T cells expressing chimeric antigen receptors

Abstract

Described herein are methods for generating and utilizing T cells comprising a chimeric antigen receptor (CAR) comprising an extracellular domain that binds to CD79b, or CD79b and CD 19. Additionally, the present invention relates to methods for treating cancer, plasma cell dyscrasias or disorders, or autoimmune diseases or disorders. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] The technology described herein relates to immunotherapy. [Background technology]

[0002] Chimeric antigen receptors (CARs) provide a method for driving cytotoxic T cell responses against target cells expressing a selected target antigen, most often a tumor antigen or tumor-associated antigen. CARs are an adaptation of a T cell receptor in which the antigen-binding domain is replaced with the antigen-binding domain of an antibody that specifically binds to the target antigen. Engagement of the target antigen on the surface of the target cell by the CAR expressed on the T cell ("CAR T cell") facilitates killing of the target cell.

[0003] Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course that is highly resistant to currently available therapies in many patients. Despite recent advances in treatment, MCL remains an incurable disease. Adoptive immunotherapy using T cells genetically engineered to express chimeric antigen receptors (CARs) targets CD19 + It has shown great promise as a treatment for B-cell malignancies, however, treatment failure due to antigen escape has been described in patients receiving CD19 CAR therapy.

[0004] New approaches to treating B-cell malignancies, including MCL, would be advantageous. Summary of the Invention

[0005] CAR T cells are a cutting-edge therapy that shows great potential in treating cancer. The technology has proven particularly effective against various non-solid cancers, such as leukemia, lymphoma, and myeloma. One challenge encountered in designing CAR T-based therapies is tumor escape through loss of the targeted antigen or tumor-associated factor recognized by the CAR. When a tumor downregulates or otherwise loses cell surface expression of the targeted antigen or factor, it is no longer effectively attacked by CAR T cells designed to target that antigen or factor. This has been observed, for example, in CAR T therapies targeting B-cell maturation antigen (BCMA), which is expressed in B-cell malignancies, leukemia, lymphoma, and multiple myeloma. It has also been observed in the context of CD19-targeted CAR T therapies.

[0006] The present invention provides chimeric antigen receptor (CAR) polypeptides, each comprising an extracellular domain comprising a sequence that specifically binds to CD79b, e.g., the antigen-binding region of an antibody against CD79b. In certain embodiments, the antigen-binding region is a single-chain antibody (scFv) against CD79b, optionally comprising a light chain and a heavy chain. The light chain can be N-terminal to the heavy chain, or the heavy chain can be N-terminal to the light chain.

[0007] The CAR polypeptide may further comprise one or more, or all, of a hinge domain, a transmembrane domain, a costimulatory domain, and a signaling domain. In various embodiments, the hinge and transmembrane domain are CD8 hinge and transmembrane domains; the costimulatory domain is a 4-1BB costimulatory domain; and / or the signaling domain is a CD3ζ signaling domain. Thus, in one embodiment, a CAR of the invention comprises an anti-CD79b scFv, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

[0008] In various embodiments, the extracellular domain of the CAR polypeptide further comprises a sequence that specifically binds to CD19, e.g., an antigen-binding region of an antibody to CD19. In certain embodiments, the sequence that binds to CD19 comprises a single-chain antibody (scFv) to CD19. The scFv can optionally comprise a light chain and a heavy chain. The light chain can be N-terminal to the heavy chain, or the heavy chain can be N-terminal to the light chain. In various further embodiments, the sequence that binds to CD79b is N-terminal to the sequence that binds to CD19, while in other embodiments, the sequence that binds to CD19 is N-terminal to the sequence that binds to CD79b.

[0009] In various embodiments, the CAR polypeptide comprises the sequence of SEQ ID NO: 1, 2, 10, or 11, or a variant thereof, wherein the sequence optionally does not include the CD8 leader sequence of SEQ ID NO: 3.

[0010] In certain embodiments, the CAR polypeptide comprises a CD8 leader sequence of SEQ ID NO: 3, or a variant thereof; an anti-CD79b light chain sequence of SEQ ID NO: 4, or a variant thereof; an anti-CD79b heavy chain sequence of SEQ ID NO: 6, or a variant thereof; a linker sequence of SEQ ID NO: 5, or a variant thereof; a CD8 transmembrane and hinge sequence of SEQ ID NO: 7, or a variant thereof; a 4-1BB ICD sequence of SEQ ID NO: 8, or a variant thereof; a CD3ζ ICD sequence of SEQ ID NO: 9, or a variant thereof; and / or an anti-CD19 scFv sequence of SEQ ID NO: 13, or a variant thereof. CARs comprising any combination of these sequences are encompassed by the present invention.

[0011] The present invention also provides nucleic acid molecules, each comprising a sequence encoding a CAR polypeptide as described herein, and vectors comprising such nucleic acid molecules. Additionally, the present invention includes cells (e.g., T cells, e.g., primary T cells (e.g., human T cells, which may be autologous or allogeneic)) comprising a CAR polypeptide as described herein, or a nucleic acid molecule or vector as described herein. The present invention further includes pharmaceutical compositions comprising a CAR polypeptide, nucleic acid molecule, vector, or cell as described herein.

[0012] The present invention also provides methods of treating a subject having or at risk of developing cancer (e.g., a B-cell malignancy) by administering to the subject a pharmaceutical composition as described herein. In various embodiments, the cancer is a lymphoma (e.g., a non-Hodgkin's lymphoma, such as mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBCL), chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL); see also below). The present invention further includes the use of the pharmaceutical compositions described herein in treating a subject (e.g., a subject having or at risk of developing a cancer as described herein).

[0013] The present invention further provides a method of treating a subject experiencing a relapse of CD19-negative lymphoma after receiving CD19 CAR therapy by administering to the subject a pharmaceutical composition as described herein. The present invention further includes the use of a pharmaceutical composition as described herein to treat such a subject.

[0014] The present invention further provides methods of generating CAR T cells that express a CAR polypeptide specific for CD79b, or CD79b and CD19. The methods include introducing a nucleic acid molecule or vector as described herein into a T cell (e.g., a primary T cell, e.g., a human primary T cell, which can be autologous or allogeneic).

[0015] Each of the CAR components described in this summary and elsewhere herein, as defined herein, can optionally have the sequence of the respective component listed in Example 2 or Example 3, or can be a variant thereof.

[0016] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise specified, or as suggested by context, the following terms and phrases include the meanings provided below. Since the scope of the technology is not limited solely by the claims, definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed technology. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0017] Definitions of common terms in immunology and molecular biology are given in The Merck Manual of Diagnosis and Therapy, 19 thEdition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited,2014(ISBN0815345305,9780815345305);Lewin's Genes XI,Published by Jones & Bartlett Publishers,2014(ISBN-1449659055);Michael Richard Green and Joseph Sambrook,Molecular Cloning: A Laboratory Manual,4 thed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,USA(2012)(ISBN1936113414);Davis et al.,Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(2012)(ISBN044460149X);Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.) Elsevier,2013(ISBN0124199542);Current Protocols in Molecular Biology(CPMB), Frederick M.Ausubel(ed.), John Wiley and Sons,2014(ISBN047150338X,9780471503385), Current Protocols in Protein Science(CPPS), John E.Coligan(ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan et al. (eds.), John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of each of which are incorporated herein by reference in their entirety.

[0018] The terms "reduce," "decreased," "reduction," or "inhibit" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "reduce," "reduction," "reduce," or "inhibit" typically refers to a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. Where applicable, the decrease can be to a level recognized as within the normal range in individuals without the given disorder.

[0019] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or an increase of up to and including 100%, or any increase between 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold or more compared to a reference level. In the context of a marker or condition, an "increase" is a statistically significant increase in such level.

[0020] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate such as a primate, rodent, livestock, or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock or game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, avian species, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0021] In various embodiments, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can advantageously be used as subjects that represent animal models of disease, such as cancer. The subject may be an adult, a pup, or a cub, and may be male or female.

[0022] The subject may be a subject who has previously been diagnosed or identified as suffering from or having a condition in need of treatment (e.g., lymphoma, leukemia, or another type of cancer, among others) or one or more complications associated with such a condition, optionally having already received treatment for the condition or one or more complications associated with the condition. Alternatively, the subject may also be a subject who has not previously been diagnosed with such a condition or associated complication. For example, the subject may be a subject who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or a subject who does not exhibit risk factors.

[0023] A subject "in need" of treatment for a particular condition can be a subject who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0024] A "disease" is a health condition in an animal, e.g., a human, in which the animal is unable to maintain homeostasis and, if the disease is not remitted, the animal's health continues to deteriorate. In contrast, a "disorder" in an animal is a health condition in which the animal is able to maintain homeostasis, but in which the animal's health state is less favorable than would be expected in the absence of the disorder. A disorder, left untreated, does not necessarily result in a further decline in the animal's health state.

