, the hinge region polypeptide comprises a CD8α hinge region.

[033] In other embodiments, the CAR comprises a hinge region polypeptide and a PD1 transmembrane domain

[034] In other embodiments, the CAR comprises a hinge region polypeptide and a CD152 transmembrane domain.

[035] In other embodiments, the CAR comprises a hinge region polypeptide and a CD8α transmembrane domain.

[036] In some embodiments, a CAR comprises a spacer region.

[037] In additional embodiments, the spacer region polypeptide comprises CH2 and CH3 regions of IgG1 or IgG4.

[038] In particular embodiments, a CAR comprises a signal peptide.

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

[040] In one embodiment, a CAR comprises an amino acid sequence as set forth in any of SEQ ID NOs: 15 to 29, 71 and 73.

[041] In another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 15.

[042] In a particular embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 16.

[043] In a certain embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 17.

[044] In a further embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 18.

[045] In another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 19.

[046] In a particular embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 20.

[047] In one embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 21.

[048] In another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 22.

[049] In yet another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 23.

[050] In yet another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 24.

[051] In a particular embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 25.

[052] In a certain embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 26.

[053] In a further embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 27.

[054] In another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 28.

[055] In another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 29.

[056] In another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 71.

[057] In another embodiment, a CAR comprises an amino acid sequence as shown in SEQ ID NO: 73.

[058] In various embodiments, a polynucleotide encoding a CAR considered here is provided.

[059] In several particular embodiments, a polynucleotide encoding a CAR is provided, wherein the polynucleotide sequence is presented in any of SEQ ID NOs: 30 to 44, 70 and 72.

[060] In various certain embodiments, a vector comprising a polynucleotide encoding a CAR considered herein or as set forth in any of SEQ ID NOs: 30 to 44, 70 and 72 is provided.

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

[062] In additional embodiments, the vector is an episomal vector.

[063] In particular embodiments, the vector is a viral vector.

[064] In other embodiments, the vector is a retroviral vector.

[065] In other embodiments, the vector is a lentiviral vector.

[066] In additional 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).

[067] In particular embodiments, a vector comprises a retroviral LTR (5') on the left, a Psi packaging signal (^), a central polypurine tract/DNA flap (cPPT/FLAP), a retroviral export element; a promoter operably linked to the polynucleotide encoding a CAR considered herein; and a retroviral LTR (3') on the right.

[068] In other embodiments, a CAR comprises a heterologous polyadenylation sequence.

[069] In some embodiments, a CAR comprises a post-transcriptional regulatory element (HPRE) or woodchuck post-transcriptional regulatory element (WPRE) of the hepatitis B virus.

[070] In certain embodiments, the 5' LTR promoter is replaced with a heterologous promoter.

[071] In other embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a Simian Virus 40 (SV40) promoter.

[072] In particular embodiments, the 5' LTR or 3' LTR is a lentivirus LTR.

[073] In particular embodiments, the LTR 3' comprises one or more modifications.

[074] In some embodiments, the 3' LTR comprises one or more deletions.

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

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

[077] In additional embodiments, a polynucleotide encoding a CAR considered herein comprises an optimized Kozak sequence.

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

[079] In various embodiments, an immune effector cell is provided comprising a vector considered here.

[080] In other embodiments, the immune effector cell is selected from the group consisting of: a T lymphocyte and a natural killer (NK) cell.

[081] In various embodiments, a composition is provided comprising an immune effector cell considered herein and a physiologically acceptable excipient.

[082] In various embodiments, a method of generating an immune effector cell comprising a CAR considered herein is provided, comprising introducing into an immune effector cell a vector comprising a polynucleotide encoding the CAR.

[083] In additional embodiments, the method further comprises stimulating the immune effector cell and inducing the cell to proliferate by contacting the cell with antibodies that bind to CD3 and antibodies that bind to CD28; thereby generating a population of immune effector cells.

[084] In particular embodiments, the immune effector cell is stimulated and induced to proliferate before introducing the vector.

[085] In certain embodiments, the immune effector cells comprise T lymphocytes.

[086] In particular embodiments, the immune effector cells comprise NK cells.

[087] In various embodiments, a method of treating a condition related to B cells in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a composition comprising anti-BCMA CAR T cells considered herein and optionally, a pharmaceutically acceptable excipient.

[088] In other embodiments, the B cell-related condition is multiple myeloma, non-Hodgkin's lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, an immunoregulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, anti-phospholipid syndrome, Chagas disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA-associated vasculitis , Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, or monoclonal gammopathy of undetermined significance.

[089] In other embodiments, the B cell-related condition is a B cell malignancy.

[090] In certain embodiments, the B-cell malignancy is multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).

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

[092] In some embodiments, the NHL is selected from the group consisting of: Burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic lymphoma of large cell, B precursor lymphoblastic lymphoma and mantle cell lymphoma.

[093] In particular embodiments, the B cell-related condition is a plasma cell malignancy.

[094] In other embodiments, the condition related to B cells is an autoimmune disease.

[095] In additional embodiments, the autoimmune disease is systemic lupus erythematosus.

[096] In certain embodiments, the condition related to B cells is rheumatoid arthritis.

[097] In particular embodiments, the condition related to B cells is idiopathic thrombocytopenic purpura, or myasthenia gravis or autoimmune hemolytic anemia.

BRIEF DESCRIPTION OF VARIOUS VIEWS OF THE DRAWINGS

[098] Figure 1 shows a schematic of humanized anti-B cell maturation antigen CAR (anti-BCMA) constructs.

[099] Figure 2 shows the amount of IFNY released from anti-BCMA CAR T cells after the cells were co-cultured for 24 hours with BCMA-expressing K562 cells.

[0100] Figure 3 shows the cytolytic activity of anti-BCMA CAR T cells co-cultured for four hours with BCMA-expressing K564 cells.

[0101] Figure 4 shows anti-BCMA CAR expression in T cells transduced with mouse (anti-BCMA-02) or humanized (anti-BCMA, anti-BCMA-10, anti-anti-BCMA-11, anti- anti-BCMA-31).

[0102] Figure 5 shows the amount of IFNY released from anti-BCMA CAR T cells after the cells were co-cultured for 24 hours with K562-BCMA cells expressing low or high amounts of BCMA, cell lines of multiple myeloma (RPMI-8226, NCI-H929), or BCMA-negative cell lines (K562, HDLM-2).

[0103] Figure 6 shows the cytolytic activity of T cells expressing anti-BCMA CARs co-cultured with K564-BCMA cells for four hours.

[0104] Figure 7 shows antigen-independent cytokine release by T cells expressing the humanized anti-BCMA-10 CAR construct cultured in media containing human serum but lacking BCMA.

[0105] Figure 8 shows antigen-independent cytokine release by T cells expressing the humanized anti-BCMA-10 CAR construct cultured in media containing human serum, but lacking BCMA.

[0106] Figure 9 shows a schematic of the anti-BCMA-02, anti-BCMA-10, anti-BCMA-10.2 and anti-BCMA-10.5 CAR constructs.

[0107] Figure 10 shows the amount of IFNY released from anti-BCMA-10, anti-BCMA-10.2 or anti-BCMA-10.5 CAR T cells after the cells were co-cultured for 24 hours with BCMA-expressing K562 cells .

[0108] Figure 11 shows a comparison of the amount of tonic IFNY release from T cells expressing mouse anti-BCMA-02 CAR, or expressing anti-BCMA-10, anti-BCMA-10.2 or anti-BCMA CAR -10.5 humanized after 24 hours in culture.

BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS

[0109] SEQ ID NOs: 1 to 3 present amino acid sequences of exemplary light chain CDR sequences for anti-BCMA CARs considered here.

[0110] SEQ ID NOs: 4 to 6 present amino acid sequences of exemplary heavy chain CDR sequences for anti-BCMA CARs considered here.

[0111] SEQ ID NOs: 7 to 9 present amino acid sequences of exemplary light chain sequences for anti-BCMA CARs considered here.

[0112] SEQ ID NOs: 10 to 14 present amino acid sequences of exemplary heavy chain sequences for anti-BCMA CARs considered here.

[0113] SEQ ID NOs: 15 to 29, 71 and 73 present amino acid sequences of exemplary anti-BCMA CARs considered here.

[0114] SEQ ID NOs: 30 to 44, 70 and 72 present polynucleotide sequences encoding exemplary anti-BCMA CARs considered here.

[0115] SEQ ID NO: 45 presents the amino acid sequence of human BCMA.

[0116] SEQ ID NOs: 46 to 56 present the amino acid sequence of various ligands.

[0117] SEQ ID NOs: 57 to 69 present the amino acid sequence of protease cleavage sites and self-cleaving polypeptide cleavage sites.

DETAILED DESCRIPTION A. Overview

[0118] The invention generally relates to improved compositions and methods for treating conditions related to B cells. As used herein, the term “B cell related conditions” refers to conditions involving inappropriate B cell activity and malignancies of B cells.

[0119] In particular embodiments, the invention relates to improved adoptive cell therapy of B cell-related conditions using genetically modified immune effector cells. Genetic approaches offer a potential means to enhance immune recognition and elimination of cancer cells. One promising strategy is to genetically engineer immune effector cells to express chimeric antigen receptors (CAR) that redirect cytotoxicity toward cancer cells. However, existing adoptive cell immunotherapies to treat B cell disorders present a serious risk of compromising humoral immunity because the cell-targeting antigens expressed on all, or most, B cells. Consequently, such therapies are not clinically desirable and thus , a need remains in the art for more efficient therapies for B cell-related conditions that spare humoral immunity.

[0120] The improved adoptive cell therapy compositions and methods disclosed herein provide genetically modified immune effector cells that can be easily expanded, exhibit long-term persistence in vivo, and reduce deficiency of humoral immunity by targeting antigen-expressing B cells of B cell maturation (BCMA, also known as CD269 or tumor necrosis factor receptor superfamily, member 17; TNFRSF17).

[0121] BCMA is a member of the tumor necrosis factor receptor superfamily (see, for example, Thompson et al., J. Exp. Medicine, 192(1): 129-135, 2000, and Mackay et al., Annu. Rev.Immunol, 21: 231-264, 2003. BCMA binds to B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) (see, for example, Mackay et al., 2003 and Kalled et al., Immunological Reviews, 204:43-54, 2005). Among nonmalignant cells, BCMA has been reported to be expressed primarily in plasma cells and mature B cell subsets (see, for example, Laabi et al., EMBO J., 77(1): 3897-3904, 1992; Exp. Medicine, 199(1): 91-97, 2004; and Ng et al., J. Immunol., 73(2): 807-817, 2004. BCMA-deficient mice are healthy and have normal numbers of B cells , but the survival of long-lived plasma cells is impaired (see, e.g., O'Connor et al., 2004; Xu et al., Mol. Cell. Biol, 21(12): 40674074, 2001; and Schiemann et al., Science, 293(5537): 2111-2114, 2001). BCMA RNA has been detected universally in multiple myeloma cells and other lymphomas, and BCMA protein has been detected on the surface of plasma cells from patients with multiple myeloma by several investigators (see, for example, Novak et al., Blood, 103 (2): 689-694, 2004; Neri et al., Clinical Cancer Research, 73(19): 5903-5909, 2007; et al., Blood, 703(8): 3148-3157, 2004.

[0122] In various embodiments, CARs comprising humanized anti-BCMA antibody sequences are highly effective compared to anti-BCMA CARs comprising mouse antibody sequences; undergo robust in vivo expansion; and recognize BCMA-expressing human B cells and show cytotoxic activity against BCMA expressing B cells. The present inventors have unexpectedly discovered that substantially modified portions of the mouse anti-human BCMA antibody sequences, including minor and structural antigen recognition domains, did not substantially alter the function of humanized anti-BCMA CAR T cells, and that the cytokine release properties of humanized anti-BCMA CAR T cells can be increased or decreased compared to a T cell expressing a reference anti-BCMA CAR humanized by altering at least the transmembrane domain of the humanized anti-BCMA CAR.

[0123] In particular embodiments, a humanized anti-BCMA CAR leads to surprisingly high tonic or antigen-independent cytokine release. In particular embodiments, the transmembrane domain of a humanized anti-BCMA CAR that generates antigen-independent cytokine release is altered such that the signaling properties (e.g., cytokine release) of the humanized anti-BCMA CAR T cells are altered. become substantially antigen-dependent compared to a T cell expressing the unaltered humanized anti-BCMA CAR.

[0124] In certain embodiments, the transmembrane domain of a humanized anti-BCMA CAR that generates antigen-independent cytokine release is altered such that the altered humanized anti-BCMA CAR T cells become substantially antigen-dependent or release substantially less cytokine in the absence of antigen compared to a T cell expressing unaltered humanized anti-BCMA CAR.

[0125] In one embodiment, a CAR comprising a humanized anti-BCMA antibody or antigen-binding fragment, a transmembrane domain, and one or more intracellular signaling domains is provided.

[0126] In one embodiment, an immune effector cell is genetically modified to express a CAR considered here is provided. T cells that express a CAR are referred to herein as CAR T cells or CAR-modified T cells.

[0127] In various embodiments, the genetically modified immune effector cells considered herein are administered to a patient with a B cell-related condition, for example, an autoimmune disease associated with B cells or a B cell malignancy.

[0128] The practice of the invention will utilize, unless specifically indicated otherwise, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the practitioner. technique, many of which are described below for illustration purposes. Such techniques are fully explained in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in journals such as Advances in Immunology.

B. Definitions

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present invention, the following terms are defined below.

[0130] The articles “one” and “the” are used here to refer to one or more than one (that is, at least one, or one or more) of the grammatical object of the article. By way of example, “an element” means an element or one or more elements.

[0131] The use of the alternative (e.g., “or”) should be understood to mean one, both or any combination of these alternatives.

[0132] The term “and/or” should be understood to mean one or both of the alternatives.

[0133] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity , weight or length. In one embodiment, the term “about” or “approximately” refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length ± 15%, ± 10% , ± 9 %, ± 8 %, ± 7 %, ± 6 %, ± 5 %, ± 4 %, ± 3 %, ± 2 %, or ± 1 % about a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length.

[0134] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprise” and “comprising” will be understood to imply the inclusion of an established step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant to include, and limited to, everything that follows the phrase “consisting of”. Additionally, the phrase “consisting of” indicates that the elements listed are necessary or required, and that no other elements may be present. By “consisting essentially of” is meant to include any element listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Additionally, the phrase “consisting essentially of” indicates that the listed elements are necessary or required, but that no other elements are present that materially affect the activity or action of the listed elements.

[0135] Reference throughout this specification to “an embodiment”, “an embodiment”, “a particular embodiment”, “a related embodiment”, “a certain embodiment”, “a additional embodiment", or "another embodiment" or combinations thereof means that a particular feature, structure or characteristic described in relation to the embodiment is included in at least one embodiment of the present invention. Furthermore, the appearances of the preceding phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any one or more embodiments. It is also understood that positive recitation of a feature in an embodiment serves as a basis for excluding the feature in a particular embodiment.

C. Chimeric antigen receptors

[0136] In various embodiments, genetically engineered receptors that redirect cytotoxicity from immune effector cells toward B cells are provided. These genetically engineered receptors are referred to here as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., BCMA) with an intracellular domain that activates the T cell receptor to generate a chimeric protein that exhibits specific anti-BCMA cellular immune activity. As used here, the term “chimeric” describes being composed of parts of different proteins or DNAs of different origins.

[0137] CARs considered here comprise an extracellular domain (also referred to as a binding domain or antigen-specific binding domain) that binds to BCMA, a transmembrane domain and an intracellular signaling domain. Engagement of the anti-BCMA antigen-binding domain of the CAR with BCMA on the surface of a target cell results in clustering of the CAR and delivers an activating stimulus to the CAR-containing cell. The main features of CARs are their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility (MHC)-independent manner. ), exploiting the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific coreceptors.

[0138] In various embodiments, a CAR comprises an extracellular binding domain comprising a humanized BCMA-specific binding domain; a transmembrane domain; one or more intracellular costimulatory signaling domains; and a primary signaling domain.

[0139] In particular embodiments, a CAR comprises an extracellular binding domain comprising a humanized anti-BCMA antibody or antigen-binding fragment thereof; one or more hinge domains or spacer domains; a transmembrane domain including; one or more intracellular costimulatory signaling domains; and a primary signaling domain.