[0025] As used herein, the terms "tumor antigen" and "cancer antigen" are used interchangeably to refer to antigens differentially expressed by cancer cells and can therefore be used to target cancer cells. Cancer antigens are antigens that can potentially stimulate distinct tumor-specific immune responses. Some of these antigens are encoded, but not necessarily expressed, by normal cells. These antigens can be characterized as those that are normally silent (i.e., not expressed) in normal cells, those that are expressed only at specific stages of differentiation, and those that are transiently expressed, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Numerous tumor antigens have been defined in terms of multiple solid tumors: immune-defined MAGE1, 2, and 3; MART-1 / Melan-A, gp100, carcinoembryonic antigen (CEA), HER2, mucins (i.e., MUC-1), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). Furthermore, viral proteins such as those encoded by hepatitis B virus (HBV), Epstein-Barr virus (EBV), and human papillomavirus (HPV) have each been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer.

[0026] As used herein, the term "chimera" refers to the product of a fusion between portions of at least two or more different polynucleotide molecules. In one embodiment, the term "chimera" refers to a gene expression element produced through the manipulation of known elements or other polynucleotide molecules.

[0027] In some embodiments, "activation" can refer to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. In some embodiments, activation can refer to induced cytokine production. In other embodiments, activation can refer to detectable effector function. At a minimum, "activated T cells," as used herein, are proliferating T cells.

[0028] As used herein, the terms "specific binding" and "specifically bind" refer to a physical interaction between two molecules, compounds, cells, and / or particles in which a first entity binds to a second, target, entity with greater specificity and affinity than it binds to a third, non-target, entity. In some embodiments, specific binding can refer to an affinity of a first entity for a second target entity that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more, greater than its affinity for a third, non-target, entity under the same conditions. A reagent specific for a given target is one that exhibits specific binding to that target under the conditions being utilized in the assay. Non-limiting examples include antibodies or ligands that recognize and bind to cognate binding partner proteins (e.g., stimulatory and / or costimulatory molecules present on T cells).

[0029] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (APC, e.g., macrophage, dendritic cell, B cell, artificial APC, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule" or "co-stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, proliferation, activation, initiation of an immune response, etc. Stimulatory ligands are well known in the art and include, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0030] "Stimulatory molecule," as the term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0031] "Co-stimulatory ligand," as the term is used herein, includes a molecule on an APC that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands can include, but are not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll-like receptors, and ligands that specifically bind B7-H3. Costimulatory ligands can also include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0032] For example, 4-1BBL is a type 2 transmembrane glycoprotein belonging to the TNFR / TNF ligand superfamily. 4-1BBL is a costimulatory ligand that binds to the receptor 4-1BB (CD137) expressed on T cells. 4-1BBL is expressed on professional APCs, including dendritic cells, macrophages, and activated B cells. The 4-1BBL sequence is known in several species, e.g., human 4-1BBL, and is also known as TNFSF9 (NCBI gene ID: 8744) polypeptide (e.g., NCBI Reference Sequence NP_003802.1) and mRNA (e.g., NCBI Reference Sequence NM_003811.3). 4-1BBL can refer to human 4-1BBL, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in animal applications, 4-1BBL can refer to 4-1BBL from, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human 4-1BBL are readily identified in such species by one of skill in the art using, for example, the NCBI ortholog search function or available sequence data to search in a given species for sequences similar to the reference 4-1BBL sequence.

[0033] A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll-like receptors, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0034] In one embodiment, the term "modified" and its grammatical equivalents, as used herein, can refer to one or more human-designed changes to a nucleic acid, e.g., a nucleic acid within the genome of an organism. In another embodiment, modified can refer to a genetic change, addition, and / or deletion. A "modified cell" can refer to a cell that has an added, deleted, and / or modified gene. The terms "cell" or "modified cell" and their grammatical equivalents, as used herein, can refer to a cell derived from a human or a non-human animal.

[0035] As used herein, the term "operably linked" refers to the connection of a first polynucleotide molecule, e.g., a promoter, with a second transcribable polynucleotide molecule, e.g., a gene of interest, where the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single, contiguous polynucleotide molecule and may or may not be contiguous. For example, a promoter is operably linked to a gene of interest if the promoter controls or mediates transcription of the gene of interest in a cell.

[0036] It is further contemplated that the various embodiments described herein encompass variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described. With respect to amino acid sequences, those skilled in the art will understand that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small percentage of amino acids within the encoded sequence are "conservatively modified variants" where the alteration results in the replacement of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants do not, in addition, exclude polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure. Variants of the sequences provided herein (see, e.g., Examples 2 and 3) are encompassed by the present invention.

[0037] A given amino acid can be substituted with a residue having similar physiochemical characteristics, such as substituting one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala for each other), or one polar residue for another (e.g., Lys for Arg; Glu for Asp; or Gln for Asn). Other such conservative substitutions, such as substitutions of entire regions with similar hydrophobic characteristics, are known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, e.g., ligand-mediated receptor activity and specificity of the native or reference polypeptide, is retained.

[0038] Amino acids can be grouped according to similarities in the properties of their side chains (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would involve exchanging a member of one of these classes for another. Particular conservative substitutions include, for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0039] In some embodiments, a polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to assays known in the art or described herein below. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0040] In some embodiments, the polypeptides described herein can be variants of such polypeptides or molecules (see, e.g., the sequences in Examples 2 and 3). In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of a native nucleotide sequence. A "variant," as referred to herein, is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide encompasses sequences that encode a variant protein or fragment thereof that contains one or more additions, deletions, or substitutions of nucleotides compared to the native or reference DNA sequence but retains the activity of the non-variant polypeptide. A wide variety of PCR-based site-directed mutagenesis techniques are known in the art and can be applied by those skilled in the art.

[0041] The amino acid or DNA sequence of the variant can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a native or reference sequence (see, e.g., sequences in Example 2). The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0042] Alterations of the native amino acid sequence can be achieved by any of several techniques known to those of skill in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutant sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-directed mutagenesis techniques can be used to provide altered nucleotide sequences with specific codons altered according to the desired substitution, deletion, or insertion. Techniques for making such modifications are well established and include, for example, those disclosed in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, each of which is incorporated by reference in its entirety. Any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, adding cysteine ​​bond(s) to a polypeptide can improve its stability and promote oligomerization.

[0043] As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term "polynucleotide" is used interchangeably herein with "nucleic acid" to refer to a polymer of nucleosides. Typically, polynucleotides are composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However, the term also encompasses molecules containing nucleosides or nucleoside analogs having chemically or biologically modified bases, modified backbones, and the like, whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for particular applications. When this application refers to polynucleotides, it is understood that both DNA and RNA, and in each case both single- and double-stranded forms (and the complementary strand of each single-stranded molecule) are provided. "Polynucleotide sequence," as used herein, can refer to the polynucleotide material itself and / or the sequence information (i.e., the series of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are shown in the 5' to 3' direction unless otherwise indicated.

[0044] The term "polypeptide" as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. As used herein, a polypeptide typically has amino acids, such as the 20 amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide can be modified, for example, by the addition of a chemical entity, such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, or a functional group. A polypeptide having a non-polypeptide moiety covalently or non-covalently associated therewith is still considered a "polypeptide." Exemplary modifications include glycosylation and palmitoylation. A polypeptide can be, for example, purified from a natural source, produced using recombinant DNA technology, or synthesized by chemical means, such as conventional solid-phase peptide synthesis. The terms "polypeptide sequence" or "amino acid sequence," as used herein, can refer to the polypeptide material itself and / or to the sequence information that biochemically characterizes the polypeptide (i.e., the series of letters or three-letter codes used as abbreviations in amino acid names). Polypeptide sequences presented herein are presented in an N-terminal to C-terminal direction unless otherwise indicated.

[0045] In some embodiments, a nucleic acid encoding a polypeptide (e.g., a CAR polypeptide) as described herein is contained by a vector. In some aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector," as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that, when associated with appropriate regulatory elements, is capable of replication and can introduce a genetic sequence into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virions, etc.

[0046] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional control sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the cell. Expression vectors may contain additional sequences; for example, an expression vector may have two replication systems, allowing its maintenance in two organisms, such as human cells for expression and prokaryotic hosts for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and protein, and, if necessary, protein secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, if applicable. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed (from DNA) into RNA in vitro or in vivo when operably linked to appropriate control sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0047] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one sequence of viral origin and is capable of being packaged into a viral vector particle. Viral vectors may contain nucleic acids encoding polypeptides as described herein in place of non-essential viral genes. The vectors and / or particles may be utilized to introduce nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0048] "Recombinant vector" means a vector comprising a heterologous nucleic acid sequence or "transgene" capable of expression in vivo. It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and treatments. In some embodiments, the vector is episomal. Use of a suitable episomal vector provides a means of maintaining a nucleotide of interest in a subject in high copy number extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0049] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatment in a subject to reverse, alleviate, ameliorate, inhibit, slow, or halt the progression or severity of a condition associated with a disease or disorder, e.g., acute lymphoblastic leukemia or other cancer, disease, or disorder. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if disease progression is reduced or halted. That is, "treatment" includes not only improvement in symptoms or markers, but also a cessation of, or at least a slowing of, the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of disease, delay or slowing of disease progression, remission or palliation of the condition, remission (whether partial or complete), and / or reduced mortality (whether detectable or undetectable). The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including symptomatic treatment).

[0050] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment and commensurate with a reasonable risk-benefit ratio. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be an artificial or modified carrier, e.g., a carrier not found in nature in which the active ingredient occurs.

[0051] As used herein, the term "administering" refers to the placement of a therapeutic or pharmaceutical composition as disclosed herein in a subject by a method or route that results in at least partial delivery of the agent to a desired site. Pharmaceutical compositions containing agents as disclosed herein can be administered by any suitable route that results in effective treatment in the subject.

[0052] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0053] Except in the experimental examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about," which, when used in connection with percentages, can mean ±1%.

[0054] As used herein, the term "comprising" means that other elements may be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0055] The term "consisting of" refers to compositions, methods, and each component thereof as described herein, excluding any element not recited within that description of an embodiment.

[0056] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the technology.

[0057] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the term "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia," and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0058] In some embodiments of any of the aspects, the disclosures described herein do not relate to processes for cloning humans, processes for modifying the genetic identity of a human germline, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of an animal that is prone to disease and does not involve any substantial medical benefit to the human or animal, nor to the animals resulting from such processes.

[0059] Other terms are defined within the description of various aspects and embodiments of the technology below. [Brief explanation of the drawings]

[0060] [Figure 1] 1 shows the surface expression of CD79b, CD79a, CD19, CD37, BCMA, TACI, Fas, CD38, and CD138 on the MCL cell line Jeko-1. [Figure 2] Constructs encoding (i) CD79b and (ii) CD79b and CD19 constructs are shown. [Figure 3] Graph showing transduction efficiency of the indicated CAR molecules in primary T cells (n=3). [Figure 4] Growth curves of untransduced (UTD) and indicated CAR-transduced cells. [Figure 5] Graph showing the level of activation of CAR-transduced cells. [Figure 6]Graph showing in vitro cytotoxic efficacy of CAR-transduced T cells on Jeko-1 cells (n=2). CD19(H / L)CAR - closed circles; CD79b(L / H)CAR - pentagons; CD79b(H / L) - triangles; UTC - open circles. [Figure 7] Graph showing the levels of effector cytokines produced by CAR-transduced cells. [Figure 8A] Timeline of a mouse xenograft model receiving Jeko-1 cells followed by CAR T cells. [Figure 8B] Graph showing the cytotoxic efficacy of CAR T cells against Jeko-1 cells, measured as FLUX. [Figure 8C] Graph showing the number of CAR T cells present in the blood 14 days after injection. [Figure 9A] Timeline of a mouse xenograft model receiving MCL PDX cells followed by CAR T cells. [Figure 9B] Graph showing the cytotoxic efficacy of CAR T cells against PDX tumors, measured as FLUX. [Figure 10] Graph showing percent activation of bispecific CARs activated by cells expressing CD19 and CD79b (n=3). DETAILED DESCRIPTION OF THE INVENTION

[0061] As described herein, CAR molecules specific for CD79b are described that can be used in the prevention and treatment of, for example, cancer (e.g., lymphomas, e.g., mantle cell lymphoma (MCL) and other non-Hodgkin's lymphomas (NHL), e.g., diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBCL), chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL)).

[0062] Also described is a bispecific CAR specific for CD79b and CD19. The bispecific CAR described herein can be advantageously used to reduce the possibility of tumor escape due to loss of target antigen. In particular, a CAR that binds to two different tumor-associated antigens or factors (such as CD79b and CD19) will not lose efficacy if one or the other of the antigens or factors is downregulated by the targeted cell. Similarly, a CD79b CAR can be advantageously used in the treatment of subjects who have previously been treated with a CD19 CAR but have experienced a CD19-negative relapse.

[0063] Embodiments of the technology described herein relate to the discovery that CD79b is expressed on cancer cells, including lymphoma cells. Thus, CARs specific for CD79b (and optionally CD19) are effective therapies for treating cancers, such as lymphomas, including MCL and other NHLs, such as DLBCL, PMBCL, CLL, and SLL.

[0064] Thus, one aspect of the invention described herein relates to a CAR polypeptide comprising: (a) an extracellular domain comprising (i) a sequence that specifically binds to CD79b or (ii) a sequence that specifically binds to CD79b and a sequence that specifically binds to CD19 (e.g., a single-chain antibody sequence; scFv), (b) a hinge and transmembrane domain, and (c) an intracellular signaling domain. Optionally, the CAR polypeptide also comprises a costimulatory domain, as described herein.

[0065] Considerations intended for use in constructing and utilizing these and other aspects of the technology are set forth below.

[0066] Chimeric Antigen Receptor The technology described herein provides improved CARs intended for use in immunotherapy. CARs and various improvements are discussed below.

[0067] The term "chimeric antigen receptor" or "CAR" or "CARs," as used herein, refers to an engineered T cell receptor that transfers ligand or antigen specificity into a T cell (e.g., a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0068] CARs position a chimeric extracellular target binding domain that specifically binds to a target, e.g., a polypeptide, expressed on the surface of a cell so as to be a target in a T cell response to a construct comprising the transmembrane and intracellular domains (including signaling domains) of a T cell receptor molecule. In one embodiment, the chimeric extracellular target binding domain comprises the antigen binding domain of an antibody that specifically binds to an antigen expressed on a cell to be targeted in a T cell response. In another embodiment, the chimeric extracellular target binding domain comprises the antigen binding domain of a first antibody that specifically binds to a first antigen expressed on a cell to be targeted by a T cell response, as well as the antigen binding domain of a second antibody that specifically binds to a second antigen expressed on a cell to be targeted by a T cell response. While the properties of the intracellular signaling domain of a CAR can vary as known in the art and as disclosed herein, the chimeric target / antigen binding domain sensitizes the receptor to signaling activation when the chimeric target / antigen binding domain binds to the target / antigen on the surface of a target cell.

[0069] With respect to intracellular signaling domains, so-called "first generation" CARs include those that provide a CD3 zeta (CD3ζ) signal solely upon antigen binding. So-called "second generation" CARs include those that provide both costimulatory (e.g., CD28 or CD137) and activation (CD3ζ) domains, and so-called "third generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and an activation domain (e.g., CD3ζ). In various embodiments, CARs are selected to have high affinity or avidity for the target / antigen. For example, target or antigen binding domains derived from antibodies will generally have higher affinity and / or avidity for the target antigen than naturally occurring T cell receptors. This property, combined with the high specificity that allows for selection for antibodies, results in highly specific T cell targeting by CAR T cells.

[0070] As used herein, "CAR T cells" or "CAR-T" refers to T cells that express a CAR. When expressed in a T cell, a CAR has the ability to redirect the specificity and reactivity of the T cell toward a selected target in a non-MHC-restricted manner, utilizing the antigen-binding properties of a monoclonal antibody. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing a major mechanism of tumor escape.

[0071] As used herein, the term "extracellular target binding domain" refers to a polypeptide found outside a cell sufficient to facilitate binding to a target. An extracellular target binding domain will specifically bind to its binding partner. Generally, an extracellular target binding domain may comprise an antigen binding domain of an antibody or a ligand that recognizes and binds to a cognate binding partner protein. In this context, a ligand is a molecule that specifically binds to a protein and / or portion of a receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein may generally be found on the surface of a cell. Ligand:cognate partner binding may result in modification of the receptor with the ligand or activate a physiological response, e.g., activate a signaling pathway or cascade. In one embodiment, the ligand may be non-native to the genome. Optionally, the ligand has a conserved function across at least two species.

[0072] antibody reagents In various embodiments, the CARs described herein comprise an antibody reagent or an antigen-binding domain thereof as the extracellular target-binding domain.

[0073] As used herein, the term "antibody reagent" refers to a polypeptide that comprises at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and that specifically binds to a given antigen. An antibody reagent may comprise an antibody or a polypeptide comprising the antigen-binding domain of an antibody. In some embodiments of any of the aspects, an antibody reagent may comprise a monoclonal antibody or a polypeptide comprising the antigen-binding domain of a monoclonal antibody. For example, an antibody may comprise a heavy (H) chain variable region (referred to herein as V H ), and a light (L) chain variable region (abbreviated as V LIn another example, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur J. Immunol. 26(3):629-639, 1996; incorporated herein by reference in its entirety)), as well as complete antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primates (human and non-human primates), and can be primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like. Fully human antibody binding domains can be selected using methods known to those skilled in the art, for example from phage display libraries.