1. Binding Domain

[0140] In particular embodiments, CARs considered herein comprise an extracellular binding domain comprising a humanized anti-BCMA antibody or antigen-binding fragment thereof that specifically binds to a human BCMA polypeptide expressed on a B cell. used herein, the terms “binding domain”, “extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain” and “extracellular antigen-specific binding domain”, are used interchangeably and provide a CAR with the ability to specifically bind to the target antigen of interest, e.g. BCMA. The binding domain may be derived from a natural, synthetic, semi-synthetic or recombinant source.

[0141] The terms “specific binding affinity” or “specifically binds” or “specifically bound” or “specific binding” or “specifically targeted” as used herein describe binding of an anti-BCMA antibody or antigen-binding fragment of this (or a CAR comprising the same) the BCMA in greater binding affinity than the preceding binding. A binding domain (or a CAR comprising a binding domain or a fusion protein containing a binding domain) “specifically binds” to a BCMA if it binds or associates the BCMA with an affinity or Ka (i.e. an equilibrium association constant of a particular binding interaction with units of 1/M), for example, greater than or equal to about 105 M-1. In certain embodiments, a binding domain (or a fusion protein thereof) binds to a target with a Ka greater than or equal to about 106 M-1, 107 M-1, 108 M-1, 109 M-1, 1010 M-1, 1011 M-1, 1012 M-1, or 1013 M-1. “High affinity” binding domains (or single-chain fusion proteins thereof) refer to those binding domains with a Ka of at least 107 M-1, at least 108 M-1, at least 109 M-1, at least 1010 M-1, at least 1011 M-1, at least 1012 M-1, at least 1013 M-1 or greater.

[0142] Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with M units (e.g., 10-5 M to 10-13 M or less). Affinities of binding domain polypeptides and CAR proteins according to the present disclosure can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or displacement assays. using labeled ligands, or using a surface plasmon resonance device such as the Biacore T100, which is available from Biacore, Inc., Piscataway, NJ, or optical biosensor technology such as the EPIC or EnSpire system which are available from Corning and Perkin Elmer respectively (see also, for example, Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173; 5,468,614 or equivalent).

[0143] In one embodiment, the specific binding affinity is about 2 times greater than the preceding binding, about 5 times greater than the preceding binding, about 10 times greater than the preceding binding, about 20 times larger than the preceding bond, about 50 times larger than the preceding bond, about 100 times larger than the preceding bond, or about 1000 times larger than the preceding bond or more.

[0144] In particular embodiments, the extracellular binding domain of a CAR comprises an antibody or antigen-binding fragment thereof. An "antibody" refers to a binding agent that is a polypeptide comprising at least one light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a peptide, lipid, polysaccharide or nucleic acid containing an antigenic determinant, such as that recognized by an immune cell.

[0145] An “antigen (Ag)” refers to a compound, composition or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions (such as one that includes a cancer-specific protein ) that are injected or absorbed into an animal. An antigen reacts with products of specific cellular or humoral immunity, including those induced by heterologous antigens, such as disclosed antigens. In particular embodiments, the target antigen is an epitope of a BCMA polypeptide.

[0146] An “epitope” or “antigenic determinant” refers to the region of an antigen to which a binding agent binds. Epitopes can be formed either from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually at least 5, about 9, or about 8 to 10 amino acids in a unique spatial conformation.

[0147] Antibodies include antigen-binding fragment thereof, such as Camel Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv proteins (scFv) Bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (“dsFv”) and single-domain antibodies (sdAb, Nanobody) and full-length antibody portions responsible for binding to the antigen. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies) and antigen-binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.

[0148] As would be understood by the skilled person and as described elsewhere here, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, Y, and μ. Mammalian light chains are classified as À or K. Immunoglobulins comprising α, δ, ε, Y, and μ heavy chains are classified as immunoglobulin (Ig)A, IgD, IgE, IgG, and IgM. The complete antibody forms a “Y” shape. The stem of the Y consists of the second and third constant region (and for IgE and IgM, the fourth constant region) of two heavy chains linked together and disulfide (inter-chain) bonds are formed at the hinge. Y, α, and δ heavy chains have a constant region composed of three tandem (in a row) Ig domains, and a hinge region for added flexibility; μ and ε heavy chains have a constant region composed of four immunoglobulin domains. The second and third constant regions are referred to as the “CH2 domain” and “CH3 domain”, respectively. Each stem of the Y includes the variable region and first constant region of a single heavy chain linked to the variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for binding to the antigen.

[0149] Variable regions of light and heavy chains contain a “structure” region interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. CDRs can be defined or identified by conventional methods, such as by sequence according to Kabat et al (Wu, TT and Kabat, E. A., J Exp Med. 132(2): 211-50, (1970); Borden, P . and Kabat E. A., PNAS, 84: 2440-2443 (1987); by structure according to Chothia et al. (Chothia, C. and Lesk, A.M., J Mol. Biol., 196(4): 901-917 (1987), Chothia, C. et al., Nature, 342: 877 -883 (1989)).

[0150] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional spaces. CDRs are mainly responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, the CDRs located in the variable domain of the antibody heavy chain are referred to as CDRH1, CDRH2, and CDRH3, while the CDRs located in the variable domain of the antibody light chain are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although the 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 called specificity-determining residues (SDRs). Illustrative examples of light chain CDRs that are suitable for constructing humanized anti-BCMA CARs considered herein include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 1 to 3. Illustrative examples of heavy chain CDRs that are suitable for construct humanized anti-BCMA CARs considered herein include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 4 to 6.

[0151] References to “VH” or “VH” refer to the variable region of a heavy chain immunoglobulin, including that of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment as disclosed herein. References to "VL" or "VL" refer to the variable region of a light chain immunoglobulin, including that of an antibody, Fv, scFv, dsFv, Fab or other antibody fragment as disclosed herein.

[0152] A “monoclonal antibody” is an antibody produced by a single clone of B lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for example, by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0153] A “chimeric antibody” has structural residues from one species, such as human, and CDRs (which generally confer binding and antigen) from another species, such as a mouse. In particular preferred embodiments, a CAR considered herein comprises an antigen-specific binding domain that is a chimeric antibody or antigen-binding fragment thereof.

[0154] In preferred embodiments, the antibody is a humanized antibody (such as a humanized monoclonal antibody) or fragment thereof that specifically binds to a human BCMA polypeptide. A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is called a “donor”, and the human immunoglobulin that provides the structure is called an “acceptor”. In one embodiment, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, that is, at least about 85 to 90%, such as about 95% or more identical. Consequently, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions, which have no substantial effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed through genetic engineering (see, for example, U.S. Patent No. 5,585,089).

[0155] In particular embodiments, a humanized anti-BCMA antibody or antigen-binding fragment thereof, includes but is not limited to a Camel Ig (a camelid antibody (VHH)), NAR Ig, Fab fragments, Fab' fragments , F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv antibody (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, stabilized Fv protein by disulfide (“dsFv”), and single domain antibody (sdAb, Nanobody).

[0156] “Camel Ig” or “VHH camelid” as used herein refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte, et al, FASEB J., 21: 3490-3498 (2007)). A “heavy chain antibody” or a “camelid antibody” refers to an antibody that contains two VH domains and no light chain (Riechmann L. et al, J. Immunol., Methods 231:25-38 (1999); WO94/04678; WO94/25591; U.S. Patent No. 6,005,079).

[0157] “IgNAR” from “immunoglobulin novel antigen receptor” refers to the class of antibodies from the shark immune repertoire that consists of homodimers of a variable novel antigen receptor (VNAR) domain and five receptor domains. constant new antigen (CNAR). IgNARs represent some of the least known immunoglobulin-based protein scaffolds and are highly stable and possess efficient binding characteristics. The inherent stability can be attributed to both (i) the underlying Ig scaffolds, which feature a considerable number of charged and hydrophilic surface-exposed residues compared to the conventional antibody VH and VL domains found in murine antibodies; as to (ii) stabilizing structural features in the complementarity determining region (CDR) loops including inter-loop disulfide bridges, and intra-loop hydrogen bonding patterns.

[0158] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a unique antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize easily . Pepsin treatment produces an F(ab')2 fragment that has two antigen-combining sites and is further capable of cross-linking to antigen.

[0159] “Fv” is the minimal antibody fragment that contains a complete antigen-binding site. In one embodiment, a double-chain Fv species consists of a dimer of a heavy and light chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, a heavy and light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structural analogue to those in a kind of double-chain Fv. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

[0160] The Fab fragment contains the variable domains of the heavy and light chains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of some residues at the carboxy terminus of the CH1 domain of the heavy chain including one or more cysteines of the antibody hinge region. Fab'-SH is the designation here for Fab' in which the cysteine residue(s) of the constant domains have a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0161] The term “diabodies” refers to antibody fragments with two antigen-binding sites, whose fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same chain of polypeptide (VH-VL). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described, 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). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0162] “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).

[0163] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of the antibody, where these domains are present on a single polypeptide chain and in any orientation (e.g., VL-VH or VH-VL ). Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0164] In preferred embodiments, a CAR considered herein comprises an antigen-specific binding domain that is an scFv and may be a murine, human or humanized scFv. Single-chain antibodies can be cloned from the V region genes of a desired target-specific hybridoma. The production of such hybridomas has become routine. A technique that can be used for cloning the variable region heavy chain (VH) and the variable region light chain (VL) has been described, for example, in Orlandi et al., PNAS, 1989; 86:3833-3837.

[0165] In particular embodiments, the antigen-specific binding domain is a humanized scFv that binds to a human BCMA polypeptide. Illustrative examples of variable heavy chains that are suitable for constructing anti-BCMA CARs contemplated herein include, but are not limited to, the amino acid sequences set forth in SEQ ID NO: 10-14. Illustrative examples of variable light chains that are suitable for constructing anti-BCMA CARs contemplated herein include, but are not limited to, the amino acid sequences set forth in SEQ ID NO: 7-9.

[0166] An exemplary humanized anti-BCMA specific binding domain is a BCMA-specific immunoglobulin variable region comprising at least one human framework region. A “human framework region” refers to a wild-type (i.e., naturally occurring) framework region of a human immunoglobulin variable region, an altered framework region of a human immunoglobulin variable region less than about than 50% (e.g., preferably less than about 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) of the amino acids in the region are deleted or replaced (e.g. , with one or more amino acid residues of a non-human immunoglobulin framework region at corresponding positions), or an altered framework region of a non-human immunoglobulin variable region by less than about 50% (e.g., less than 45% , 40%, 30%, 25%, 20%, 15%, 10% or 5%) of the amino acids in the deleted or substituted region (e.g., at exposed residue positions and/or with one or more amino acid residues of a human immunoglobulin framework region at corresponding positions) so that, in one aspect, immunogenicity is reduced.

[0167] In certain embodiments, a human framework region is a wild-type framework region of a human immunoglobulin variable region. In some other embodiments, a human framework region is an altered framework region of a human immunoglobulin variable region with amino acid deletions or substitutions in one, two, three, four, five, six, seven, eight, nine, ten or more positions. In other embodiments, a human framework region is an altered framework region of a non-human immunoglobulin variable region with amino acid deletions or substitutions in one, two, three, four, five, six, seven, eight, nine, ten or more positions.

[0168] In particular embodiments, a BCMA-specific binding domain comprises at least one, two, three, four, five, six, seven or eight human framework regions (FR) selected from human light chain FR1, FR1 of human heavy chain, 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.

[0169] Human FRs that may be present in a specific binding domain of BCMA 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 replaced or deleted.

[0170] In certain embodiments, a humanized anti-BCMA specific binding domain comprises (a) a humanized light chain variable region comprising a human light chain FR1, a human light chain FR2, a human light chain FR3 human light chain, and a human light chain FR4, and (b) a humanized heavy chain variable region comprising a human heavy chain FR1, a human heavy chain FR2, a human heavy chain FR3, and a heavy chain FR4 human.

[0171] BCMA-specific binding domain provided here also comprises one, two, three, four, five or six CDRs. Such CDRs may be non-human CDRs or altered non-human CDRs selected from CDRL1, CDRL2 and CDRL3 of the light chain and CDRH1, CDRH2 and CDRH3 of the heavy chain. In certain embodiments, a BCMA-specific binding domain comprises (a) a light chain variable region comprising a light chain CDRL1, a light chain CDRL2, and a light chain CDRL3, and (b) a light chain variable region. heavy chain comprising a heavy chain CDRH1, a heavy chain CDRH2 and a heavy chain CDRH3.

2. Ligands

[0172] In certain embodiments, the CARs considered here may comprise linker residues between the various domains, for example, added for appropriate conformation and spacing of the molecule. In particular embodiments, the linker is a variable region binding sequence. A “variable region binding sequence” is an amino acid sequence that connects the VH and VL domains and provides a spacer function compatible with the interaction of the two binding subdomains so that the resulting polypeptide retains a specific binding affinity. to the same target molecule as an antibody comprising the same light and heavy chain variable regions. CARs considered here may comprise one, two, three, four or five, or more ligands. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more amino acids long.

[0173] Illustrative examples of linkers include glycine (G)n polymers; glycine-serine polymers (G1-5S1-5)n, where n is an integer of at least one, two, three, four or five; glycine-alanine polymers; alanine-serine polymers; and other flexible binders known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be capable of serving as a neutral boundary between domains of fusion proteins such as the CARs described here. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). The skilled artisan will recognize that the design of a CAR in particular embodiments may include linkers that are all or partially flexible, such that the linker may include a flexible linker as well as one or more less flexible structure-conferring moieties to provide a structure of desired CAR.

[0174] Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 46); TGEKP (SEQ ID NO: 47) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 48) (Pomerantz et al. 1995, supra); (GGGGS)n where = 1, 2, 3, 4 or 5 (SEQ ID NO: 49) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: 50) (Chaudhary et al., 1990, Proc. Natl. Sci. U.S.A. 87:10661070); 52); LRQRDGERP (SEQ ID NO: 53); LRQKDGGGSERP (SEQ ID NO: 54); LRQKD(GGGS)2 ERP (SEQ ID NO: 55). modeling either DNA binding sites and peptides alone (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:1109911103 (1994) or by phage display methods. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 56) (Cooper et al., Blood, 101(4): 16371644 (2003)).

3. Spacer Domain

[0175] In particular embodiments, the CAR binding domain is followed by one or more “spacer domains,” which refer to the region that moves the antigen-binding domain away from the effector cell surface to enable contact. appropriate cell/cell antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0176] In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG1 or IgG4.

4. Hinge Domain

[0177] The CAR binding domain is generally followed by one or more “hinge domains”, which play a role in positioning the antigen binding domain away from the effector cell surface to enable appropriate cell/cell contact, binding and activation to the antigen. A CAR generally comprises one or more hinge domains between the binding domain and the transmembrane (TM) domain. The hinge domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0178] An “altered hinge region” refers to (a) a naturally occurring hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10% or 5 % amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region that is at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) in length with up to 30% of amino acid changes (e.g., up to 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions), or (c) a portion of a naturally occurring hinge region comprising the hinge region of core (which can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, or 15 amino acids in length). In certain embodiments, one or more cysteine residues in a naturally occurring immunoglobulin hinge region may be replaced by one or more other amino acid residues (e.g., one or more serine residues). An altered immunoglobulin hinge region may alternatively or additionally have a proline residue from a wild-type immunoglobulin hinge region replaced by another amino acid residue (e.g., a serine residue).

[0179] Other illustrative hinge domains suitable for use in the CARs described here include the hinge region derived from extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, PD1, CD152 and CD7, which can be wild-type hinge regions from these molecules or can be altered. In another embodiment, the hinge domain comprises a hinge region, PD1, CD152 or CD8α.

5. Transmembrane domain (TM)

[0180] The “transmembrane domain” is the portion of the CAR that fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. The TM domain may be derived from (i.e., comprises at least the transmembrane regions of) T cell receptor alpha, beta, or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154 and PD1. In a particular embodiment, the TM domain is synthetic and predominantly comprises hydrophobic residues such as leucine and valine.