[0074] V H and V L Regions can be further subdivided into regions of hypervariability termed "complementarity-determining regions" ("CDRs") interspersed with more conserved regions termed "framework regions" ("FRs"). The extent of framework regions and CDRs has been previously defined (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al., J. Mol. Biol. 196:901-917, 1987; which are incorporated herein by reference in their entireties). H and V L Each of these is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0075] In one embodiment, the antibody or antibody reagent is not human (i.e., the antibody or antibody reagent is murine), but is humanized. A "humanized antibody or antibody reagent" refers to a non-human antibody or antibody reagent that has been modified at the protein sequence level to increase its similarity to variants of antibodies or antibody reagents naturally produced in humans. One approach to humanizing antibodies utilizes the grafting of murine or other non-human CDRs onto a human antibody framework.

[0076] In one embodiment, the extracellular target binding domain of the CAR is linked to the V domain of an antibody, typically a monoclonal antibody, via a flexible linker peptide. H and V L and / or consisting essentially of a single-chain Fv (scFv) fragment created by fusing domains, in various embodiments, the scFv is fused to a transmembrane domain and a T cell receptor intracellular signaling domain, such as a modified intracellular signaling domain as described herein.

[0077] Antibody binding domains and methods for selecting and cloning them are well known to those skilled in the art.

[0078] In one embodiment, the extracellular domain of the CAR polypeptide comprises an antibody reagent or antigen-binding domain thereof as an extracellular target binding domain that is specific for CD79b. In another embodiment, the extracellular domain of the CAR polypeptide comprises (i) an antibody reagent or antigen-binding domain thereof as an extracellular target binding domain that is specific for CD79b, and (ii) an antibody reagent or antigen-binding domain thereof as an extracellular target binding domain that is specific for CD19.

[0079] Thus, for example, in one embodiment, the extracellular domain of a CAR polypeptide comprises, consists essentially of, or consists of the light chain sequence of SEQ ID NO: 4 and / or the heavy chain sequence of SEQ ID NO: 6, or comprises, consists essentially of, or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 4 and / or SEQ ID NO: 6. The light and heavy chain sequences can be in either order, e.g., the light chain sequence can be N-terminal to the heavy chain sequence, or the heavy chain sequence can be N-terminal to the light chain sequence. In various embodiments, the light and heavy chain sequences are separated from each other by a linker sequence (e.g., a glycine-rich sequence; e.g., SEQ ID NO: 5).

[0080] In another example, the extracellular domain of a CAR polypeptide comprises, consists essentially of, or consists of (i) a sequence comprising an scFv against CD79b (SEQ ID NO: 12), comprising a light chain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a heavy chain (SEQ ID NO: 6), (ii) an optional linker (SEQ ID NO: 5), and (iii) a sequence comprising an scFv against CD19 (SEQ ID NO: 13). The extracellular domain of a CAR polypeptide can optionally have a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one or more of these sequences. Furthermore, the order of the light and heavy chains, for example in a CD79b scFv, can be reversed.

[0081] In one embodiment, the CAR polypeptide contains one or more mutations in its coding region, thereby generating a variant sequence as described herein. One skilled in the art will be able to introduce mutations into the nucleic acid sequence of a gene or gene product using standard techniques. For example, point mutations can be introduced via site-directed point mutagenesis, PCR techniques. Site-directed mutagenesis kits are commercially available, for example, through New England Biolabs; Ipswich, MA. Alternative methods for introducing point mutations into the nucleic acid sequence of a gene or gene product include, but are not limited to, cassette mutagenesis or whole-plasmid mutagenesis.

[0082] In one embodiment, a CAR useful in the technology described herein comprises at least two antigen-specific targeting regions (e.g., SEQ ID NO: 12 and / or 13) in the extracellular domain, transmembrane domain, and intracellular signaling domain. In such an embodiment, the two or more antigen-specific targeting regions of such a bispecific CAR target at least two different antigens and may be arranged in tandem and separated by a linker sequence (e.g., SEQ ID NO: 5).

[0083] target / antigen Generally, any cell surface moiety can be targeted by a CAR. Most often, the target will be a cell surface polypeptide that is differentially or preferentially expressed on cells that one desires to target for a T cell response. In this regard, tumor antigens or tumor-associated antigens provide attractive targets, providing a means to target tumor cells while avoiding or at least limiting collateral damage to non-tumor cells or tissues. CARs specific for CD79b, or both CD79b and CD19, are described herein. Non-limiting examples of additional tumor antigens or tumor-associated antigens include CEA, immature laminin receptor, TAG-72, HPV E6 and E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, BRCA1 / 2, CDK4, MART-2, p53, Ras, MUC1, and TGF-βRII. CARs against one or more of these antigens can be used in combination with CARs against CD79b, or CD79b and CD19, as described herein, as determined appropriate by one of skill in the art.

[0084] Hinge and TM domains Each CAR as described herein can include a hinge domain that separates the extracellular target binding domain from the T cell membrane.

[0085] As used herein, "hinge domain" refers to an amino acid region that allows for separation and flexibility of the binding moiety and the T cell membrane. A flexible hinge length also allows for better binding to relatively remote epitopes, e.g., a longer hinge region allows for optimal binding. One skilled in the art will be able to determine an appropriate hinge for a given CAR target. In one embodiment, the transmembrane domain or fragment thereof of any of the CAR polypeptides described herein comprises a CD8 or 4-1BB hinge domain.

[0086] Each CAR as described herein comprises a transmembrane domain that links an extracellular target binding domain to an intracellular signaling domain.

[0087] As used herein, "transmembrane domain" (TM domain) refers to the generally hydrophobic region of a CAR that crosses the plasma membrane of a cell. The TM domain can be a transmembrane region or fragment thereof of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are presented herein and used in the examples, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternative embodiments of the technology. The selected transmembrane region or fragment thereof will preferably not interfere with the intended function of the CAR. "Fragment thereof," when used in reference to the transmembrane domain of a protein or polypeptide, refers to a portion of the transmembrane domain sufficient to anchor or attach the protein to the cell surface.

[0088] In one embodiment, the transmembrane domain or fragment thereof of any of the CAR polypeptides described herein comprises a transmembrane domain selected from the transmembrane domains of CD8 or 4-1BB. In alternative embodiments of any aspect, the transmembrane domain or fragment thereof of a CAR described herein comprises a transmembrane domain selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KL RF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1 , ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0089] CD8 is an antigen found preferentially on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and acts as a T cell coreceptor. CD8 is composed of α (CD8a) and β (CD8b) chains. CD8a sequences are known in several species, including human CD8a (NCBI Gene ID:925) polypeptide (NCBI Ref Seq NP_001139345.1) and mRNA (e.g., NCBI Ref Seq NM_000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to CD8 from, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human CD8 are readily identified for such species by one skilled in the art using, for example, the NCBI ortholog search function or by searching available sequence data for a given species for sequences similar to a reference CD8 sequence.

[0090] In one embodiment, the CD8 hinge and transmembrane sequence comprises the sequence of SEQ ID NO:7; or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:7.

[0091] Costimulatory domain Each CAR described herein can optionally include one or more intracellular domains of a costimulatory molecule, or costimulatory domain. As used herein, the term "co-stimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen. 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, the intracellular domain is the intracellular domain of 4-1BB.

[0092] Thus, in one embodiment, the CAR polypeptide further comprises an intracellular domain. As used herein, "intracellular domain" refers to a sequence that is completely contained within the cell. In one embodiment, the intracellular domain refers to the intracellular domain of a receptor. The intracellular domain can interact with the interior of the cell. In relation to the intracellular domain of a receptor, the intracellular domain can function to relay a signal to be transmitted. The intracellular domain of a receptor can include enzymatic activity.

[0093] In one embodiment, the intracellular domain is the intracellular domain (ICD) of 4-1BB. In one embodiment, the 4-1BB intracellular domain comprises the sequence of SEQ ID NO: 8; or comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 8.

[0094] Intracellular signaling domains A CAR as described herein comprises an intracellular signaling domain. "Intracellular signaling domain" refers to the portion of a CAR polypeptide that is involved in transducing the message of effective CAR binding to a target antigen inside an immune effector cell and inducing effector cell function, such as activation, cytokine production, proliferation, and cytotoxic activity, such as release of cytotoxic factors into the CAR-bound target cell, or other cellular response elicited after antigen binding to the extracellular CAR domain.

[0095] CD3 is a T cell coreceptor that promotes T lymphocyte activation when simultaneously engaged with appropriate costimulation (e.g., binding of costimulatory molecules). The CD3 complex consists of four distinct chains; mammalian CD3 consists of the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) and CD3ζ to generate an activation signal in T lymphocytes. The complete TCR complex includes the TCR, CD3ζ, and the complete CD3 complex.

[0096] In some embodiments of any aspect, a CAR polypeptide described herein comprises an intracellular signaling domain that includes an immunoreceptor tyrosine-based activation motif, or ITAM, from CD3 zeta (CD3ζ). In some embodiments of any aspect, the ITAM includes the ITAM triad motif of CD3ζ (ITAM3). In some embodiments of any aspect, the ITAM triad motif of CD3ζ is mutated.