[0181] In one embodiment, the CARs considered here comprise a TM domain derived from PD1, CD152 or CD8α. In another embodiment, a CAR considered herein comprises a TM domain derived from PD1, CD152 or CD8α and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length that binds to the TM domain and intracellular signaling domain of CAR. A glycine-serine-based linker provides a particularly suitable linker.

6. Intracellular Signaling Domain

[0182] In particular embodiments, CARs considered here comprise an intracellular signaling domain. An “intracellular signaling domain” refers to that part of a CAR that participates in transducing the effective anti-BCMA CAR message that binds a human BCMA polypeptide within the immune effector cell to evoke effector cell function, e.g. , activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses induced with antigen that binds to the extracellular CAR domain.

[0183] The term “effector function” refers to a specialized function of an immune effector cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including secretion of a cytokine. Furthermore, the term “intracellular signaling domain” refers to the portion of a protein that transduces the signal for effector function and that directs the cell to perform a specialized function. Although usually the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain means to include any truncated portion of the intracellular signaling domain sufficient to transduce effector function signal.

[0184] It is known that signals generated through the TCR alone are insufficient for complete activation of the T cell and that a secondary or co-stimulatory signal is also necessary. Furthermore, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation via the TCR (e.g., a TCR/CD3 complex) and costimulatory signaling systems that act in an antigen-independent manner to provide a secondary or costimulatory signal. In preferred embodiments, a CAR considered herein comprises an intracellular signaling domain comprising one or more "costimulatory signaling domain" and a "primary signaling domain".

[0185] Primary signaling domains regulate primary activation of the TCR complex in a stimulatory manner, or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs that are known as tyrosine-based immunoreceptor activation motifs or ITAMs.

[0186] Illustrative examples of ITAM containing primary signaling domains that are of particular use in the invention include those derived from TCRZ, FCRY, FcRβ, CD3Y, CD3δ, CD3ε, CD3Z, CD22, CD79a, CD79b and CD66d. In particular preferred embodiments, a CAR comprises a CD3Z Primary Signaling Domain and one or more costimulatory signaling domains. The primary intracellular signaling and costimulatory signaling domains can be linked in any order together with the carboxyl terminus of the transmembrane domain.

[0187] CARs considered here comprise one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing CAR receptors. As used herein, the term “costimulatory signaling domain” or “costimulatory domain” refers to an intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal necessary for efficient activation and function of T lymphocytes upon antigen binding. Illustrative examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4 ), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70. In one embodiment, a CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137 and CD134 and a CD3Z primary signaling domain.

[0188] In another embodiment, a CAR comprises costimulatory signaling domains CD28 and CD137 and a primary signaling domain CD3Z.

[0189] In yet another embodiment, a CAR comprises CD28 and CD134 costimulatory signaling domains and a CD3Z primary signaling domain.

[0190] In one embodiment, a CAR comprises costimulatory signaling domains CD137 and CD134 and a primary signaling domain CD3Z.

[0191] In one embodiment, a CAR comprises a CD137 costimulatory signaling domain and a CD3Z primary signaling domain.

[0192] In one embodiment, a CAR comprises a CD134 costimulatory signaling domain and a CD3Z primary signaling domain.

[0193] In one embodiment, a CAR comprises a CD28 costimulatory signaling domain and a CD3Z primary signaling domain.

[0194] In particular embodiments, CARs considered here comprise a humanized anti-BCMA antibody or antigen-binding fragment thereof specifically linked to a BCMA polypeptide expressed in B cells.

[0195] In one embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA polypeptide; a transmembrane domain derived from a polypeptide selected from the group consisting of: T cell receptor alpha, beta, or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33 , CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154 and PD1; and one or more intracellular costimulatory signaling domains of a costimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40 ), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70; and a primary signaling domain of TCRZ, FcRY, FcRβ, CD3Y, CD3δ, CD3ε, CD3Z, CD22, CD79a, CD79b, and CD66d.

[0196] In one embodiment, a CAR comprises a humanized anti-BCMA scFv that binds to a BCMA polypeptide; a hinge domain selected from the group consisting of: IgG1 hinge/CH2/CH3, IgG4 hinge/CH2/CH3, a PD1 hinge, a CD152 hinge, and a CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of: T cell receptor alpha, beta, or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33 , CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154 and PD1; and one or more intracellular costimulatory signaling domains of a costimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40 ), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70; and a primary signaling domain of TCRZ, FCRY, FcRβ, CD3Y, CD3δ, CD3ε, CD3Z, CD22, CD79a, CD79b and CD66d.

[0197] In one embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain selected from the group consisting of: IgG1 hinge/CH2/CH3, IgG4 hinge/CH2/CH3, a PD1 hinge, a CD152 hinge, and a CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of: T cell receptor alpha, beta, or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33 , CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154 and PD1; a short polypeptide or oligo linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length that links the TM domain to the intracellular signaling domain of the CAR; and one or more intracellular costimulatory signaling domains of a costimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40 ), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70; and a primary signaling domain of TCRZ, FcRY, FcRβ, CD3Y, CD3δ, CD3ε, CD3Z, CD22, CD79a, CD79b, and CD66d.

[0198] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA 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 CD3Z primary signaling domain.

[0199] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA 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 CD3Z primary signaling domain.

[0200] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA 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 CD3Z primary signaling domain.

[0201] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain comprising a PD1 hinge polypeptide; a PD1 or CD152 transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD137 intracellular costimulatory signaling domain; and a CD3Z primary signaling domain.

[0202] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain comprising a PD1 hinge polypeptide; a PD1 or CD152 transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD134 intracellular costimulatory signaling domain; and a CD3Z primary signaling domain.

[0203] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain comprising a PD1 hinge polypeptide; a PD1 or CD152 transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD28 intracellular costimulatory signaling domain; and a CD3Z primary signaling domain.

[0204] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain comprising a CD 152 hinge polypeptide; a PD1 or CD152 transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD137 intracellular costimulatory signaling domain; and a CD3Z primary signaling domain.

[0205] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain comprising a CD 152 hinge polypeptide; a PD1 or CD152 transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD134 intracellular costimulatory signaling domain; and a CD3Z primary signaling domain.

[0206] In a particular embodiment, a CAR comprises a humanized anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain comprising a CD 152 hinge polypeptide; a PD1 or CD152 transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD28 intracellular costimulatory signaling domain; and a CD3Z primary signaling domain.

[0207] Furthermore, the design of the CARs considered here allows for improved expansion, long-term persistence, and cytotoxic properties in T cells expressing the CARs compared to unmodified T cells or T cells modified to express other CARs.

D. Polypeptides

[0208] The present invention contemplates, in part, CAR polypeptides and fragments thereof, cells and compositions comprising the same, and vectors that express polypeptides. In preferred embodiments, a polypeptide comprising one or more CARs as set forth in any of SEQ ID NOs: 15 to 29, 71 and 73 is provided.

[0209] “Polypeptide”, “polypeptide fragment”, “peptide” and “protein” are used interchangeably, unless otherwise specified, and in accordance with conventional meaning, that is, as a sequence of amino acids. Polypeptides are not limited to a specific length, for example, they may comprise a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphosrylations and the like, as well as other modifications known in the art, both naturally occurring and naturally occurring. In various embodiments, the CAR polypeptides considered herein comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-transductionally or post-transductionally directs transfer of the protein. Illustrative examples of suitable signal sequences useful in CARs disclosed herein include, but are not limited to, an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha signal polypeptide. Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides considered herein specifically encompass the CARs of the present disclosure, or sequences that have deletions of additions and/or substitutions of one or more amino acids of a CAR as disclosed herein.

[0210] An “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refers to in vitro isolation and/or purification of a peptide or polypeptide molecule from a cellular environment, and association with other components of the cell, that is, it is not significantly associated with substances in vivo. Similarly, an “isolated cell” refers to a cell that was obtained from a tissue or organ in vivo and is substantially free of extracellular matrix.

[0211] Polypeptides include “polypeptide variants”. Polypeptide variants may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the binding affinity and/or other biological properties of CARs by introducing one or more substitutions, deletions, additions and/or insertions into a binding domain, hinge, TM domain. , costimulatory signaling domain or primary signaling domain of a CAR polypeptide. Preferably, polypeptides of the invention include polypeptides having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% identity thereof.

[0212] Polypeptides include “polypeptide fragment”. Polypeptide fragment refers to a polypeptide, which may be monomeric or multimeric, that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion or substitution of a recombinantly produced or naturally occurring polypeptide . In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 500 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Particularly useful polypeptide fragments include functional domains, including antigen-binding domains or antibody fragments. In the case of a humanized anti-BCMA antibody, useful fragments include, but are not limited to: a CDR region, a light or heavy chain CDR3 region; a variable region of a light or heavy chain; a portion of an antibody chain or variable region including two CDRs; and the like.

[0213] The polypeptide can also be fused in structure or conjugated to a ligand or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.

[0214] As noted above, polypeptides of the invention can be altered in various ways including amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence changes 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), Pat. U.S. No. 4,873,192, Watson, J. D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and references cited herein. Guidance on appropriate amino acid substitutions that do not affect biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.) .

[0215] In certain embodiments, a variant will contain conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted in place of another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications can be made to the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a polypeptide variant or derivative with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, polypeptide variant of the invention, one skilled in the art, for example, can change one or more of the codons of the coding DNA sequence, e.g. , according to Table 1. TABLE 1 - Amino Acid Codons

[0216] Guidance in determining which amino acid residues can be replaced, inserted or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTARTM software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, that is, substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and polar amino acids uncharged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In a peptide or protein, suitable conservative amino acid substitutions are known to those of skill in the art and generally can be made without altering the biological activity of a resulting molecule. Those of skill in this art recognize that, in general, 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). Exemplary conservative substitutions are described in U.S. Provisional Patent Application No. 61/241,647, the disclosure of which is incorporated herein by reference.

[0217] When making such changes, the hydropathic amino acid index can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function in a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8); tryptophan (0.9); tyrosine (1.3); proline (1.6); Histidine (3.2); glutamate (3.5); glutamine (3.5); aspartate (3.5); asparagine (3.5); Lysine (3.9); and arginine (4.5).

[0218] It is known in the art that certain amino acids can be replaced by other amino acids having a similar index or hydropathic point and still result in a protein with similar biological activity, that is, still obtain a functionally biological equivalent protein. When making such changes, substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that substitution of similar amino acids can be done effectively on the basis of hydrophilicity.

[0219] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted in place of another having a similar hydrophilicity value and still obtain a biologically equivalent protein, and in particular, an immunologically equivalent one. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0220] As outlined above, amino acid substitutions can be based on the relative similarity of the amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size and the like.

[0221] Polypeptide variants further include glycosylated forms, aggregation conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). Covalent variants can be prepared by linking functionalities to groups that are found in the amino acid chain or in the N- or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants and muteins. Truncations or deletions of regions that do not affect the functional activity of proteins are also variants.

[0222] In one embodiment, where expression of two or more polypeptides is desired, the polynucleotide sequences that encode each other can be separated by an IRES sequence as discussed elsewhere here. In another embodiment, two or more polypeptides can be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences.

[0223] Polypeptides of the present invention include fusion polypeptides. In preferred embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided, e.g., CARs. Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten or more polypeptide segments. Fusion polypeptides are typically linked C-terminal to N-terminal, although they can also be linked C-terminal to C-terminal, N-terminal to N-terminal, or N-terminal to C-terminal. The fusion protein polypeptides may be in any order or a specified order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, as long as the desired transcriptional activity of the fusion polypeptide is preserved. Fusion polypeptides can be produced by synthetic chemical methods or by chemical linkage between the two moieties or can generally be prepared using other standard techniques. Linked DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as discussed elsewhere herein.

[0224] In one embodiment, a fusion partner comprises a sequence that assists in expressing the protein (an expression enhancer) in higher yields than the native recombinant protein. Other fusion partners can be selected to increase the solubility of the protein or to allow the protein to be targeted to desired intracellular compartments or to facilitate transport of the fusion protein across the cell membrane.

[0225] Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. Furthermore, the polypeptide site can be placed 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, ) and self-cleaving viral oligopeptides (see de Felipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0226] Suitable protease cleavage sites and self-cleaving peptides are known to the skilled person (see, for example, in Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al (2004) Nature Biotech. 5, 589594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for NIa proteases of potyviruses (e.g., tobacco etch virus protease), HC proteases of potyviruses, (P35)P1 proteases of potyviruses, NIa proteases of bioviruses, proteases encoding biovirus RNA-2, L proteases of aptaviruses, 2A proteases of enteroviruses, 2A proteases of rhinoviruses, 3C proteases of picorna, 24k proteases of comoviruses, 24k proteases of nepoviruses, 3C-like protease of RTSV (virus rice tungro spherical), PYVF (parsnip yellow spot virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase. Due to their high cleavage stringency, TEV (tobacco attack virus) protease cleavage sites are preferred in one embodiment, for example, EXXYXQ(G/S) (SEQ ID NO: 57), e.g. ENLYFQG (SEQ ID NO: 58) and ENLYFQS (SEQ ID NO: 59), where X represents any amino acid (TEV cleavage occurs between Q and G or Q and S).

[0227] In a particular embodiment, self-cleaving peptides include those polypeptide sequences obtained from peptides of potyvirus and cardiovirus 2A, FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus and porcine teschovirus.

[0228] In certain embodiments, the self-cleavage polypeptide site comprises a 2A or 2A-like sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). TABLE 2: Exemplary 2A sites include the following sequences:

[0229] In preferred embodiments, a polypeptide considered herein comprises a CAR polypeptide.

E. Polynucleotides

[0230] In preferred embodiments, a polynucleotide encoding one or more CAR polypeptides is provided, for example, SEQ ID NOs: 30 to 44, 70 and 72. As used herein, the terms “polynucleotide” or “nucleic acid” refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA ) or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99% or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the variant maintains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates, in part, polynucleotides comprising expression vectors, viral vectors, and transfer plasmids, and compositions, and cells comprising the same.

[0231] In particular embodiments, polynucleotides are provided by this invention that encode at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500 , 1750, or 2000 or more contiguous amino acid residues of a polypeptide of the invention, as well as all intermediate lengths. It will be readily understood that “intermediate lengths” in this context mean any length between the cited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

[0232] As used herein, the terms “polynucleotide variant” and “variant” and the like refer to polynucleotides that exhibit substantial sequence identity to a polynucleotide reference sequence or polynucleotides that hybridize to a reference sequence under stringent conditions. which are defined below. These terms include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides compared to a reference polynucleotide. In this regard, it is well understood in the art that certain changes including mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0233] The phrases “sequence identity” or, for example, comprising a “sequence 50% identical to”, as used herein, refers to the extent that sequences are identities on a nucleotide-by-nucleotide basis or an amino acid basis. per amino acid over a comparison window. Additionally, a “percent sequence identity” can be calculated by comparing two optimally aligned sequences over the comparison window by determining the number of positions at which the nucleic acid base of identity (e.g., A, T, C , G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln , Cys, and Met) occurs in both sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0234] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence identity”, “percent sequence identity”, and “substantial identity”. A “reference sequence” is at least 12, but often 15 to 18 and often at least 25 monomer units, inclusive of nucleotide and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150, in which a sequence is compared to a reference sequence. of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which do not comprise additions or deletions) for optimal alignment of the two sequences. Aligning a comparison window can be conducted by computerizing algorithm implementations (GAP, BESTFIT, FASTA, and TFASTA o Wisconsin Genetics Software Package Version 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of several selected methods. Reference can also be made to the BLAST family of programs as, for example, disclosed by Altschul et al., 1997, Nucl. Acid Res 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 19941998, Chapter 15.

[0235] As used herein, “isolated polynucleotide” refers to a polynucleotide that has been purified from the flanking sequences in a naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment. An “isolated polynucleotide” also refers to a complementary DNA (cDNA), a recombinant DNA, or another polynucleotide that does not exist in nature and was made by human hands.

[0236] Terms that describe the orientation of polynucleotides include: 5' (typically the end of the polynucleotide having a free phosphate group) and 3' (typically the end of the polynucleotide having a free hydroxyl group (OH)). Polynucleotide sequences can be annotated in a 5' to 3' orientation or a 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the “sense,” “positive,” or “coding” strand because its sequence is identical to the premessenger (premRNA) sequence [except for uracil (U) in RNA, instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand that is the strand transcribed by RNA polymerase is designated as the “template,” “antisense,” “negative,” or “non-coding” strand. As used herein, the term “reverse orientation” refers to a 5' to 3' sequence written in the 3' to 5' orientation or a 3' to 5' sequence written in the 5' to 3' orientation.