[0097] ITAMs are known as primary signaling domains that regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Non-limiting examples of ITAMs with intracellular signaling domains that have particular use in the technology include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0098] One skilled in the art will be able to introduce mutations into the nucleic acid sequence of a gene or gene product, e.g., an ITAM, using standard techniques. For example, point mutations can be introduced via site-directed point mutagenesis, PCR techniques. Site-directed mutagenesis kits are commercially available, for example, through New England Biolabs; Ipswich, MA. Alternative methods for introducing point mutations into the nucleic acid sequence of a gene or gene product include, but are not limited to, cassette mutagenesis or whole-plasmid mutagenesis.

[0099] In one embodiment, the ITAM used in the CAR is based on alternatives to CD3ζ, such as mutated ITAMs from CD3ζ (which has three ITAM motifs), truncations of CD3ζ, and alternative splice variants known as CD3ε, CD3θ, and artificial constructs engineered to express fusions between CD3ε or CD3θ and CD3ζ.

[0100] In one embodiment, the CD3 zeta intracellular signaling sequence corresponds to or comprises the amino acid sequence of SEQ ID NO:9; or comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:9.

[0101] A more detailed description of CARs and CAR T cells can be found in Maus et al., Blood 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039-3047, 2014; Maher et al., Cancer Res 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012 (each of which is incorporated by reference in its entirety).

[0102] In one embodiment, the CAR polypeptide further comprises a CD8 leader sequence. As used herein, "leader sequence," also known as leader RNA, refers to the region of an mRNA that is immediately upstream of the start codon. Leader sequences can be important for regulating translation of a transcript.

[0103] In one embodiment, the CD8 leader sequence corresponds to the amino acid sequence of SEQ ID NO:3; or comprises SEQ ID NO:3; or comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:3.

[0104] In one embodiment, the CAR further comprises a linker domain. As used herein, "linker domain" refers to an oligo- or polypeptide region, approximately 2-100 amino acids in length, that links together any of the domains / regions of the CAR as described herein. In some embodiments, the linker can comprise or consist of flexible residues, such as glycine and serine, to allow adjacent protein domains to move freely relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. The linker may be cleavable or non-cleavable. Examples of cleavable linkers include a 2A linker (e.g., T2A), a 2A-like linker, or functional equivalents thereof, and combinations thereof. In one embodiment, the linker region is T2A from the Thosea asigna virus. Non-limiting examples of linkers include a linker derived from Thosea asigna virus and a linker derived from an internal ribosome entry site (IRES) sequence.

[0105] In one embodiment, a CAR as described herein further comprises a reporter molecule, for example, to allow for non-invasive imaging (e.g., positron emission tomography (PET) scans). In bispecific CARs that comprise a reporter molecule, the first extracellular binding domain and the second extracellular binding domain can comprise different or the same reporter molecule. In bispecific CAR T cells, the first CAR and the second CAR can express different or the same reporter molecule. In another embodiment, a CAR as described herein can be coupled to a substrate or chemical (e.g., 9-[4-[ 18 F]fluoro-3-(hydroxymethyl)butyl]guanine ([ 18 In another embodiment, the CAR as described herein further comprises a reporter molecule (e.g., hygromycin phosphotransferase (hph)) that is imageable in combination with a nanoparticle (e.g., [F]FHBG) that is readily imageable using non-invasive techniques. 64 Cu 2+and further including gold nanoparticles (GNPs) functionalized with . Labeling of CAR T cells for non-invasive imaging is reviewed, for example, in Bhatnagar et al., Integr. Biol. (Camb) 5(1):231-238, 2013, and Keu et al., Sci. Transl. Med.;9(373), 2017, which are incorporated by reference in their entireties.

[0106] GFP and mCherry are presented herein as useful fluorescent tags for imaging CARs expressed on T cells (e.g., CAR T cells). It is contemplated that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical use, CARs do not need to contain a fluorescent tag or fluorescent protein.

[0107] Another aspect of the invention relates to a CAR polypeptide comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1, 2, 10, and 11 (optionally excluding the CD8 leader sequence of SEQ ID NO: 3). Another aspect of the invention relates to a CAR polypeptide comprising a sequence selected from SEQ ID NOs: 1, 2, 10, and 11 (optionally excluding the CD8 leader sequence of SEQ ID NO: 3).

[0108] Another aspect of the invention described herein pertains to a polypeptide complex comprising two or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of any of the CAR polypeptides described herein. In one embodiment, the polypeptide complex comprises three of any of the CAR polypeptides described herein.

[0109] Another aspect of the invention relates to a mammalian cell comprising any of the CAR polypeptides described herein; or a nucleic acid encoding any of the CAR polypeptides described herein. In one embodiment, the mammalian cell comprises an antibody, an antibody reagent, an antigen-binding portion thereof, or any of the CAR polypeptides described herein, or a nucleic acid encoding such an antibody, antibody reagent, an antigen-binding portion thereof, or any of the CAR polypeptides described herein. The mammalian cell or tissue can be from a human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat, although any other mammalian cell may be used. In a preferred embodiment of any aspect, the mammalian cell is human.

[0110] In one embodiment, the cell is a T cell. In alternative embodiments of any aspect, the cell is an immune cell. As used herein, "immune cell" refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cell is a T cell; a NK cell; a NKT cell; a lymphocyte, such as a B cell and a T cell; and a myeloid cell, such as a monocyte, macrophage, eosinophil, mast cell, basophil, and granulocyte.

[0111] In one embodiment, the cells are obtained from an individual who has or has been diagnosed with cancer, a plasma cell disorder, or an autoimmune disease.

[0112] "Cancer," as used herein, can refer to the hyperproliferation of cells when a reduction in characteristic traits (normal cellular controls) leads to uncontrolled growth, lack of differentiation, local tissue invasion, and metastasis, and can be, for example, lymphoma, leukemia, multiple myeloma, or solid tumors. In particular examples, the cancer is any type of B-cell malignancy. Non-limiting examples of B-cell malignancies include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, hairy cell leukemia (HCL), Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), and leukemia-associated lymphoid tissue lymphoma (LET). ), lymphoplasmacytic lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granuloma, primary central nervous system lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, and unclassifiable B-cell lymphoma.

[0113] Non-limiting examples of leukemias include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In one embodiment, the cancer is ALL or CLL. Non-limiting examples of solid tumors include adrenocortical tumors, alveolar soft part sarcoma, carcinoma, chondrosarcoma, colorectal carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumors, yolk sac tumor, epithelioid hemangioendothelioma, Ewing's sarcoma, germ cell tumors (solid tumors), giant cell tumor of bone and soft tissue, hepatoblastoma, hepatocellular carcinoma, melanoma, renal tumor, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcomas (NRSTS), osteosarcoma, paraspinal sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms' tumor. Solid tumors can be found in bone, muscle, or an organ and can be sarcomas or cancers. It is contemplated that any embodiment of the invention described herein can be used to treat any type of cancer, including cancers not enumerated herein. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue, e.g., of a malignant or benign type.

[0114] As used herein, an "autoimmune disease or disorder" is characterized by the inability of one's own immune system to distinguish between foreign and healthy cells. As a result, one's own immune system targets one's own healthy cells through programmed cell death. Non-limiting examples of autoimmune diseases or disorders include inflammatory arthritis, type 1 diabetes, multiple sclerosis, psoriasis, inflammatory bowel disease, SLE, and vasculitis, allergic inflammation such as allergic asthma, atopic dermatitis, and contact hypersensitivity, rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, Graves' disease (overactive thyroid), Hashimoto's thyroiditis (underactive thyroid), chronic graft-versus-host disease, hemophilia with antibodies to clotting factors, celiac disease, Crohn's disease and ulcerative colitis, Guillain-Barré syndrome, primary biliary sclerosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, and leukoencephalopathy. These include: rheumatic syndrome, scleroderma, Sjögren's syndrome, Good Pasteur's disease, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), psoriasis, autoimmune Addison's disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anemia, canine polyarthritis, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, and fibromyalgia (FM).

[0115] In one embodiment, the mammalian cells are obtained in a patient with an immune system disorder that results in abnormally low activity in the immune system, or an immunodeficiency disorder, that inhibits the patient's ability to fight foreign cells (i.e., viral or bacterial cells).

[0116] Plasma cells are white blood cells that develop from B lymphocytes and function to produce and release antibodies necessary to fight infection. As used herein, a "plasma cell disorder or disease" is characterized by abnormal proliferation of plasma cells. Abnormal plasma cells have the ability to "crowd out" healthy plasma cells, thereby resulting in a reduced ability to fight foreign targets, such as viral or bacterial cells. Non-limiting examples of plasma cell disorders include amyloidosis, Waldenstrom's macroglobulinemia, osteosclerotic myeloma (POEMS syndrome), monoclonal gammopathy of undetermined significance (MGUS), and plasma cell myeloma.

[0117] T cells can be obtained from a subject using standard techniques known in the art, for example, T cells are isolated from peripheral blood taken from a patient.