[0237] The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the rules of base pairing. For example, the complementary strand of the DNA sequence 5' A G T C A T G 3' is 3' T C A G T A C 5'. The last sequence is often written as the reverse complement with the 5' end on the left and the 3' end on the right, 5' C A T G A C T 3'. A sequence that is equal to its reverse complement is said to be a palindromic sequence. Complementarity can be “partial”, in which only some of the nucleic acids’ bases are combined according to the rules of base pairing. Or, it may be “complete” or “total” complementarity between the nucleic acids.

[0238] Furthermore, it will be appreciated by one of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide, or variant fragment thereof, as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are specifically considered by the present invention, for example, polynucleotides that are optimized by human and/or primate codon selection. Furthermore, alleles of the genes comprising the polynucleotide sequences provided herein can also be used. Alleles are endogenous genes that are changed as a result of one or more mutations, such as deletions, additions and/or nucleotide substitutions.

[0239] The term “nucleic acid cassette” as used herein refers to the genetic sequences within a vector that can express an RNA, and subsequently a protein. The nucleic acid cassette contains the gene of interest, for example, a CAR. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergoes appropriate post-translational modifications necessary for activity in the transformed cell. , and be translocated to the appropriate compartment by biological activity targeting the appropriate intracellular compartments or secretion into extracellular compartments. Preferably, the cassette has its 3' and 5' ends adapted for ready insertion into a vector, for example, it has restriction endonuclease sites at each end. In a preferred embodiment of the invention, the nucleic acid cassette contains the sequence of a chimeric antigen receptor used to treat a B cell malignancy. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.

[0240] In particular embodiments, polynucleotides include at least one polynucleotide of interest. As used herein, the term “polynucleotide-of-interest” refers to a polynucleotide that encodes a polypeptide (i.e., a polypeptide of interest), inserted into an expression vector that is desired to be expressed. A vector may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 polynucleotides of interest. In certain embodiments, the polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or disorder. Polynucleotides of interest, and polypeptides encoded therefrom, include both polynucleotides that encode wild-type polypeptides, as well as functional variants and fragments thereof. In particular embodiments, a functional variant has 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, a functional variant or fragment has at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of a corresponding wild-type polypeptide.

[0241] In one embodiment, the polynucleotide of interest does not encode a polypeptide but serves as a template for transcribing miRNA, siRNA, or shRNA, ribozyme, or other inhibitory RNA. In various other embodiments, a polynucleotide comprises a polynucleotide-of-interest encoding a CAR and one or more additional polynucleotides of interest including, but not limited to, an inhibitory nucleic acid sequence including, but not limited to: an siRNA, an miRNA, an shRNA and a ribozyme.

[0242] As used herein, the terms “siRNA” or “Short interfering RNA” refers to a short polynucleotide sequence that mediates a process of sequence-specific post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetic RNAi in animals (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al. , 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13, 139-141; and Strauss, 1999, Science, 286, 886). In certain embodiments, an siRNA comprises a first strand and a second strand that have the same number of nucleosides; however, the first and second strands are displaced such that the two terminal nucleosides on the first and second strands are not paired with a residue on the complementary strand. In certain cases, the two nucleosides that are unpaired are thymidine residues. The siRNA must include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the siRNA, or a fragment thereof, can mediate downregulation of the target gene. Furthermore, an siRNA includes a region that is at least partially complementary to the target RNA. It is not necessary that there be perfect complementarity between the siRNA and the target, but the correspondence must be sufficient to allow the siRNA, or a cleavage product thereof, for direct sequence-specific silencing, such as by RNAi cleavage of the target RNA. Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. Although perfect complementarity, particularly on the antisense strand, is often desired, some embodiments include one or more, but preferably 10, 8, 6, 5, 4, 3, 2, or fewer mismatches with respect to the target RNA. Discrepancies are more tolerated in the terminal regions, and if present are preferably in a terminal region or regions, for example, within 6, 5, 4 or 3 nucleotides of the 5' and/or 3' termini. The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded character of the molecule.

[0243] Additionally, an siRNA can be modified or include nucleoside analogues. Single-stranded regions of an siRNA may be modified or include nucleoside analogs, e.g., the unpaired region or regions of a hairping structure, e.g., a region linking two complementary regions, may have modifications or nucleoside analogs. Modification to stabilize one or more 3'- or 5'-termini of an siRNA, for example, against exonucleases, or to favor the antisense siRNA agent to enter RISC are also useful. Modifications may include C3 (or C6, C7, C12) amino ligands, thiol ligands, carboxyl ligands, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), biotin or special fluorescein reagents that come as phosphoramidites and which have another hydroxyl group protected by DMT, allowing multiple couplings during RNA synthesis. Each strand of an siRNA can be equal to or less than 30, 25, 24, 23, 22, 21 or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred siRNAs have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24 or 25 nucleotide pairs, and one or more overhangs of 2 to 3 nucleotides, preferably one or two 3' overhangs, of 2 to 3 nucleotides.

[0244] As used herein, the terms “miRNA” or “microRNA” refer to small non-coding RNAs of 20 to 22 nucleotides, typically excised ~70 nucleotides do not translate RNA precursor structures known as pre-miRNAs. MiRNAs negatively regulate their targets in one of two ways depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind with perfect or near-perfect complementarity to protein-coding mRNA sequences induce the RNA-mediated interference (RNAi) pathway. MiRNAs, which exert their regulatory effects by binding to imperfect complementary sites within the 3' untranslated regions (UTRs) of their mRNA targets, repress target gene expression post-transcriptionally, apparently at the level of translation, through a complex of RISC that is similar to, or possibly identical to, one that is used for the RNAi pathway. Consistent with translational control, miRNAs that use these mechanisms reduce the protein levels of their target genes, but the mRNA levels of these genes are only minimally affected. MiRNAs encompass both naturally occurring miRNAs as well as artificially engineered miRNAs that can specifically target any mRNA sequence. For example, in one embodiment, the skilled artisan can design Short hairping RNA constructs expressed as human miRNA (e.g., miR-30 or miR-21) primary transcripts. This design adds a Drosha processing site to the hairping construct and has been shown to significantly increase knockdown efficiency (Pusch et al., 2004). Hairpin stem consisting of 22-nt dsRNA (e.g., antisense has perfect complementarity to the desired target) and a 15-19-nt loop of a human miR. Adding the miR loop and miR30 flanking sequences on both sides of the hairpin results in more than a 10-fold increase in Drosha and Dicer processing of the expressed hairpins when compared to conventional shRNA designs without microRNA. Increased Drosha and Dicer processing translates into greater siRNA/miRNA production and greater potency for expressed hairpins.

[0245] As used here, the terms “shRNA” or “Short hairping RNA” refers to the double-stranded structure that is formed by a single strand of self-complementary RNA. shRNA constructs containing a nucleotide sequence identical to a portion, coding or non-coding sequence, of the target gene are preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the target gene portion is preferred. In certain preferred embodiments, the length of the duplex-forming portion of an shRNA is at least 20, 21, or 22 nucleotides in length, e.g., corresponding in size to the RNA products produced by Dicer-dependent cleavage. In certain embodiments, the shRNA construct is at least 25, 50, 100, 200, 300 or 400 bases in length. In certain embodiments, the shRNA construct is 400-800 bases in length. shRNA constructs are highly tolerant of variation in loop sequence and loop size.

[0246] As used herein, the term “ribozyme” refers to a catalytically active RNA molecule capable of site-specific cleavage of target mRNA. Several subtypes have been described, for example, hammerhead and hairping ribozymes. Ribozyme catalytic activity and stability can be improved by substituting deoxyribonucleotides for ribonucleotides in noncatalytic bases. While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at sites dictated by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two-base sequence: 5‘-UG-3‘. The construction and production of hammerhead ribozymes is well known in the art.

[0247] A preferred method of releasing a polynucleotide of interest comprising an siRNA, a miRNA, an shRNA, or a ribozyme comprises one or more regulatory sequences, such as, for example, a strong constitutive pol III, e.g. , human U6 snRNA promoter, the mouse U6 snRNA promoter, the mouse and human H1 RNA promoter, and the human tRNA-val promoter, or a strong constitutive pol II promoter, as described elsewhere place here.

[0248] The polynucleotides of the present invention, regardless of the length of their coding sequence, can be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals , and self-cleaving polypeptides encoding polynucleotides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length can vary considerably. It is therefore considered that a polynucleotide fragment of practically any length can be used, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0249] Polynucleotides can be prepared, manipulated and/or expressed using any of a variety of well-established techniques known and available in the art. In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40 ). Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the chimeric protein coding sequences disclosed herein may be ligated into such expression vectors for expression of the chimeric protein in mammalian cells.

[0250] In particular embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term “episomal” refers to a vector that is capable of replicating without integration into host chromosomal DNA and without gradual loss of a dividing host cell also meaning that said vector replicates extrachromosomally or episomally. The vector is engineered to harbor the coding sequence for the origin of DNA replication or "ori" of a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papillomavirus, or a yeast, specifically an origin of replication of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to EBV oriP. In a particular aspect, the lymphotrophic herpes virus may be Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes saimiri virus (HS), or Marek's disease virus (MDV). Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus. Typically, the host cell comprises the viral replication transactivator protein that activates replication.

[0251] The “control elements” or “regulatory sequences” present in an expression vector are those untranslated regions of the vector—origin of replication, selection cassettes, promoters, enhancers, intron translation initiation signals (Shine sequence Dalgarno or Kozak sequence), a polyadenylation sequence, 5' and 3' untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector and host system used, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters, can be used.

[0252] In particular embodiments, a vector for the practical use of the invention including, but not limited to, expression vectors and viral vectors, will include exogenous, endogenous or heterologous control sequences such as promoters and/or enhancers. An “endogenous” control sequence is one that is naturally linked to a given gene in the genome. An “exogenous” control sequence is one that is placed in juxtaposition to a gene through genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter. A “heterologous” control sequence is an exogenous sequence that is from a different species than the cell being genetically manipulated.

[0253] The term “promoter” as used herein refers to a recognition site for a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In particular embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from from the start of transcription, a CNCAAT region where N can be any nucleotide.

[0254] The term “enhancer” refers to a segment of DNA that contains sequences capable of providing improved transcription and in some cases can function independently of its orientation relative to another control sequence. An enhancer may function cooperatively or additively with promoter elements and/or another enhancer. The term “promoter/enhancer” refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.

[0255] The term “operationally linked” refers to a juxtaposition in which the described components are in a relationship allowing them to function in their intended manner. In one embodiment, the term refers to a functional link between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide. interest, in which the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0256] As used herein, the term “constitutive expression control sequence” refers to a promoter, enhancer, or promoter/enhancer that continuously or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence can be a “ubiquitous” promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell and tissue types, or a “cell-specific” promoter, enhancer, or promoter/enhancer. cell type-specific”, “cell lineage-specific”, or “tissue-specific” which allows expression in a restricted range of cell and tissue types, respectively.

[0257] Illustrative ubiquitous expression control sequences suitable for use in particular embodiments of the invention include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a simian virus viral 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) promoter (thymidine kinase), H5, P7 promoters ,5, and vaccinia virus P11, a promoter of elongation factor 1-alpha (EF1a), early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH ), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 70KDa 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β -KIN), the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus/chicken β-actin enhancer (CAG), a β-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, promoter substituted in the dl587rev primer binding site (MND) (Challita et al ., J Virol. 69(2):748-55 (1995)).

[0258] In one embodiment, a vector of the invention comprises an MND promoter.

[0259] In one embodiment, a vector of the invention comprises an EF1a promoter comprising the first intron of the human EF 1a gene.

[0260] In one embodiment, a vector of the invention comprises an EF1a promoter that lacks the first intron of the human EF 1a gene.

[0261] In a particular embodiment, it may be desirable to express a polynucleotide comprising a CAR from a T cell-specific promoter.

[0262] As used herein, “conditional expression” may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological state, or disease, etc. This definition is not intended to exclude specific cell type or tissue expression. Certain embodiments of the invention provide conditional expression of a polynucleotide of interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide of interest.

[0263] Illustrative examples of inducible promoters/systems include, but are not limited to, steroid inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionin promoter (inducible by treatment with various heavy metals), MX-1 promoter (interferon inducible), the mifepristone-regulatable “GeneSwitch” system (Sirin et al., 2003, Gene, 323:67), the cumate inducible switch gene (WO 2002/088346) , tetracycline-dependent regulatory systems, etc.

[0264] Conditional expression can also be achieved using a site-specific DNA recombinase. According to certain embodiments of the invention the vector comprises at least one (typically two) site(s) for recombination mediated by a site-specific recombinase. As used herein, the terms “recombinase” or “site-specific recombinase” include excisive or integrative proteins, enzymes, cofactors, or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), 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 sequences or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention 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.

[0265] Vectors may comprise one or more recombination sites for any of a wide variety of site-specific recombinases. It should be understood that the target site for a site-specific recombinase is in addition to any site required for integration of a vector, for example, a retroviral vector or lentiviral vector. As used herein, the terms “recombination sequence”, “recombination site”, or “site-specific recombination site” refers to a particular nucleic acid sequence to which a recombinase recognizes and binds.

[0266] For example, a recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprising two pairs of 13 inverted repeat bases (serving as the recombinase binding sites) flanking an 8 pair core sequence base (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0267] Suitable recognition sites for FLP recombinase include, but are not limited to: FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode , 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).

[0268] Other examples of recognition sequences are the sequences attB, attP, attL and attR, which are recognized by the enzyme À integrase recombinase, for example, phi-c31. The ΦC31 SSR mediates recombination only between the attB (34 bp in length) and attP (39 bp in length) heterotypic sites (Groth et al., 2000). attB and attP, named for the binding sites for phage integrase in bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are likely bound by homodimers of ΦC31 (Groth et al., 2000). The product sites, attL and attR, are effectively inert to further ΦC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. To catalyze insertions, attB-containing DNA has been found to insert into a genomic attP site more easily than an attP site into a genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Furthermore, typical strategies for recombination homologs position an attP-containing “docking site” at a defined locus, which is then associated with an incoming sequence containing attB for insertion.

[0269] As used herein, an “internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a region encoding protein), thereby leading to cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In particular embodiments, the vectors considered by the invention include one or more polynucleotides of interest that encode one or more polypeptides. In particular embodiments, to obtain efficient translation of each of the plurality of polypeptides, the polynucleotide sequences may be separated by one or more self-cleaving polypeptides that encode IRES sequences or polynucleotide sequences.

[0270] As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation. The Kozak consensus sequence is (GCC)RCCATGG, 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 particular embodiments, the vectors considered by the invention comprise polynucleotides that have a Kozak consensus sequence and that encode a desired polypeptide, for example, a CAR.

[0271] In some embodiments of the invention, a polynucleotide or cell harboring the polynucleotide utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and/or uncontrolled proliferation. In specific aspects, the suicide gene is not immunogenic to the host harboring the polynucleotide or cell. A certain example of a suicide gene that can be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0272] In certain embodiments, vectors comprise gene segments that cause the immune effector cells of the invention, e.g., T cells, to be susceptible to negative selection in vivo. By “negative selection” we mean that the infused cell may be eliminated as a result of a change in the in vivo condition of the individual. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, e.g., a compound. Negative selectable genes are known in the art, and include, inter alia the following: the Herpes simplex virus type I thymidine kinase gene (HSV-I TK) (Wigler et al., Cell. 11:223, 1977) which confers sensitivity to ganciclovir; the cellular hypoxanthine phosfribosyltransferase gene (HPRT), the cellular adenine phosphoribosyltransferase gene (APRT), and bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0273] In some embodiments, genetically modified immune effector cells, such as T cells, comprise a polynucleotide further comprising a positive marker that allows the selection of cells of the negative selectable phenotype in vitro. The positive selectable marker can be a gene that, even when introduced into the host cell, expresses a dominant phenotype allowing positive selection of cells carrying the gene. Genes of this type are known in the art, and include, inter alia, the hygromycin B phosphotransferase (hph) gene that confers resistance to hygromycin B, the Tn5 aminoglycoside phosphotransferase (neo or aph) gene that codes for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR), the adenosine deaminase gene (ADA), and the multidrug resistance gene (MDR).