[0118] Cells, e.g., T cells, can be engineered to contain any of the CAR polypeptides described herein; or a nucleic acid encoding any of the CAR polypeptides described herein. In one embodiment, the CAR polypeptides described herein are contained within a lentiviral vector. The lentiviral vector is used to express the CAR polypeptide in the cell using standard infection techniques.

[0119] Retroviruses, such as lentiviruses, provide a convenient platform for delivering genes, i.e., nucleic acid sequences encoding chimeric genes of interest. A selected nucleic acid sequence can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, for example, in vitro or ex vivo. Retroviral systems are well known in the art and are described, for example, in U.S. Pat. No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu and Pathak Pharmacological Reviews 2000 52:493-512 (incorporated herein by reference in their entirety). A lentiviral system for efficient DNA delivery can be purchased from OriGene, Rockville, MD. In other embodiments, the CAR polypeptide of any of the CARS described herein is expressed in mammalian cells via transfection or electroporation of an expression vector containing a nucleic acid encoding the CAR. Methods for transfection or electroporation are known in the art.

[0120] Efficient expression of any of the CAR polypeptides described herein can be assessed using standard assays that detect mRNA, DNA, or gene products of the nucleic acid encoding the CAR, including, for example, RT-PCR, FACS, Northern blotting, Western blotting, ELISA, or immunohistochemistry.

[0121] In one embodiment, the CAR polypeptide of any of the CAR polypeptides described herein is constitutively expressed. In one embodiment, the CAR polypeptide of any of the CAR polypeptides described herein is encoded by a recombinant nucleic acid sequence.

[0122] One aspect of the invention described herein relates to a method of treating cancer, a plasma cell disorder, an amyloidosis, or an autoimmune disease in a subject, the method comprising modifying T cells to comprise on their surface any of the CAR polypeptides described herein; and administering the modified T cells to the subject.

[0123] Another aspect of the invention described herein relates to a method of treating cancer, a plasma cell disorder, or an autoimmune disease in a subject, comprising administering any of the CAR polypeptides described herein, or a cell comprising a nucleic acid encoding any of the CAR polypeptides described herein.

[0124] In one embodiment, the method further comprises activating or stimulating the CAR-T prior to administering the cells to the subject, e.g., according to a method as described elsewhere herein.

[0125] In one embodiment, the cancer cells comprise the tumor antigen CD79b, or both the tumor antigens CD79b and CD19.

[0126] Administration In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer, a plasma cell dyscrasia or disorder, or an autoimmune disease or disorder with a mammalian cell comprising any of the CAR polypeptides described herein or a nucleic acid encoding any of the CAR polypeptides described herein. As used herein, "CAR T cells as described herein" refers to any of the CAR polypeptides described herein or a mammalian cell comprising a nucleic acid encoding any of the CAR polypeptides described herein. As used herein, "condition" refers to cancer, a plasma cell dyscrasia or disorder, or an autoimmune disease or disorder. Subjects having a condition can be identified by a physician using current methods for diagnosing the condition. Symptoms and / or complications of the condition characterize these conditions and aid in diagnosis and are well known in the art and include, but are not limited to, fatigue, persistent infection, and persistent bleeding. For example, tests that may aid in the diagnosis of a condition are known in the art for a given condition, including, but not limited to, blood screening and bone marrow examination. Family history of a condition or exposure to risk factors for a condition can also aid in determining whether a subject is likely to have a condition or in making a diagnosis of a condition.

[0127] The compositions described herein can be administered to a subject having or diagnosed with a condition. In some embodiments, the methods described herein comprise administering to a subject an effective amount of activated CAR T cells described herein to reduce the symptoms of the condition. As used herein, "reducing the symptoms of a condition" refers to ameliorating any condition or symptom associated with the condition. When compared to a matched untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, as measured by any standard technique. Various means for administering the compositions described herein to a subject are known to those skilled in the art. In one embodiment, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered to the site of a tumor.

[0128] The term "effective amount," as used herein, refers to the amount of activated CAR T cells necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to an amount of a cell preparation or composition sufficient to produce the desired effect. Thus, the term "therapeutically effective amount" refers to an amount of activated CAR T cells sufficient to produce a specific anti-conditioning effect when administered to a typical subject. Effective amount, as used herein, also encompasses an amount sufficient to delay the onset of disease symptoms, alter the course of symptoms or disease (such as, but not limited to, slowing the progression of a condition), or reverse symptoms of a condition, under various circumstances. Thus, it is generally not feasible to specify an exact "effective amount." However, in any particular case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0129] Effective doses, toxicity, and therapeutic effects can be assessed by standard pharmaceutical procedures in cell cultures or experimental animals. Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compositions and methods that exhibit a large therapeutic index are preferred. The therapeutically effective amount can be initially estimated from cell culture assays. A dose can also be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of activated CAR T cells that achieves half-maximal inhibition of symptoms) as determined in cell culture or an appropriate animal model. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dose can be monitored, inter alia, by a suitable bioassay, such as a bone marrow biopsy assay. The dose can be determined by a physician and adjusted, if necessary, to suit the observed effect of treatment.

[0130] In one aspect of the present invention, the technology described herein relates to a pharmaceutical composition comprising activated CAR T cells as described herein and, optionally, a pharmaceutically acceptable carrier. The active ingredient of the pharmaceutical composition comprises at least activated CAR T cells as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of activated CAR T cells as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of activated CAR T cells as described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulations include saline and aqueous buffers, Ringer's solution, and serum components, such as serum albumin, HDL, and LDL. Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein.

[0131] In some embodiments, pharmaceutical compositions comprising activated CAR T cells as described herein can be in a parenteral dosage form. Because administration of a parenteral dosage form typically bypasses the patient's natural defenses against contaminants, components other than the CAR T cells themselves are preferably sterile or can be sterilized before administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry products that are easily dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions for injection, and emulsions. Any of these can be added to the activated CAR T cell preparation before administration.

[0132] Suitable vehicles that can be used to provide parenteral dosage forms of activated CAR T cells as disclosed herein are well known to those of skill in the art. Examples include, but are not limited to, saline; glucose solutions; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0133] dose "Unit dosage form," as the term is used herein, refers to a suitable single dose. For example, a unit dosage form may be the amount of therapeutic agent dispensed in a delivery device, such as a syringe or intravenous bag. In one embodiment, a unit dosage form is administered in a single dose. In another embodiment, two or more unit dosage forms may be administered simultaneously.

[0134] In some embodiments, the activated CAR T cells described herein are administered as a monotherapy, i.e., no other therapeutic agent for the condition is administered to the subject at the same time.

[0135] Pharmaceutical compositions comprising the T cells described herein generally contain 10 4 ~10 9cells / kg body weight, in some cases 10 5 ~10 6 The T cell compositions may be administered at a dose of 1000 cells / kg body weight (including all integer values ​​within these ranges). If necessary, the T cell compositions may also be administered multiple times at these doses. The cells may be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 319:1676, 1988).

[0136] In certain embodiments, it may be desirable to administer activated CAR T cells to a subject, then subsequently re-draw blood (or perform apheresis), activate T cells therefrom as described herein, and re-infuse these activated and expanded T cells into the patient. This process may be performed multiple times, every few weeks. In certain embodiments, T cells may be activated from a blood draw of 10 cc to 400 cc. In certain embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

[0137] Modes of administration may include, for example, intravenous (iv) injection or infusion. The compositions described herein may be administered intraarterially, intratumorally, intranodally, or intramedullarily to a patient. In some embodiments, the T cell compositions may be injected directly into a tumor, lymph node, or site of infection. In one embodiment, the compositions described herein are administered into a body cavity or fluid (e.g., ascites, pleural effusion, peritoneal fluid, or cerebrospinal fluid).

[0138] In certain exemplary aspects, subjects may undergo leukapheresis, in which leukocytes are collected, enriched, or ex vivo depleted, and cells of interest, e.g., T cells, are selected and / or isolated. These T cell isolates may be expanded by contact with artificial antigen-presenting cells (aAPCs), e.g., aAPCs expressing anti-CD28 and anti-CD3 CDRs, and treated to generate CAR T cells by introduction of one or more CAR constructs of the invention. Subjects in need thereof may then undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. After or concurrently with transplantation, subjects may receive an infusion of the expanded CAR T cells. In one embodiment, the expanded cells are administered pre- or post-operatively.

[0139] In some embodiments, the subject is lymphodepleted prior to administration of one or more CAR T cells as described herein, hi such embodiments, lymphodepletion can include administering one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0140] The dosage of the therapeutic agent to be administered to a patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed according to art-recognized practices.

[0141] In some embodiments, a single treatment regimen is required. In others, one or more subsequent doses or treatment regimens may be administered. For example, after three months of treatment every other week, treatment may be repeated once every month, six months, or a year or more. In some embodiments, no additional treatment is administered after the initial treatment.

[0142] The dosage of the compositions described herein can be determined by a physician and adjusted as necessary to suit the observed effects of treatment. It is typical for a skilled clinician to monitor the subject to determine when the treatment is producing a therapeutic effect, in relation to the duration and frequency of treatment, and to determine whether additional cells should be administered, whether treatment should be discontinued, whether treatment should be resumed, or whether changes should be made to the treatment regimen. The dosage should not be so high as to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary depending on the age, condition, and sex of the patient and can be determined by those skilled in the art. The dosage can also be adjusted by an individual physician if any complications arise.

[0143] Combination therapy The activated CAR T cells described herein can be used in combination with other known drugs and therapies. In one embodiment, the subject is administered anti-CD19 therapy and anti-CD79b therapy. In another embodiment, the subject is further administered anti-BCMA therapy. "Combined" administration, as used herein, refers to the delivery of two (or more) different therapeutic agents to a subject during the course of the subject's affliction with a disorder, e.g., the delivery of two or more therapeutic agents between the time the subject is diagnosed with the disorder and the time the disorder is cured or eliminated, or treatment is otherwise terminated. In some embodiments, there is an overlap in administration, where the delivery of one therapeutic agent is still ongoing when the delivery of the second begins. This is sometimes referred to herein as "simultaneous" or "combined delivery." In other embodiments, the delivery of one therapeutic agent ends before the delivery of the other therapeutic agent begins. In some embodiments in either case, the therapeutic agents are more effective due to the combined administration. For example, the second therapeutic agent may be more effective, e.g., an equivalent effect may be seen with a reduction in the second therapeutic agent, or the second therapeutic agent may reduce symptoms to a greater extent than would be seen if the second therapeutic agent were administered in the absence of the first therapeutic agent, or a similar situation may be seen in the presence of the first therapeutic agent. In some embodiments, delivery is such that the degree of reduction in symptoms, or other parameter related to the disorder, is greater than that seen when one therapeutic agent is delivered in the absence of the other. The effects of the two therapeutic agents may be partially additive, fully additive, or greater than additive. Delivery may be such that the effect of the first therapeutic agent being delivered remains detectable when the second therapeutic agent is delivered. The activated CAR T cells described herein and at least one additional therapeutic agent may be administered simultaneously, in the same composition or in separate compositions, or sequentially. In sequential administration, the cells expressing a CAR described herein may be administered first and the additional agent may be administered second, or the order of administration may be reversed. CAR T therapy and / or other therapeutic agents, procedures, or modalities can be administered during active disease or during periods of remission or less active disease. CAR T therapy can be administered before, alongside, after another treatment, or during remission of the disorder.

[0144] When administered in combination, the activated CAR T cells and additional agent (e.g., second or third agent), or all, can be administered in amounts or dosages that are higher, lower, or the same as the amount or dosage of each agent used individually, e.g., as monotherapy. In certain embodiments, the administered amount or dosage of the activated CAR T cells, additional agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually. In other embodiments, the amount or dosage of the activated CAR T cells, additional agent (e.g., second or third agent), or all, that produces a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent required individually to achieve the same therapeutic effect. In further embodiments, the activated CAR T cells described herein can be used in a treatment regimen in combination with surgery, chemotherapy, radiation, mTOR pathway inhibitors, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapeutics, cytoxin, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, or peptide vaccines, such as those described in Izumoto et al., J. Neurosurg. 108:963-971, 2008.

[0145] In one embodiment, the activated CAR T cells described herein may be used in combination with checkpoint inhibitors. Exemplary checkpoint inhibitors include anti-PD-1 inhibitors (nivolumab, MK-3475, pembrolizumab, pidilizumab, AMP-224, AMP-514), anti-CTLA4 inhibitors (ipilimumab and tremelimumab), anti-PDL1 inhibitors (atezolizumab, avelumab, MSB0010718C, MEDI4736, and MPDL3280A), and anti-TIM3 inhibitors.

[0146] In one embodiment, the activated CAR T cells described herein may be combined with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), antimetabolites, anti-cancer drugs (including, for example, antifolates, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulatory agents such as thalidomide or thalidomide derivatives (e.g., lenalidomide). Common chemotherapy agents being considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platin®), and cisplatin (Platin®). nol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®),Doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), , methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium 90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®,Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenetriamine (Tetrafluriazole), thiazolinone ... These include thiazolidine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (actinomycin-D, also known as Cosmegen®); melphalan (L-PAM, L-sarcolysin, and phenylalanine mustard, also known as Alkeran®); altretamine (hexamethylmelamine (HMM), also known as Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (CCNU, also known as CeeNU®); cisplatin (CDDP,also known as Platinol® and Platinol®-AQ; chlorambucil (Leukeran®); cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (DTIC, DIC, and imidazole carboxamide, also known as DTIC-Dome®); altretamine (hexamethylmelamine (HMM), also known as Hexalen®); ifosfamide (Ifex®); prednumustine; procarbazine (Matul®) ane®); mechlorethamine (nitrogen mustard, mustine and mechlorethamine hydrochloride, also known as Mustargen®); streptozocin (Zanosar®); thiotepa (thiophosphamide, TESPA and TSPA, also known as Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine HCl (Treanda®). Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimus (formerly known as deferolimus, (lR,2R,45)-4-[(2R)-2[(lR,95,125,15R,16E,18R,19R,21R,235,24E,26E,28Z,305,325,35R)-l,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-ll, 36-dioxa-4-azatricyclo[30.3.1.04'9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383; everolimus (Afinitor® or RADOOl); rapamycin (AY22989,Sirolimus®; simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-bis[(35,)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-JJpyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-l-benzo ...

[00100] [zopyran-2-yl]morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-La-aspartyl L-serine, inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplary immunomodulatory agents include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (a mixture of human cytokines including interleukin-1, interleukin-2, and interferon-γ, CAS 951209-71-5, IRX Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®,Idamycin PFS®; mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin. Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®). vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). Exemplary proteosome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, (5)-4-methyl-N-((5)-l-(((5)-4-methyl-l-((R)-2-methyloxiran-2-yl)-l-oxopentan-2-yl)amino)-l-oxo-3-phenylpropan-2-yl)-2-((5,)-2-(2-morpholinoacetamido)-4-phenylbutane amide)-pentanamide); marizomib (NPT0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(11S')-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0147] Those skilled in the art can easily identify the chemotherapeutic agent to be used (see, for example, Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18 thedition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer DS (ed): The Cancer Chemotherapy Handbook, 4th ed., St. Louis, Mosby-Year Book, 2003).

[0148] In one embodiment, the activated CAR T cells described herein are administered to a subject in combination with a molecule that reduces the activity and / or levels of a molecule that targets GITR and / or modulates GITR function, a molecule that reduces the Treg cell population, an mTOR inhibitor, a GITR agonist, a kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a CDK4 inhibitor, and / or a BTK inhibitor.

[0149] Effectiveness For example, the effectiveness of activated CAR T cells in treating a condition described herein or for inducing a response as described herein (e.g., a reduction in cancer cells) can be determined by a skilled clinician. However, a treatment is considered "effective treatment," as the term is used herein, when one or more of the signs or symptoms of a condition described herein are modified in a beneficial manner, other clinically recognized symptoms are improved or even ameliorated, or a desired response is induced (e.g., at least 10% after treatment in accordance with the methods described herein). Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence of a condition treated in accordance with the methods described herein, or any other suitable measurable parameter. Treatment in accordance with the methods described herein can reduce the level of a marker or symptom of a condition, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0150] Efficacy may also be assessed by the individual's failure to deteriorate as assessed by hospitalization or the need for medical intervention (i.e., halting of disease progression). Methods for measuring these indicators are known to those of skill in the art and / or are described herein.

[0151] Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals), including (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing the amelioration of symptoms. An effective amount in the treatment of a disease means an amount that, when administered to a subject in need thereof, is sufficient to provide effective treatment for the disease, as defined herein. The effectiveness of an agent can be determined by assessing physical indicators of the condition or desired response. It is well within the capabilities of one of ordinary skill in the art to monitor the effectiveness of administration and / or treatment by measuring any one or any combination of such parameters. The effectiveness of a given procedure can be evaluated in animal models of the conditions described herein, e.g., the treatment of lymphoma described herein. When using experimental animal models, the effectiveness of the treatment is demonstrated when a statistically significant change in a marker is observed.

[0152] All patents and other publications, including, for example, literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described therein that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and in no way represent an admission as to the accuracy of the dates or contents of these documents.

[0153] The description of the embodiments of the present disclosure is not intended to be precise or to limit the disclosure to the precise form disclosed. While specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as will be understood by those skilled in the art. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or may perform the functions substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods as appropriate. Further embodiments can be combined to provide additional embodiments. Aspects of the present disclosure can be modified, if necessary, to utilize the compositions, functions, and concepts of the above-mentioned references and applications to provide further embodiments of the present disclosure. Furthermore, considerations of biological and functional equivalence allow for some changes in protein structure without affecting biological or chemical activity in type or amount. These and other changes can be made in the present disclosure, given the detailed description. All such modifications are not intended to be included within the scope of the appended claims.