[0274] Preferably, the positive selectable marker and the negative selectable element are linked such that the loss of the negative selectable element is necessarily also accompanied by the loss of the positive selectable marker. Even more preferably, the positive and negative selectable markers are fused so that the loss of one necessarily leads to the loss of the other. An example of a fused polynucleotide that produces as an expression product a polypeptide that confers both the desired negative and positive selection characteristics described above is a hygromycin phosphotransferase thymidine kinase (HyTK) fusion gene. Expression of this gene produces a polypeptide that confers resistance to hygromycin B for positive selection in vitro, and sensitivity to ganciclovir for negative selection in vivo. See Lupton S. D., et al, Mol. and Cell. Biology 1 1:3374-3378, 1991. Furthermore, in preferred embodiments, the polynucleotides of the invention that encode the chimeric receptors are retroviral vectors containing the fused gene, particularly those that confer resistance to hygromycin B for positive selection in vitro, and sensitivity to ganciclovir for negative selection in vivo, for example, the HyTK retroviral vector described in Lupton, S. D. et al. (1991), supra. See also PCT publications US91/08442 and PCT/US94/05601, by S. D. Lupton, describing the use of bifunctional selectable fusion genes derived from fusing dominant positive selectable markers with negative selectable markers.

[0275] Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco and gpt, and preferred negative selectable markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK , HPRT, APRT and gpt. Especially preferred markers are bifunctional selectable fusion genes in which the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene or selectable marker. Inducible suicide genes

F. Viral vectors

[0276] In particular embodiments, a cell (e.g., an immune effector cell) is transduced with a retroviral vector, e.g., a lentiviral vector, that encodes a CAR. For example, an immune effector cell is transduced with a vector encoding a CAR comprising a humanized anti-BCMA antibody or antigen-binding fragment that binds to a BCMA polypeptide, with an intracellular signaling domain of CD3Z, CD28, 4- 1BB, Ox40, or any combinations of these. Furthermore, these transduced cells can evoke a CAR-mediated cytotoxic response.

[0277] Retroviruses are a common tool for gene delivery (Miller, 2000, Nature. 357: 455-460). In particular embodiments, a retrovirus is used to deliver a polynucleotide encoding a chimeric antigen receptor (CAR) to a cell. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a “provirus”. The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules that encode the structural proteins and enzymes necessary to produce new viral particles.

[0278] Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Moloney murine sarcoma virus Harvey (HaMuSV), murine mammary tumor virus (MuMTV), gibbon monkey leukemia virus (GaLV), feline leukemia virus (FLV), foamvirus, Friend's murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)) and lentivirus.

[0279] As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV); the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector structures (i.e., HIV cis-acting sequence elements) are preferred. In particular embodiments, a lentivirus is used to deliver a polynucleotide comprising a CAR to a cell.

[0280] Retroviral vectors and more particularly lentiviral vectors can be used in the practice of particular embodiments of the present invention. Consequently, the term “retrovirus” or “retroviral vector” as used herein is meant to include “lentivirus” and “lentiviral vectors” respectively.

[0281] The term “vector” used here refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, for example, inserted into the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to permit integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0282] As will be evident to one of skill in the art, the term “viral vector” is widely used to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes nucleic acid elements derived from viruses that typically facilitate the transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s).

[0283] The term viral vector may refer to a virus or viral particle capable of transferring a nucleic acid into a cell or to its transferred nucleic acid. Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus. The term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus. The term “hybrid vector” refers to a vector, LTR or other nucleic acid containing both retroviral, e.g., lentiviral, and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a transfer vector or plasmid comprising retroviral, e.g., lentiviral sequences for reverse transcription, replication, integration and/or packaging.

[0284] In particular embodiments, the terms “lentiviral vector”, “lentiviral expression vector” may be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles. Where reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that sequences of these elements are present in RNA form in the lentiviral particles of the invention and are present in RNA form. DNA in the DNA plasmids of the invention.

[0285] At each end of the provirus are structures called “long terminal repeats” or “LTRs”. The term “long terminal repeat (LTR)” refers to base pair domains located at the ends of retroviral DNAs that, in their natural sequence context, are direct repeats and contain U3, R, and U5 regions. LTRs generally provide fundamental functions for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and for viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals, and sequences required for replication and integration of the viral genome. The viral LTR is divided into three regions called U3, R and U5. The U3 region contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. The LTR is composed of U3, R, and U5 regions and appears at both the 5' and 3' ends of the viral genome. Adjacent to the 5' LTR are sequences required for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site).

[0286] As used herein, the term “packaging signal” or “packaging sequence” refers to sequences located within the retroviral genome that are necessary for insertion of the viral RNA into the viral capsid or particle, see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pages 2101-2109. Several retroviral vectors use the minimal packaging signal (also referred to as the psi[^] sequence) required for encapsidation of the viral genome. Additionally, as used herein, the terms “packaging sequence”, “packaging signal”, “psi” and the symbol “Φ”, are used in reference to the non-coding sequence required for encapsidation of retroviral RNA strands during formation of viral particle.

[0287] In various embodiments, vectors comprise 5' LTRs and/or modified 3' LTRs. One or both LTRs may comprise one or more modifications including, but not limited to, one or more deletions, insertions or substitutions. Modifications of the 3' LTR are often made to improve the safety of lentiviral or retroviral systems by becoming the virus replication defect. As used herein, the term “replication defect” refers to viruses that are not capable of complete, effective replication such that infectious virions are not produced (e.g., replication-defective lentiviral progeny). The term “replication competent” refers to wild-type virus or mutant virus that is capable of replication, such that viral replication of the virus is capable of producing infectious virons (e.g., replication-competent lentiviral progeny).

[0288] “Self-inactivating” (SIN) vectors refer to replication defect vectors, for example, retroviral or lentiviral vectors, in which the right LTR (3') enhancer region promoter, known as the U3 region , has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right U3 LTR (3') region is used as a template for the left U3 LTR (5') region during viral replication and thus viral transcription cannot be done without the U3 enhancer promoter. In another embodiment of the invention, the 3' LTR is modified such that the U5 region is replaced, for example, with an ideal poly(A) sequence. It should be noted that modifications to the LTRs such as modifications to the 3' LTR, the 5' LTR, or both the 3' and 5' LTRs, are also included in the invention.

[0289] An additional safety enhancement is provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, 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) promoters (thymidine kinase). Typical promoters are capable of driving high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to generate replication-competent virus because there is no complete U3 sequence in the virus production system. In certain embodiments, the heterologous promoter has additional advantages in controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter may be inducible, such that transcription of all or part of the viral genome will occur only when inducible factors are present. Induction factors include, but are not limited to, one or more chemical compounds or physiological conditions, such as temperature or pH, in which host cells are cultured.

[0290] In some embodiments, viral vectors comprise a TAR element. The term “TAR” refers to the “transactivation response” genetic element located in the LTRs of the lentiviral (e.g., HIV) R region. This element interacts with the lentiviral trans-activating genetic element (TAT) to enhance viral replication. However, this element is not necessary in embodiments in which the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0291] The “R region” refers to the region within retroviral LTRs beginning at the beginning of the capping group (i.e., the start of transcription) and ending immediately before the start of the poly A tract. The R region is also defined as being flanked by regions U3 and U5. The R region plays a role during reverse transcription by allowing the transfer of nascent DNA from one end of the genome to the other.

[0292] As used herein, the term “FLAP element” refers to a nucleic acid whose sequence includes the core polypurine tract and core termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV -two. Suitable FLAP elements are described in U.S. Pat. U.S. No. 6,682,907 and in Zennou, et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, central initiation of the positive-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the central flap DNA of HIV-1. Although not intended to be linked by any theory, flap DNA may act as a cis-active determinant of nuclear import of the lentiviral genome and/or may increase virus titer. In particular embodiments, the retroviral or lentiviral vector structures comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. For example, in particular embodiments a transfer plasmid includes a FLAP element. In one embodiment, a vector of the invention comprises a FLAP element isolated from HIV-1.

[0293] In one embodiment, retroviral or lentiviral transfer vectors comprise one or more export elements. The term “export element” refers to a cis-acting post-transcriptional regulatory element that regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, human immunodeficiency virus (RRE) rev (HIV) response elements (see, for example, 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). Generally, the RNA export element is placed within the 3' UTR of a gene, and can be inserted as one or multiple copies.

[0294] In particular embodiments, expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors. A variety of post-transcriptional regulatory elements can increase expression of a heterologous nucleic acid protein, for example, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886 ); the post-transcriptional regulatory element present in the hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766). In particular embodiments, vectors of the invention comprise a post-transcriptional regulatory element such as a WPRE or HPRE.

[0295] In particular embodiments, vectors of the invention lack or do not comprise a post-transcriptional regulatory element such as a WPRE or HPRE because in some cases these elements increase the risk of cellular transformation and/or do not substantially or significantly increase the amount of mRNA transcription or increase mRNA stability. Therefore, in some embodiments, vectors of the invention lack or do not comprise a WPRE or HPRE as an added security measure.

[0296] Elements that direct the termination and efficient polyadenylation of heterologous gene expression increase heterologous nucleic acid transcriptions. Transcription termination signals are generally found downstream of the polyadenylation signal. In particular embodiments, vectors comprise a 3' polyadenylation sequence of a polynucleotide encoding a polypeptide to be expressed. The term “polyA site” or “polyA sequence” as used herein denotes a DNA sequence that directs both termination and polyadenylation of nascent RNA transcription by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the sequence they encode and then contribute to increased translational efficiency. Efficient polyadenylation of the recombinant transcript is desirable as in the absence of a poly A tail transcripts are unstable and are rapidly degraded. Illustrative examples of polyA signals that can be used in a vector of the invention include an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA), a bovine growth hormone polyA sequence (BGHpA), a β- rabbit globin (rβgpA), or another suitable heterologous or endogenous polyA sequence known in the art.

[0297] In certain embodiments, a retroviral or lentiviral vector further comprises one or more insulator elements. Insulator elements may contribute to protecting lentivirus-expressed sequences, e.g., therapeutic polypeptides, from integration site effects, which may be mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences (i.e., of position; see, for example, Burgess-Beusse et al., 2002, Proc. Natl., USA, 99:16433; In some embodiments, transfer vectors comprise one or more insulator elements in the 3' LTR and after integration of the provirus into the host genome, the provirus comprises one or more insulators in either the 5' LTR or 3' LTR, by virtue of duplication. the LTR 3'. Suitable insulators for use in the invention include, but are not limited to, the chicken β-globin insulator (see Chung et al., 1993. Cell 74:505; Chung et al., 1997. PNAS 94:575; and Bell et al. al., 1999. Cell 98:387, incorporated by reference here). Examples of insulator elements include, but are not limited to, an insulator of a β-globin locus, such as chicken HS4.

[0298] According to certain specific embodiments of the invention, more or all of the viral vector framework sequences are derived from a lentivirus, for example, HIV-1. However, it should be understood that many different sources of retroviral and/or lentiviral sequences can be used, or combined, and numerous substitutions and changes in certain of the lentiviral sequences can be accommodated without impairing the ability of a transfer vector to perform the functions described herein. . Furthermore, a variety of lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, Pat. U.S. Nos. 6,013,516; and 5,994,136, many of which can be adapted to produce a viral vector or transfer plasmid of the present invention.

[0299] In various embodiments, the vectors of the invention comprise a promoter operatively linked to a polynucleotide encoding a CAR polypeptide. Vectors may have one or more LTRs, wherein LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vectors may further comprise one of more accessory elements to increase transduction efficiency (e.g., a cPPT/FLAP), viral packaging (e.g., a Psi(^) packaging signal, RRE), and/or other elements that increase therapeutic gene expression (e.g., poly(A) sequences), and may optionally comprise a WPRE or HPRE.

[0300] In a particular embodiment, the transfer vector of the invention comprises a retroviral LTR (5') on the left; a central flap polypurine/DNA tract (cPPT/FLAP); a retroviral export element; an active promoter in a T cell, operatively linked to a CAR polypeptide encoding polynucleotide considered herein; and a retroviral LTR (3') on the right; and optionally a WPRE or HPRE.

[0301] In a particular embodiment, the transfer vector of the invention comprises a retroviral LTR (5') on the left; a retroviral export element; an active promoter in a T cell, operatively linked to a CAR polypeptide encoding polynucleotide considered herein; a retroviral LTR (3') on the right; and a poly(A) sequence; and optionally a WPRE or HPRE. In another particular embodiment, the invention provides a lentiviral vector comprising: an LTR (5') on the left; a cPPT/FLAP; an RRE; an active promoter in a T cell, operatively linked to a CAR polypeptide encoding polynucleotide considered herein; an LTR (3’) on the right; and a polyadenylation sequence; and optionally a WPRE or HPRE.

[0302] In a certain embodiment, the invention provides a lentiviral vector comprising: an HIV-1 LTR (5') on the left; a Psi (^) packing signal; a cPPT/FLAP; an RRE; an active promoter in a T cell, operatively linked to a CAR polypeptide encoding polynucleotide considered herein; a self-inactivating (SIN) HIV-1 LTR (3') on the right; and a rabbit β-globin polyadenylation sequence; and optionally a WPRE or HPRE.

[0303] In another embodiment, the invention provides a vector comprising: at least one LTR; a central flap polypurine/DNA tract (cPPT/FLAP); a retroviral export element; and a promoter active in a T cell, operatively linked to a CAR polypeptide encoding polynucleotide considered herein; and optionally a WPRE or HPRE.

[0304] In a particular embodiment, the present invention provides a vector comprising at least one LTR; a cPPT/FLAP; an RRE; an active promoter in a T cell, operatively linked to a CAR polypeptide encoding polynucleotide considered herein; and a polyadenylation sequence; and optionally a WPRE or HPRE.

[0305] In a certain embodiment, the present invention provides at least one SIN HIV-1 LTR; a Psi (^) packing signal; a cPPT/FLAP; an RRE; an active promoter in a T cell, operatively linked to a CAR polypeptide encoding polynucleotide considered herein; and a rabbit β-globin polyadenylation sequence; and optionally a WPRE or HPRE.

[0306] A “host cell” includes cells electroporated, transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or a polynucleotide of the invention. Host cells may include packaging cells, producer cells, and cells infected with viral vectors. In particular embodiments, host cells infected with the viral vector of the invention are administered to a subject in need of therapy. In certain embodiments, the term "target cell" is used interchangeably with host cell and refers to transfected, infected, or transduced cells of a desired cell type. In preferred embodiments, the target cell is a T cell.

[0307] Large-scale viral particle production is often necessary to obtain a reasonable viral titer. Viral particles are produced by transfecting a transfer vector into a packaging cell line that comprises viral structural and/or accessory genes, e.g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes. or other retroviral genes.

[0308] As used herein, the term “packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide that encodes one, two, three, four or more structural genes and /or viral accessories. Typically, packaging vectors are included in a packaging cell, and are introduced into the cell through transfection, transduction or infection. Methods for transfection, transduction or infection are well known to those of skill in the art. A retroviral/lentiviral transfer vector of the present invention can be introduced into a packaging cell line, through transfection, transduction or infection, to generate a producer cell or cell line. The packaging vectors of the present invention can be introduced into human cells or cell lines by standard methods including, for example, transfection, lipofection or calcium phosphate electroporation. In some embodiments, packaging vectors are introduced into cells along with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the drug. appropriate and isolation of clones. A selectable marker gene can be physically linked to the genes it encodes by the packaging vector, for example, by IRES or self-cleaving viral peptides.

[0309] Viral envelope (env) proteins determine the range of host cells that can be ultimately infected and transformed by recombinant retroviruses generated from the cell lines. In the case of lentiviruses, such as HIV-1, HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by packaging cells of the invention are encoded in a separate vector from the viral gag and pol genes, as previously described.

[0310] Illustrative examples of retroviral-derived env genes that can be used in the invention include, but are not limited to: MLV envelopes, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV ( avian plague virus), and influenza virus envelopes. Similarly, genes encoding RNA virus envelopes (e.g., RNA virus families of Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Buniaviridae, Arenaviridae, Reoviridae, Birnaviridae, Retroviridae) as well as from DNA viruses (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxiiridae and Iridoviridae) can be used. Representative examples include, FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10 and EIAV.

[0311] In other embodiments, envelope proteins for pseudotyping the viruses of the present invention include, but are not limited to, any of the following viruses: Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu), Influenza B , Influenza C virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rotavirus, any virus in the Norwalk virus group, enteric adenovirus, parvovirus, Dengue virus, smallpox Monkey, Mononegavirales, Lyssaviruses such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat virus, Ephemerovirus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesvirus (HHV), human herpesvirus types 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus , Arenaviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filoviruses) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, viruses of Omsk hemorrhagic fever, Tick-borne encephalitis causing viruses and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (pox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus , SARS-associated coronavirus (SARS-CoV), West Nile virus, any virus causing encephalitis.

[0312] In one embodiment, the invention provides packaging cells that produce recombinant retroviruses, e.g., lentiviruses, pseudotyped with the VSV-G glycoprotein.

[0313] The terms “pseudotype” or “pseudotyping” as used herein refers to a virus whose viral envelope proteins have been replaced with those of another virus possessing preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G protein (VSV-G) envelope proteins, which allows HIV to infect a broader range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4+ cells. In a preferred embodiment of the invention, lentiviral envelope proteins are pseudotyped with VSV-G. In one embodiment, the invention provides packaging cells that produce recombinant retroviruses, e.g., lentiviruses, pseudotyped with the VSV-G envelope glycoprotein.

[0314] As used herein, the term “packaging cell lines” is used in reference to cell lines that do not contain a packaging signal, but stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol and env) which are necessary for the correct packaging of viral particles. Any suitable cell line can be used to prepare packaging cells of the invention. Generally, the cells are mammalian cells. In a particular embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293 T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos- cells 2, Huh7 cells, HeLa cells, W163 cells, 211 cells and 211A cells. In preferred embodiments, the packaging cells are 293 cells, 293 T cells or A549 cells. In another preferred embodiment, the cells are A549 cells.

[0315] As used herein, the term “producer cell line” refers to a cell line that is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal. The production of infectious viral particles and viral stock solutions can be accomplished using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated by, for example, Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau et al. (1992) J. Virol. 66:5110-5113. Infectious virus particles can be collected from the packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis, or collection of cell culture supernatant, as is known in the art. Optionally, the collected virus particles can be purified if desired. Suitable purification techniques are well known to those skilled in the art.

[0316] The release of a gene or other polynucleotide sequence using a retroviral or lentiviral vector through viral infection rather than through transfection is referred to as “transduction”. In one embodiment, retroviral vectors are transduced into a cell through infection and integration of the provirus. In certain embodiments, a target cell, for example, a T cell, is “transduced” if it comprises a gene or other polynucleotide sequence delivered to the cell by infection using a viral or retroviral vector. In particular embodiments, a transduced cell comprises one or more genes or other polynucleotide sequences released by a retroviral or lentiviral vector into its cellular genome.

[0317] In particular embodiments, host cells transduced with a viral vector of the invention that express one or more polypeptides are administered to a subject to treat and/or prevent a B cell malignancy. viral gene therapy, which can be used in accordance with certain embodiments of the present invention, can be found in, for example, Kay, M. A. (1997) Chest 111(6 Supp.): 138S-142S; Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9:1975-81; Shiratory, Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opinion. Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther. 7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M. (1995) J. Hepatol. 23:746-58; Brody, S. L. and Cristal, R. G. (1994) Ann. N.Y. Acad. Sci. 716:90-101; Strayer, D. S. (1999) Expert Opinion. Investigation. Drugs 8:2159-2172; Smith-Arica, J. R. and Bartlett, J. S. (2001) Curr. Cardiol. Rep. 3:43-49; and Lee, H.C. et al. (2000) Nature 408:483-8.

G. Genetically Modified Cells

[0318] The present invention contemplates, in particular embodiments, cells genetically modified to express the CARs considered herein, for use in treating conditions related to B cells. As used herein, the term “genetically engineered” or “genetically engineered” modified” refers to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms, “genetically modified cells”, “engineered cells”, and “repurposed cells”, are used interchangeably. As used herein, the term “gene therapy” refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material in a cell that restores, corrects or modifies expression of a gene, or for the purpose of expressing a therapeutic polypeptide, for example, a CAR.

[0319] In particular embodiments, the CARs considered here are introduced and expressed in immune effector cells so as to redirect their specificity to a target antigen of interest, for example, a BCMA polypeptide. An “immune effector cell” is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell death activity, cytokine secretion, induction of ADCC and/or CDC).

[0320] Immune effector cells of the invention can be autologous/autogenic (“self”) or non-autologous (“non-self”, e.g., allogeneic, syngeneic or xenogeneic).

[0321] “Autologous”, as used herein, refers to cells from the same subject.

[0322] “Allogeneic”, as used herein, refers to cells of the same species that differ genetically from the comparison cell.

[0323] “Syngeneic,” as used herein, refers to cells from a different subject that are genetically identical to the cell being compared.

[0324] “Xenogenic”, as used herein, refers to cells of a different species to the cell being compared. In preferred embodiments, the cells of the invention are allogeneic.

[0325] Illustrative immune effector cells used with the CARs considered here include T lymphocytes. The terms “T cells” or “T lymphocyte” are recognized techniques and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes , or activated T lymphocytes. A T cell can be a T helper cell (Th), for example, a T helper cell 1 (Th1) or a T helper cell 2 (Th2). The T cell may be a helper T cell (HTL; CD4+ T cells), CD4+ T cells, a cytotoxic T cell (CTL; CD8+ T cells), CD4+CD8+ T cells, CD4-CD8- T cells, or any other subset of T cells. Other illustrative T cell populations suitable for use in particular embodiments include naïve T cells and memory T cells.

[0326] As would be understood by the skilled person, other cells can also be used as immune effector cells with the CARs as described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitors of effector cells in which such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Furthermore, in particular embodiments, immune effector cell includes progenitors of immune effector cells such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from cord blood, bone marrow or mobilized peripheral blood that upon administration into a subject to differentiate into mature immune effector cells, or that can be induced in vitro to differentiate into mature immune effector cells.

[0327] As used herein, genetically engineered immune effector cells containing BCMA-specific CAR may be referred to as, “BCMA-specific redirected immune effector cells”.

[0328] The term, “CD34+ cell”, as used here refers to a cell that expresses the CD34 protein on its cell surface. “CD34” as used here refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell entry into lymph nodes. The CD34+ cell population contains hematopoietic stem cells (HSC), which upon administration to a patient differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte/macrophage lineage.

[0329] The present invention provides methods for manufacturing immune effector cells that express the CAR considered here. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CARs as described herein. In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual. In additional embodiments, immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a CAR. In this regard, immune effector cells can be cultured before and/or after being genetically modified (i.e., transduced or transfected to express a CAR considered herein).

[0330] In particular embodiments, prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the cell source is obtained from a subject. In particular embodiments, the CAR-modified immune effector cells comprise T cells. T cells can be obtained from various sources including, but not limited to, mononuclear cells from peripheral blood, bone marrow, lymph node tissue, cord blood, emission thymus, tissue from a site of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, T cells may be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLLTM separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other white blood cells, nucleated red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a buffer or appropriate media for subsequent processing. Cells can be washed with PBS or another suitable solution that lacks calcium, magnesium, and most, if not all other, divalent cations. As would be appreciated by one of ordinary skill in the art, a washing step can be carried out by methods known to those in the art, such as using a semi-automatic flow centrifuge. For example, the Cobe 2991 cellular processor, the Baxter CytoMate, or similar. After washing, cells can be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In certain embodiments, undesirable components of the apheresis sample can be removed in the cell directly placed back into suspension culture media.

[0331] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLLTM gradient. A specific subpopulation of T cells, which expresses one or more of the following markers: CD3, CD28, CD4, CD8, CD45RA, and CD45RO, can further be isolated by positive or negative selection techniques. In one embodiment, a specific subpopulation of T cells expressing CD3, CD28, CD4, CD8, CD45RA and CD45RO is further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed at surface markers unique to the negatively selected cells. One method for use here is cell separation and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, enriching for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell separation can also be used to isolate cell populations of interest for use in the present invention.

[0332] PBMC can be directly genetically modified to express CARs using methods considered here. In certain embodiments, after isolation of PBMC, T lymphocytes are further isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be separated into naïve, memory, and effector T cell subpopulations before or after modification and /or genetic expansion.

[0333] CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further separated into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of those CD8+ cell types.

[0334] In certain embodiments, naive CD8+ T lymphocytes are characterized by the expression of naive T cell phenotypic markers including CD62L, CCR7, CD28, CD3, CD 127 and CD45RA.

[0335] In particular embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC are separated into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, expression of central memory T cell phenotypic markers include CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and are negative for granzyme B. In some embodiments, central memory T cells are T cells. CD45RO+, CD62L+ and CD8+.

[0336] In some embodiments, effector T cells are negative for CD62L, CCR7, CD28 and CD127, and positive for granzyme B and perforin.

[0337] In certain embodiments, CD4+ T cells are further separated into subpopulations. For example, CD4+ helper T cells can be separated into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naïve CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, CD4+ central memory cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+ cells are CD62L and CD45RO negative.

[0338] Immune effector cells such as T cells can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro before being genetically modified. In a particular embodiment, immune effector cells, such as T cells, are genetically modified with the chimeric antigen receptors considered herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then are activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a CAR, using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and Publication of U.S. Patent Application No. 20060121005.

[0339] Generally, T cells are expanded by contact with a surface, with an agent attached to them that stimulates a signal associated with the CD3 TCR complex and a ligand that stimulates a costimulatory molecule on the surface of the T cells. Cell populations T can be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium inophore. Co-stimulation of accessory molecules on the surface of T cells is also considered.

[0340] In particular embodiments, PBMCs or isolated T cells are contacted with a stimulatory agent and co-stimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a medium. culture with appropriate cytokines, such as IL-2, IL-7, and/or IL-15. To stimulate proliferation of CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diacione, Besancon, France) can be used with other methods commonly known in the art (Berg et al., Transplant Proc. 30(8): 3975 - 3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Anti-CD3 and anti-CD28 antibodies bound to the same bead serve as a “surrogate” antigen-presenting cell (APC). In other embodiments, T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in US6040177; US5827642; and WO2012129514.

[0341] In other embodiments, artificial APC (aAPC) is engineered from K562, U937, 721,221, T2 and C1R cells to direct the stable expression and secretion of a variety of costimulatory molecules and cytokines. In a particular embodiment, K32 or U32 aAPCs are used to direct the display of one or more antibody-based stimulatory molecules on the AAPC cell surface. Expression of various combinations of genes in the aAPC allows precise determination of human T cell activation requirements, such that aAPCs can be adapted for the optimal propagation of T cell subsets with specific growth requirements and distinct functions. aAPCs support ex vivo growth and long-term expansion of functional human CD8 T cells without requiring the addition of exogenous cytokines, unlike the use of natural APCs. T cell populations can be expanded by aAPCs that express a variety of costimulatory molecules including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and/or CD80 or CD86. Finally, aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression on CD8 T cells. The aAPCs provided in WO 03/057171 and US2003/0147869 are incorporated herein by reference in their entirety.

[0342] In one embodiment, CD34+ cells are transduced with a nucleic acid construct according to the invention. In certain embodiments, the transduced CD34+ cells differentiate into mature immune effector cells in vivo after administration into a subject, generally the subject from whom the cells were originally isolated. In another embodiment, CD34+ cells can be stimulated in vitro before exposure to or after being genetically modified with a CAR as described herein, with one or more of the following cytokines: Flt-3 ligand (FLT3), cell factor Stem (SCF), megakaryocyte growth and differentiation factor (TPO), IL-3 and IL-6 according to previously described methods (Asheuer et al., 2004; Imren, et al., 2004).

[0343] The invention provides a population of modified immune effector cells for the treatment of cancer, the modified immune effector cells comprising a CAR as disclosed herein. For example, a population of modified immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient diagnosed with the B cell malignancy described here (autologous donors). PBMCs form a heterogeneous population of T lymphocytes that can be CD4+, CD8+, or CD4+ and CD8+.

[0344] PBMCs may also include other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of a CAR considered here can be introduced into a population of human donor T cells, NK cells or NKT cells. Successfully transduced T cells carrying the expression vector can be separated using flow cytometry to isolate CD3-positive T cells and then further propagated to increase the number of this CAR protein expressing T cells in addition to cell activation using anti-CD3 antibodies. CD3 and or anti-CD28 antibodies and IL-2 or any other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing CAR protein T cells for storage and/or preparation for use in a human subject. In one embodiment, in vitro transduction, culture and/or expansion of T cells are performed in the absence of non-human animal derived products such as fetal calf serum and fetal bovine serum. Since a heterogeneous population of PBMCs is genetically modified, the resulting transduced cells are a heterogeneous population of modified cells comprising a BCMA-targeted CAR as considered here.

[0345] In a further embodiment, a mixture of, for example, one, two, three, four, five or more different expression vectors can be used by genetically modifying a donor population of immune effector cells in which each vector encodes a different chimeric antigen receptor protein as considered here. The resulting modified immune effector cells form a mixed modified cell population, with a proportion of the modified cells expressing more than one different CAR protein.

[0346] In one embodiment, the invention provides a method of storing immune effector cells expressing genetically modified murine, human or humanized CAR protein that targets a BCMA protein, comprising cryopreserving the immune effector cells such that the cells remain viable until defrosting. A fraction of the immune effector cells expressing the CAR proteins can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients afflicted with the B cell-related condition. When necessary, the transformed immune effector cells Cryopreserved cells can be thawed, cultured and expanded into more such cells.

[0347] As used herein, “cryopreserving” refers to the preservation of cells by cooling to sub-zero temperatures, such as (typically) 77 K or -196 °C. (the boiling point of liquid nitrogen). Cryoprotective agents are often used at sub-zero temperatures to prevent cells being preserved from damage due to freezing at low temperatures or warming to room temperature. Cryopreservative agents and optimal cooling rates can protect against cellular injury. Cryoprotective agents 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. N.Y. Acad. Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 1° to 3°C/minute. After at least two hours, the T cells have reached a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage such as in a long-term cryogenic storage vessel.

H. Compositions and Formulations

[0348] The compositions considered here may comprise one or more polypeptides, vector polynucleotides, comprising the same, genetically modified immune effector cells, etc., as considered here. Compositions include, but are not limited to, pharmaceutical compositions. A “pharmaceutical composition” refers to a composition formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or an animal, alone, or in combination with one or more other therapy modalities. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutics, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There is practically no limit to the other components that can also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to provide the intended therapy.

[0349] The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions and/or dosage forms that are, within the scope of pertinent clinical judgment suitable for use in contact with the tissues of human beings. humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, compatible with a reasonable risk/benefit ratio.

[0350] As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, pigment/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; Tragacanth; malt; gelatin; baby powder; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and any other compatible substances used in pharmaceutical formulations.

[0351] In particular embodiments, compositions of the present invention comprise a number of CAR-expressing immune effector cells considered here. As used herein, the term “amount” refers to “an effective amount” or “an effective amount” of a genetically modified therapeutic cell, e.g., T cells, to obtain a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0352] A “prophylactically effective amount” refers to an amount of a genetically modified Therapeutic cell effective to obtain the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects before or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0353] A “therapeutically effective amount” of a genetically modified Therapeutic cell may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the stem and progenitor cells to evoke a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the virus or transduced therapeutic cells are offset by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject ). It can generally be established that a pharmaceutical composition comprising the T cells described herein can be administered at a dosage of 102 to 1010 cells/kg body weight, preferably 105 to 106 cells/kg body weight, including all integer values within those ranges. . The number of cells will depend upon the final use for which the composition is intended as the type of cells included in it. For the use provided here, the cells are generally in a volume of one liter or less, may be 500 mLs or smaller, even 250 mLs or 100 mLs or less. Accordingly, the desired cell density is typically greater than 106 cells/ml and is generally greater than 107 cells/ml, generally 108 cells/ml or greater. The clinically relevant number of immune cells can be divided into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012 cells. In some aspects of the present invention, particularly since all infused cells will be redirected to a particular target antigen, lower numbers of cells, in the range of 106/kilogram (106 to 1011 per patient) can be administered. CAR-expressing cell compositions can be administered multiple times at dosages within these ranges. The cells can be allogeneic, syngeneic, xenogeneic or autologous to the patient undergoing therapy. If desired, treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines and/or chemokines (e.g., IFN-Y, IL-2, IL-12, TNF-alpha, IL-18, and TNF -beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described here to enhance immune response induction.

[0354] Generally, compositions comprising activated and expanded cells as described herein can be used in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, compositions comprising the CAR-modified T cells considered herein are used in the treatment of B-cell malignancy. The CAR-modified T cells of the present invention can be administered alone, or as a pharmaceutical composition in combination with carriers, diluents, excipients. , and/or with other components such as IL-2 or other cytokines or cell populations. In particular embodiments, pharmaceutical compositions contemplated herein comprise a quantity of genetically modified T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0355] Pharmaceutical compositions of the present invention comprising a CAR-expressing immune effector cell population, such as T cells, may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g. aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated by parenteral administration, for example, intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration.

[0356] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, sodium chloride isotonic, fixed oils such as synthetic mono- or diglycerides that can serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The parenteral preparation may be contained in ampoules, disposable syringes or multi-dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0357] In a particular embodiment, compositions considered herein comprise an effective amount of CAR-expressing immune effector cells, alone or in combination with one or more therapeutic agents. Furthermore, CAR-expressing immune effector cell compositions can be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc. The compositions can also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer. Therapeutic agents considered exemplary include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and auxiliary agents.

[0358] In certain embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and poposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornafazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorrubicin, idarubicin , marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); 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 calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid booster such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; ansacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pyrarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizophyran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”- trichlorotriethylamine; urethane; vindesina; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, for example, paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbina; novantrona; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomitine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); Esperamycins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that regulate age or inhibit hormone action in cancers such as antiestrogens including for example tamoxifen, raloxifene, 4(5)-imidazole aromatase inhibitors, 4-hydroxytamoxifen, trioxifen, ceoxifen, LY117018, onapristone and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0359] A variety of other therapeutic agents can be used in conjunction with the compositions described here. In one embodiment, the 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 (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide, and mycophenolate.

[0360] Other exemplary NSAIDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib) and sialylates. Exemplary analgesics are chosen from the group consisting of acetaminophen, oxycodone, tramadol and proproxyphene hydrochloride. Exemplary glucocorticoids are chosen from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary 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 Biological response modifiers include monoclonal antibodies as well as exemplary recombinant forms of DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine. , gold (oral (auranofin) and intramuscular) and minocycline.

[0361] Illustrative examples of therapeutic antibodies suitable for combination with the CAR-modified T cells considered herein include but are not limited to bavituximab, bevacizumab (avastin), bivatuzumab, blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab, dalotuzumab, elotuzumab ( HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, ocaratuzumab, ofatumumab, rituximab, siltuximab, teprotumumab and ublituximab.

[0362] In certain embodiments, the compositions described here are administered in conjunction with a cytokine. By “cytokine” as used here is meant a generic term for proteins released by a population of cells that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; Mullerian inhibitory substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factors; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta and -gamma; colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural or recombinant cell culture sources, and biologically active equivalents of native sequence cytokines.

I. Therapeutic Methods

[0363] The genetically modified immune effector cells considered here provide improved methods of adoptive immunotherapy for use in treating B cell-related conditions that include, but are not limited to, immunoregulatory conditions and hematological malignancies.

[0364] In particular embodiments, the specificity of a primary immune effector cell is redirected to B cells by genetically modifying the primary immune effector cell with a CAR considered here. In various embodiments, a viral vector is used to genetically modify an immune effector cell with a particular polynucleotide encoding a CAR comprising a humanized anti-BCMA antigen binding domain that binds a BCMA polypeptide; a hinge domain; a transmembrane domain (TM), a short oligo- or polypeptide linker, which connects the TM domain to the intracellular signaling domain of the CAR; and one or more intracellular costimulatory signaling domains; and a primary signaling domain.

[0365] In one embodiment, the present invention includes a type of cell therapy where T cells are genetically modified to express a CAR that targets BCMA-expressing B cells, and the CAR T cells are infused into a recipient in need for this. The infused cell is capable of exterminating disease causing B cells in the recipient. Unlike antibody therapies, CAR T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained cancer therapy.

[0366] In one embodiment, the CAR T cells of the invention can undergo robust T cell expansion in vivo and can persist for an extended amount of time. In another embodiment, the CAR T cells of the invention evolve into specific memory T cells that can be reactivated to inhibit any further tumor formation or growth.

[0367] In particular embodiments, compositions comprising immune effector cells comprising the CARs considered here are used in the treatment of conditions associated with abnormal B cell activity.

[0368] Illustrative examples of conditions that can be treated, prevented or ameliorated using the immune effector cells comprising the CARs considered herein include, but are not limited to: systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura , anti-phospholipid syndrome, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease and rapidly progressive glomerulonephritis.

[0369] Modified immune effector cells may also have application in plasma cell disorders such as heavy chain disease, primary or immune cell-associated amyloidosis, and monoclonal gammopathy of undetermined significance (MGUS).

[0370] As used herein, “B cell malignancy” refers to a type of cancer that forms in B cells (a type of immune system cell) as discussed below.

[0371] In particular embodiments, compositions comprising CAR-modified T cells considered herein are used in the treatment of hematological malignancies, including, but not limited to, B-cell malignancies such as, for example, multiple myeloma (MM) and lymphoma of non-Hodgkin (NHL).

[0372] Multiple myeloma is a B-cell malignancy of mature plasma cell morphology characterized by neoplastic transformation of a single clone of these cell types. These plasma cells proliferate in the BM and can invade adjacent bone and sometimes the blood. Variant forms of multiple myeloma include overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma (see, for example, Braunwald, et al. (eds. ), Harrison's Principles of Internal Medicine, 15th Edition (McGraw-Hill 2001)).

[0373] Non-Hodgkin's lymphoma encompasses a large group of lymphocyte (white blood cell) cancers. Non-Hodgkin's lymphomas can occur at any age and are often marked by lymph nodes that are larger than normal, fever, and weight loss. There are many different types of non-Hodgkin's lymphoma. For example, non-Hodgkin's lymphoma can be divided into aggressive (fast-growing) and indolent (slow-growing) types. Although non-Hodgkin's lymphomas can be derived from B cells and T cells, as used herein, the terms “non-Hodgkin's lymphoma” and “B-cell non-Hodgkin's lymphoma” are used interchangeably. B-cell non-Hodgkin's lymphomas (NHL) include Burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, large cell immunoblastic lymphoma, B-precursor lymphoblastic lymphoma, and mantle cell lymphoma. Lymphomas that occur after bone marrow or stem cell transplantation are usually B-cell non-Hodgkin lymphomas.

[0374] Chronic lymphocytic leukemia (CLL) is an indolent (slow-growing) cancer that causes a slow increase in immature white blood cells called B lymphocytes or B cells. Cancer cells spread through the blood and bone marrow, and can also affect lymph nodes or other organs such as the liver and spleen. CLL eventually causes the bone marrow to fail. Sometimes, in more advanced stages of the disease, the disease is called small lymphocytic lymphoma.

[0375] In particular embodiments, methods comprising administering a therapeutically effective amount of CAR-expressing immune effector cells considered herein or a composition comprising the same, to a patient in need thereof, alone or in combination with one or more therapeutic agents, They are provided. In certain embodiments, the cells of the invention are used in treating patients at risk for developing a condition associated with abnormal B cell activity or a B cell malignancy. Additionally, the present invention provides methods for treating or preventing a condition associated with abnormal B cell activity or a B cell malignancy comprising administering to a subject in need thereof, a therapeutically effective amount of the CAR-modified cells considered herein.

[0376] As used herein, the terms “individual” and “subject” are often used interchangeably and refer to any animal that exhibits a symptom of a disease, disorder, or condition that can be treated with gene therapy vectors. , cell-based therapeutics, and methods disclosed elsewhere herein. In preferred embodiments, a subject includes any animal that exhibits symptoms of a disease, disorder, or condition of the hematopoietic system, e.g., a B-cell malignancy, that can be treated with gene therapy vector-based therapies. in cell, and methods disclosed elsewhere here. Suitable subjects (e.g., patients) include laboratory animals (such as a mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates, and preferably human patients, are included. Typical subjects include human patients who have a B-cell malignancy, have been diagnosed with a B-cell malignancy, or are at risk for or have a B-cell malignancy.

[0377] As used herein, the term “patient” refers to a subject who has been diagnosed with a particular disease, disorder, or condition that can be treated with gene therapy vectors, cell-based therapeutics, and methods published elsewhere here.

[0378] As used herein “treatment” or “treating”, includes any beneficial or desirable effect on the symptoms or pathology of a disease condition or pathology, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. . Treatment may optionally involve reducing or improving symptoms of the disease or condition, or delaying the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0379] As used herein, "prevent", and similar words such as "prevented", "preventing", etc., indicate a method to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition. Also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of symptoms of a disease or condition. As used herein, “prevention” and similar words also include reducing the intensity, effect, symptoms and/or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0380] By “enhance” or “promote,” or “increase” or “expand” we generally refer to the ability of a composition considered herein, e.g., a genetically modified T cell or vector encoding a CAR, to produce, evoke , or cause a greater physiological response (i.e., downstream effects) compared to the response caused by vehicle or a control molecule/composition. A measurable physiological response may include an increase in expansion, activation, persistence, and/or an increase in T cell capacity to kill cancer cells, among others evident from understanding the art and the description herein. An “increased” or “enhanced” quantity is typically a “statistically significant” quantity, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 15, 20, 30 or more times (e.g. 500, 1,000 times) (including all integers and decimal points between and above 1, e.g. 1.5, 1.6, 1.7 , 1.8, etc.) the response produced by vehicle or a control composition.

[0381] By “decrease” or “lower”, or “attenuate”, or “reduce”, or “abate” we generally refer to the ability of the composition considered here to produce, evoke, or cause an attenuated physiological response (i.e. , the downstream effects) compared to the response caused by vehicle or a control molecule/composition. A “decreased” or “reduced” quantity is typically a “statistically significant” quantity, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 15, 20, 30 or more times (e.g. 500, 1,000 times) (including all integers and decimal points between and above 1, e.g. 1.5, 1.6, 1.7 , 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell line.

[0382] By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial diminution” generally refers to the ability of a composition considered here to produce , evoke, or cause an attenuated physiological response (i.e., downstream effects) in a cell, as compared to the response caused by vehicle, a control molecule/composition, or the response in a particular cell line. A comparable response is one that is not significantly different or measurably different from the reference response.

[0383] In one embodiment, a method of treating a condition related to B cells in a subject in need thereof comprises administering an effective amount, for example, the therapeutically effective amount of a composition comprising genetically modified immune effector cells considered herein. The amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's illness, although appropriate dosages can be determined by clinical trials.

[0384] In certain embodiments, it may be desirable to administer activated immune effector cells to a subject and then subsequently collect the blood (or have an apheresis performed), activate immune effector cells thereof in accordance with the present invention, and reinfuse the patient with these activated and expanded immune effector cells. This process can be carried out several times in a few weeks. In certain embodiments, immune effector cells can be activated from 10cc to 400cc blood draws. In certain embodiments, immune effector cells are activated from blood collections of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Not to be limited by theory, using this multiple reinfusion/multiple blood collection protocol may serve to select certain populations of immune effector cells.

[0385] Administration of the compositions considered here can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases “parenteral administration” and “parenterally administered” as used herein refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, injection and infusion, intravascular, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-orbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal. In one embodiment, the compositions considered here are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0386] In one embodiment, a subject in need thereof is administered an effective amount of a composition to enhance a cellular immune response to a condition related to B cells in the subject. The immune response may include cellular immune responses mediated by cytotoxic T cells capable of exterminating infected cells, regulatory T cells, and helper T cell responses. Humoral immune responses, mediated mainly by helper T cells capable of activating B cells, thus leading to antibody production, can also be induced. A variety of techniques can be used to analyze the type of immune responses induced by the compositions of the present invention, which are well described in the art; for example, Current Protocols in Immunology, edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.

[0387] In the case of T cell-mediated killing, CAR ligand binding initiates CAR signaling to T cells, resulting in activation of a variety of T cell signaling pathways that induce T cells to produce or release proteins capable of to induce target cell apoptosis by several mechanisms. These T cell-mediated mechanisms include (but are not limited to) transfer of intracellular cytotoxic granules from T cells into the target cell, T cell secretion of pro-inflammatory cytokines that can induce target cell killing directly (or indirectly by through recruitment of other effector cell killers), and even regulation of death receptor ligands (e.g., FasL) on the surface of T cells that induce target cell apoptosis upon binding to their cognate death receptor (e.g., Fas ) in the target cell.

[0388] In one embodiment, the invention provides a method of treating a subject diagnosed with a B cell-related condition comprising removing immune effector cells from a subject diagnosed with a B cell-related condition expressing BCMA, genetically modifying said immune effector cells with a vector comprising a nucleic acid encoding a CAR as contemplated 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.

[0389] In certain embodiments, the present invention also provides methods for stimulating an immune effector cell-mediated immune modulator response to a target cell population in a subject comprising the steps of administering to the subject an immune effector cell population that expresses a nucleic acid construct that encodes a CAR molecule.

[0390] Methods for administering the cellular compositions described herein include any method that is effective to result in the reintroduction of ex vivo genetically modified immune effector cells that directly express a CAR of the invention into the subject or in reintroduction of genetically modified progenitors of immune effector cells which upon introduction into a subject to differentiate into mature immune effector cells that express the CAR. One method comprises transducing peripheral blood T cells ex vivo with a nucleic acid construct according to the invention and returning the transduced cells to the subject.

[0391] All publications, patent applications, and granted patents cited in this specification are incorporated herein by reference as if each individual publication, patent application, or granted patent was specifically and individually indicated to be incorporated by reference.

[0392] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made to this without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that can be altered or modified to produce essentially similar results.

Examples Example 1 Construction of anti-BCMA CARs

[0393] CARs containing humanized anti-BCMA scFv antibodies were designed to contain an MND promoter operably linked to the anti-BCMA scFv, a hinge and transmembrane domain from CD8α and CD137 co-stimulatory domains followed by the intracellular signaling domain of CD3Z chain. Figure 1. Anti-BCMA CARs comprise a CD8α signal peptide (SP) sequence for surface expression on immune effector cells. The polynucleotide sequences of the anti-BCMA CARs are presented in SEQ ID NOs: 30 to 44, 70 and 72; polypeptide sequences of anti-BCMA CARs are presented in SEQ ID NOs: 15 to 29, 71 and 73; and vector maps are shown in Figure 1. Tables 3 through 5 show the Identity, Genbank Reference, Source Name, and Citation for the various nucleotide segments of several exemplary anti-BCMA CAR lentiviral vectors. Table 3.

Example 2 Humanized Anti-BCMA CAR High Yield Screen

[0394] A microculture assay was developed to rapidly display anti-BCMA CARs. The microculture assay can compare 15 to 20 CAR-modified T cells (CAR T cells). Peripheral blood mononuclear cells (PBMC) were cultured in 24-well plates in media containing IL-2 (CellGenix) and antibodies specific for CD3 and CD28 (Miltenyi Biotec) to initiate T cell development. The expanded T cells were >99% of positive CD3 T cells after 7 days of culture.

[0395] Expanded PBMC T cells were modified by lentivirus to express anti-BCMA CARs. Transient lentiviral supernatants were added to the PBMC culture at a 1:1 (volume:volume) ratio one day after culture initiation. Cultures were split as needed with fresh media containing IL-2 after becoming optically confluent using an inverted light microscope. After twelve days of culture, CAR T cells were harvested and assessed for CAR expression and function.

[0396] Expression of CARs on the surface of T cells was assessed by flow cytometry using an antibody reactive to the single-chain variable fragment portion of CARs (Goat anti-mouse IgG (GAM), Life Technologies). The average expression of each of the 15 CARs in three separate normal donors is shown in Table 6. Expression of CARs was comparable, ranging from 47% to 63% of CAR-positive T cells. Transduction efficiency (represented as VCN in Table 6) among the various CAR constructs tested. VCN was assessed by PCR using primers that amplify the integrated virus. Table 6.

Example 3 CAR T-Cell Antigen-Specific Reactivity

[0397] Antigen-specific reactivity was examined after co-culture with BCMA-positive cell lines. Anti-BCMA CAR T cells were co-cultured with BCMA-engineered K562 cells (K562-BCMA) for 24 hours. Reactivity of anti-BCMA CAR T cells to K562-BCMA was assessed by release of IFN-gamma (IFNY) into the supernatant by ELISA. All humanized anti-BCMA CAR T cells tested released similar amounts of IFNγ and the amounts were comparable to the parental mouse anti-BCMA CAR. Figure 2. In contrast, IFNγ was not detected in cultures containing non-transduced or CD19-specific CAR T cells confirming that BCMA specificity of CAR T cells was required for reactivity to K562-BCMA cells.

[0398] In another set of experiments, tumor cytolytic activity of T cells expressing one of five humanized anti-BCMA CARs was examined. Anti-BCMA CAR T cells were co-cultured with K562-BCMA cells for four hours. The five humanized anti-BCMA CAR T cells exhibited similar cytolytic activity to K562-BCMA across three T cell:tumor ratios. Figure 3. Surprisingly, all humanized anti-BCMA CAR T cells tested showed greater cytolytic activity than the parental mouse anti-BCMA CAR T cells.

Example 4 Phenotype and Function of Humanized Anti-BCMA CAR T Cells

[0399] The phenotype and function of three humanized anti-BCMA constructs (anti-BCMA-10, anti-BCMA-11, anti-BCMA-31) were evaluated in a culture system directly scalable for large clinical manufacturing processes. Briefly, peripheral blood mononuclear cells (PBMC) were cultured in static flasks in media containing IL-2 (CellGenix) and antibodies specific for CD3 and CD28 (Miltenyi Biotec). 2 x 108 transduction units of lentivirus encoding anti-BCMA CARs were added one day after culture initiation. Anti-BCMA CAR T cells were maintained in logarithmic phase by adding fresh media containing IL-2 for a total of ten days of culture. T cells transduced with mouse anti-BCMA CAR (BCMA-02), the three humanized anti-BCMA CARs, or an anti-CD19 CAR lacking signaling capacity (CAR19Δ) were expanded from three normal donors (800290, 800309, 801269 ). CAR T cells were evaluated for CAR and antigen expression and tumor cell reactivity at the end of culture.

[0400] Expression of anti-BCMA CARs on the surface of T cells was assessed by flow cytometry using an antibody reactive to the single-chain variable fragment portion of the CAR (Goat anti-mouse Ig (GAM), Life Technologies). Figure 4. Comparable CAR expression was observed by both mouse (msBCMA-02) or humanized (anti-BCMA-10, anti-BCMA-11, anti-BCMA-31) sequences. No significant differences in mouse or humanized anti-BCMA CAR expression were observed among all three donors (Table7).Table7.

[0401] There was no significant difference in transduction efficiency between anti-BCMA CAR T cells. Vector copy number (VCN), assessed by PCR using primers that amplify integrated virus, was comparable in all anti-BCMA CAR T cells (Table 8). Table 8.

[0402] The reactivity of anti-BCMA CAR T cells to BCMA-positive cell lines and tumors was evaluated. Comparable IFNγ release was observed after coculture of anti-BCMA CAR T cells with K562-BCMA (expressing low or high amounts of BCMA) cells or multiple myeloma cell lines (RPMI-8226, NCI-H929) but not for BCMA-negative cell lines (HDLM-2, K562). Figure 5.

[0403] Cytolytic activity of T cells expressing anti-BCMA CARs was examined. Anti-BCMA CAR T cells were co-cultured with K562-BCMA cells for four hours. All anti-BCMA CAR T cells tested caused comparable K562-BCMA cell cytotoxicity. Figure 6.

Example 5 Humanized

[0404] In particular embodiments, anti-BCMA CAR molecules can release cytokines in the absence of stimulation. This antigen-independent (“tonic”) cytokine release can be observed after in vitro culture under conditions that lack the presence of this antigen.

[0405] Tonic cytokine release from T cells engineered to express a humanized anti-BCMA-10 CAR was observed. Humanized anti-BCMA10 was derived from mouse anti-BCMA-02 to reduce potential immunogenicity caused by immune reactions to mouse sequences. In total, there is a sequence difference of less than 20% between anti-BCMA-02 and anti-BCMA-10 CAR molecules. Despite these changes, no differences in CAR expression or reactivity to BCMA antigens were observed between mouse anti-BCMA-02 and humanized anti-BCMA-10 (Figure 5 and Table 7). However, in the absence of BCMA, humanized anti-BCMA-10 CAR T cells released significantly more inflammatory cytokines. Figure 7.

[0406] Mouse anti-BCMA-02 or humanized anti-BCMA-10 CAR T cells were cultured overnight in media containing human serum but lacking any BCMA. Levels of 12 cytokines released into the supernatant were determined using a multiplex bead method (Luminex assay). 5 x 104 humanized anti-BCMA-10 CAR T cells released up to 5 ng/ml of inflammatory cytokines including MIP1α, MIP1β, IFNY, GMCSF, IL-8, and TNFα. The same amount of mouse anti-BCMA-02 CAR T cells released less than 200 pg/ml. Neither CAR modification impacted the release of anti-inflammatory cytokines including IL-10 and IL-4.

[0407] Tonic cytokine release raises known toxicity issues caused by excessive cytokine release in patients treated with anti-CD19 CAR T cells. The high antigen-independent tonic cytokine release observed from humanized anti-BCMA-10 CAR T cells can cause toxic cytokine levels in patients even in the absence of BCMA antigen. The sequence changes made to mouse anti-BCMA-02 to generate humanized anti-BCMA-10 may introduce nascent reactivity to proteins in human serum not observed in mouse anti-BCMA-02 T cells. Reactivity to human serum proteins may explain “tonic” cytokine release observed in humanized anti-BCMA-10.

[0408] Mouse anti-BCMA-02 and humanized anti-BCMA-10 CAR T cells were rested for 48 hours in media containing fetal calf serum instead of human serum. CAR T cells were switched to media containing human serum and the amount of IFNY released was compared to cultures maintained in fetal calf serum. Culture media did not impact the release of IFN-γ from CAR T cells. No IFNγ was detected at the end of cultured mouse anti-BCMA-02 CAR T cells while anti-humanized BCMA10 cultures contained about 4 ng/ml of IFNγ regardless of the culture media (Figure 8). The lack of reactivity in mouse anti-BCMA-02 CAR T cells was not due to T cell dysfunction: Both anti-BCMA CAR T cells reacted to plate-immobilized BCMA (data not shown).

Example 6 Tuned Cytokine Release in Humanized anti-BCMA CAR T Cells

[0409] CARs containing mouse sequences have the potential to cause undesirable immune responses. Anecdotal evidence from CAR clinical trials has suggested that these immune responses may limit the effectiveness of CAR T cells in some patients. Humanization of mouse sequences is a strategy to reduce immune responses to mouse sequences. However, humanized anti-BCMA-10 CAR introduced tonic inflammatory release. The T cell antigen recognition domain of anti-BCMA-10 CAR is linked to the surface of T cells using a transmembrane domain of CD8α. The identity of the transmembrane domain and its impact on dimerization of CAR molecules and subsequent activation in the absence of antigen was examined.

[0410] Anti-BCMA-10 CAR molecules were constructed to replace the transmembrane domain of CD8α with a transmembrane domain of CTLA-4 (anti-BCMA-10.2) or PD-1 (anti-BCMA-10.5) (Figure 9) . Anti-BCMA-10.2 and anti-BCMA-10.5 expression was compared to anti-BCMA-10 by staining recombinant human BCMA protein conjugated to IgG1 Fc-PE and evaluating using flow cytometry. Table 9 shows results from two representative normal donor T cells that robustly express surface of all three CAR molecules that were observed. Table 9.

[0411] The reactivity of anti-BCMA CAR T cells to BCMA-positive K562 cell lines was evaluated. Comparable IFNγ release was observed after coculture anti-BCMA-10, anti-BCMA-10.2, or anti-BCMA-10.5 with K562-BCMA cells (Figure 10). These data show that changes in the transmembrane domain to CTLA-4 or PD-1 sequences do not impact anti-BCMA-10 CAR T cell reactivity.

[0412] Tonic cytokine release was assessed by measuring IFNY in overnight culture supernatants with mouse anti-BCMA-02, or humanized anti-BCMA-10, anti-BCMA-10.2 or anti-BCMA-10.5. Anti-BCMA-02 cultures contained low amounts of IFNY as previously observed. Supernatants from anti-BCMA-10 cultures contained higher amounts of IFNY. Anti-BCMA-10.2 and anti-BCMA-10.5 cultures contained levels of IFNY comparable to anti-BCMA-02 and lower levels than anti-BCMA-10 CAR T cells (Figure 11). These data suggest that change in the transmembrane domain may reduce tonic cytokine release without affecting CAR T cell antigen reactivity.

[0413] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the scope complete set of equivalents to which the claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims (13)

1. Chimeric antigen receptor (CAR) CHARACTERIZED by the fact that it comprises: an extracellular domain comprising a humanized anti-BCMA (B cell maturation antigen) antibody or antigen-binding fragment thereof that binds to one or more epitopes of a human BCMA polypeptide; a transmembrane domain and a CTLA-4 hinge or a transmembrane domain and a PD-1 hinge that reduces antigen-independent cytokine release from the CAR; one or more intracellular costimulatory signaling domains; and a primary signaling domain. 2. CAR, according to claim 1, CHARACTERIZED by the fact that the humanized anti-BCMA antibody or the antigen-binding fragment that binds to the human BCMA polypeptide: a) is selected from the group consisting of: a Camel Ig , Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv antibody (“scFv”), bis-scFv, (scFv)2, minibody , diabody, triabody, tetrabody, disulfide-stabilized Fv protein (“dsFv”) and single-domain antibody (sdAb, Nanobody), preferably a scFv; and/or b) comprises one or more CDRs as set forth in any of SEQ ID NOs: 4 to 6; and/or; c) comprises one or more CDRs as set forth in any of SEQ ID NOs: 1 to 3; and/or d) comprises a variable light chain sequence as set forth in any of SEQ ID NOs: 7 to 9, optionally wherein the variable light chain sequence comprises the CDR sequences set forth in SEQ ID NOs: 1 to 3; and/or e) comprises a variable heavy chain sequence as set forth in any of SEQ ID NOs: 10 to 14, optionally wherein the variable heavy chain sequence comprises the CDR sequences set forth in SEQ ID NOs: 4 to 6. 3. CAR, according to claim 1 or 2, CHARACTERIZED by the fact that the one or more costimulatory signaling domains are from a costimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA-4), CD223 (LAG3), CD270 (HVEM), CD273 (PD -L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70, preferably wherein the one or more costimulatory signaling domains are from a selected costimulatory molecule of the group consisting of: CD28, CD134 and CD137. 4. CAR, according to any one of claims 1 to 3, CHARACTERIZED by the fact that it further comprises a signal peptide, preferably wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide or a polypeptide human GM-CSF receptor alpha signal. 5. CAR, according to any one of claims 1 to 4, CHARACTERIZED by the fact that it comprises an amino acid sequence as presented in SEQ ID NO: 71 or SEQ ID NO: 73. 6. Polynucleotide CHARACTERIZED by the fact that it is the nucleic acid sequence of SEQ ID NO: 70 or SEQ ID NO: 72 and degenerate sequences thereof, which encode a CAR, as defined in any one of claims 1 to 5. 7. Vector CHARACTERIZED by the fact that it comprises the polynucleotide, as defined in claim 6, optionally wherein the vector is an expression vector, an episomal vector, a viral vector, a retroviral vector, a lentiviral vector, preferably wherein the vector lentiviral 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). 8. Vector, according to claim 7, CHARACTERIZED by the fact that the vector is a viral vector, retroviral vector or lentiviral vector comprising a retroviral LTR (5') on the left, a Psi packaging signal (^), a tract core polypurine/DNA flap (cPPT/FLAP), a retroviral export element; a promoter operably linked to the polynucleotide, as defined in claim 6, and a trailing retroviral LTR (3'), optionally wherein: a) the vector further comprises a heterologous polyadenylation sequence; and/or b) the vector further comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE); and/or c) the 5'LTR promoter is replaced with a heterologous promoter; and/or d) the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a Simian Virus 40 (SV40) promoter; and/or e) the 5' LTR or the 3' LTR is an LTR lentivirus; and/or f) the 3' LTR comprises one or more modifications, comprises one or more deletions and/or is a self-inactivating LTR (SIN); and/or g) the polyadenylation sequence is a bovine growth hormone polyadenylation or signal rabbit β-globin polyadenylation sequence; and/or h) the polynucleotide as defined in claim 6 comprises an optimized Kozak sequence; and/or i) the promoter operably linked to the polynucleotide as defined in claim 6 is selected from the group consisting of: a cytomegalovirus (CMV) immediate early gene promoter, an elongation factor 1 alpha (EF1-α) promoter ), a phosphoglycerate kinase-1 (PGK) promoter, a ubiquitin-C (UBQ-C) promoter, a cytomegalovirus enhancer/chicken beta-actin (CAG) promoter, polyoma enhancer/chicken beta-actin (CAG) promoter, herpes simplex (MC1), a beta actin promoter (β-ACT), a simian virus 40 (SV40) promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer promoter-binding site replaced (MND). 9. Composition CHARACTERIZED by the fact that it comprises an immune effector cell comprising the CAR, as defined in any one of claims 1 to 5, the polynucleotide, as defined in claim 6, or the vector, as defined in claim 7 or 8, preferably wherein the immune effector cell is selected from the group consisting of: a T lymphocyte and a natural killer (NK) cell, and a physiologically acceptable excipient. 10. Composition, according to claim 9, CHARACTERIZED by the fact that it is for use in treating a condition related to the B cell in an individual in need thereof, comprising formulating a therapeutically effective amount of the composition to be administered to the individual. 11. Composition, according to claim 10, CHARACTERIZED by the fact that the B cell-related condition is: a) multiple myeloma, non-Hodgkin's lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, post-Hodgkin's lymphoproliferative disorder transplant, an immunoregulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, anti-phospholipid syndrome, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, or monoclonal gammopathy of undetermined significance; b) a B-cell malignancy, preferably wherein the B-cell malignancy is multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL), optionally wherein the MM is selected from the group consisting of: overt multiple myeloma, multiple myeloma smoldering, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone and extramedullary plasmacytoma, or in which the NHL is selected from the group consisting of: Burkitt's lymphoma, chronic lymphocytic leukemia/lymphocytic lymphoma small (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, large cell immunoblastic lymphoma, B-precursor lymphoblastic lymphoma, and mantle cell lymphoma; c) a plasma cell malignancy; or d) an autoimmune disease, preferably wherein the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis or autoimmune hemolytic anemia. 12. Use of the composition, as defined in claim 9, CHARACTERIZED by the fact that it is for the preparation of a medicine to treat a condition related to the B cell in an individual in need thereof. 13. Use, according to claim 12, CHARACTERIZED by the fact that the condition related to the B cell is: a) multiple myeloma, non-Hodgkin's lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, post-Hodgkin's lymphoproliferative disorder transplant, an immunoregulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, anti-phospholipid syndrome, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, or monoclonal gammopathy of undetermined significance; b) a B-cell malignancy, preferably wherein the B-cell malignancy is multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL), optionally wherein the MM is selected from the group consisting of: overt multiple myeloma, multiple myeloma smoldering, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone and extramedullary plasmacytoma, or in which the NHL is selected from the group consisting of: Burkitt's lymphoma, chronic lymphocytic leukemia/ small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, large cell immunoblastic lymphoma, B-precursor lymphoblastic lymphoma, and mantle cell lymphoma; c) a plasma cell malignancy; or d) an autoimmune disease, preferably wherein the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, myasthenia gravis or autoimmune hemolytic anemia.