[0154] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements in other embodiments. Furthermore, while advantages associated with particular embodiments of the present disclosure are described in connection with those embodiments, other embodiments may also exhibit such advantages, and all of the embodiments need not necessarily exhibit such advantages to be included within the scope of the present disclosure.

[0155] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting. [Example]

[0156] Example 1 We have engineered CARs that target CD79b as part of the B cell receptor (BCR) complex. Using this approach, we are expanding treatment options for lymphoma patients, including those who experience relapse with CD19-negative disease after CD19 CAR therapy. We have also engineered bispecific CARs that target both CD79b and CD19.

[0157] Materials and Methods We generated CAR constructs containing scFv-based anti-CD79b fused to 4-1BB and CD3ζ via the CD8 hinge and transmembrane domains. Human primary T cells were lentivirally transduced with CD79b or CD19 CARs. Cytotoxicity against the MCL cell line Jeko-1, T cell activation, and cytokine production were evaluated. Furthermore, the cytotoxic effect of the CD79b CAR was evaluated in comparison with the CD19 CAR in xenograft experiments in Jeko-1 tumor-bearing mice and MCL PDX tumor-bearing mice.

[0158] result Figure 1 shows the results of characterization of the cell surface expression of CD79b and CD19, as well as CD79a, CD37, BCMA, TACI, Fas, CD38, and CD138, of the MCL cell line Jeko-1. Human primary T cells were effectively transduced with lentiviral constructs expressing CD19(H / L)CAR, CD79b(L / H)CAR, and CD79b(H / L)CAR (see, e.g., Figure 2) (Figure 3). Figure 4 shows the growth curves of untransduced cells and CD79b(L / H)CAR- and CD79b(H / L)CAR-transduced cells, while Figure 5 shows the level of activation of Jurkat NFAT luciferase reporter cells transduced with CD19 or CD79bCAR after overnight incubation with the indicated target cells expressing CD19 or CD79b (n=3).

[0159] In vitro studies demonstrated the cytotoxic effect of CAR-transduced T cells incubated overnight with luciferase-expressing Jeko-1 cells (Figure 6). CD19(H / L)CAR and CD79b(L / H)CAR exhibited relatively high levels of cytotoxicity. The levels of effector cytokines produced by CD19, CD79b(L / H), and CD79b(H / L)CAR after overnight incubation with Jeko-1 cells (1:1 ratio) are shown in Figure 7.

[0160] Next, we tested the CAR T cells in two in vivo animal models. 6 Jeko-1-Luc+ cells, and then 7 days later, 2 × 10 6 Figure 8B shows the timeline of the xenograft model using mice receiving intravenous injection of 10 CAR T cells. The cytotoxic effect of CAR T cells (CD79b(L / H) and CD19 CAR) compared to non-transduced cells, as measured by FLUX, is shown in Figure 8B, while the number of CAR T cells present in the blood 14 days after injection using TrueCount beads is shown in Figure 8C. Figure 9A shows the number of CAR T cells present in the blood 14 days after injection using TrueCount beads 39 days after tumor injection. 6 MCL PDX cells and 3 x 10 6The timeline for the xenograft model with mice receiving CAR T cells is shown in Figure 9B. The cytotoxic effect of CAR T cells against PDX tumor cells as measured by FLUX is shown in Figure 9B.

[0161] Figure 10 shows that bispecific CARs are activated by cells expressing both CD19 and CD79b (n=3).

[0162] conclusion CD79b CARs demonstrated high tumor clearance, cytokine production, expansion upon repeated antigen stimulation, and activation in in vitro assays. Evaluation of tumor clearance in a xenograft model of MCL across multiple healthy T cell donors demonstrated complete tumor clearance comparable to that of CD19 CARs. Furthermore, bispecific CARs were shown to be activated by cells expressing both CD19 and CD79b.

[0163] Example 2 The sequences of two CAR polypeptides of the invention that are specific for CD79b are provided and described as follows:

[0164] pMGH73 contains the following domains: CD8L, anti-CD79b L / H (separated by a linker), CD8 TM and hinge, 4-1BB, and CD3ζ, the sequences of which are shown below. TIFF0007818348000001.tif63161

[0165] The sequence of the CD8 leader is MALPVTALLLPLALLLHAARP (SEQ ID NO: 3).

[0166] The light chain sequence is The file is TIFF0007818348000002.tif15161.

[0167] The sequence of the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0168] The heavy chain sequence is The file is TIFF0007818348000003.tif20161.

[0169] The sequence of the CD8 transmembrane and hinge domain is The file is TIFF0007818348000004.tif14161.

[0170] The sequence of the 4-1BB ICD is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 8).

[0171] The sequence of CD3ζ ICD is The file is TIFF0007818348000005.tif20161.

[0172] pMGH74 contains the following domains: CD8L, anti-CD79b H / L (separated by a linker), CD8 TM and hinge, 4-1BB, and CD3ζ, the sequences of which are shown below. TIFF0007818348000006.tif62161

[0173] The sequence of the CD8 leader is MALPVTALLLPLALLLHAARP (SEQ ID NO: 3).

[0174] The heavy chain sequence is The file is TIFF0007818348000007.tif20161.

[0175] The sequence of the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0176] The light chain sequence is The file is TIFF0007818348000008.tif14161.

[0177] The sequence of the CD8 transmembrane and hinge domain is The file is TIFF0007818348000009.tif17161.

[0178] The sequence of the 4-1BB ICD is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 8).

[0179] The sequence of CD3ζ ICD is The file is TIFF0007818348000010.tif21161.

[0180] Example 3 The sequences of two CAR polypeptides of the invention, which are specific for both CD79b and CD19, are provided and described as follows:

[0181] The first CAR contains the following domains: CD8L, anti-CD79b L / H (with L and H separated by a linker), linker, anti-CD19 scFv (with a glycine-rich linker between the heavy and light chains), CD8 TM and hinge, 4-1BB, and CD3ζ, with the sequences shown below. TIFF0007818348000011.tif96161

[0182] The sequence of the CD8 leader is MALPVTALLLPLALLLHAARP (SEQ ID NO: 3).

[0183] The sequence of anti-CD79b(L / H) scFv is The file is TIFF0007818348000012.tif34161.

[0184] The sequence of the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0185] The sequence of the anti-CD19 scFv (including the glycine-rich linker separating the heavy and light chains) is: The file is TIFF0007818348000013.tif35161.

[0186] The sequence of the CD8 transmembrane and hinge domain is The file is TIFF0007818348000014.tif16161.

[0187] The sequence of the 4-1BB ICD is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 8).

[0188] The sequence of CD3ζ ICD is The file is TIFF0007818348000015.tif20161.

[0189] The second CAR contains the following domains: CD8L, anti-CD19 scFv (with a glycine-rich linker separating the heavy and light chains), linker, anti-CD79b L / H (with L and H separated by a linker), CD8 TM and hinge, 4-1BB, and CD3ζ, with the sequences shown below. TIFF0007818348000016.tif97161

[0190] The sequence of the CD8 leader is MALPVTALLLPLALLLHAARP (SEQ ID NO: 3).

[0191] The sequence of the anti-CD19 scFv (including the glycine-rich linker separating the heavy and light chains) is: The file is TIFF0007818348000017.tif35161.

[0192] The sequence of the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5).

[0193] The sequence of anti-CD79b(L / H) scFv is The file is TIFF0007818348000018.tif36161.

[0194] The sequence of the CD8 transmembrane and hinge domain is The file is TIFF0007818348000019.tif14161.

[0195] The sequence of the 4-1BB ICD is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 8).

[0196] The sequence of CD3ζ ICD is The file is TIFF0007818348000020.tif21161.

[0197] Other embodiments are within the scope of the following numbered paragraphs. 1. A chimeric antigen receptor (CAR) polypeptide comprising an extracellular domain comprising a sequence that specifically binds to CD79b. 2. The CAR polypeptide of paragraph 1, wherein the sequence that specifically binds to CD79b comprises an antigen-binding region of an antibody to CD79b. 3. The CAR polypeptide of paragraph 1 or 2, wherein the sequence that specifically binds to CD79b comprises a single chain antibody (scFv) against CD79b. 4. The CAR polypeptide of paragraph 3, wherein the scFv comprises a light chain and a heavy chain. 5. The CAR polypeptide of paragraph 4, wherein the light chain is N-terminal to the heavy chain. 6. The CAR polypeptide of paragraph 4, wherein the heavy chain is N-terminal to the light chain. 7. The CAR polypeptide of any one of paragraphs 1-6, further comprising one or more, or all of a hinge domain, a transmembrane domain, a costimulatory domain, and a signaling domain. 8. The CAR polypeptide of paragraph 7, comprising all of the hinge domain, transmembrane domain, costimulatory domain, and signaling domain. 9. The CAR polypeptide of paragraph 7 or 8, wherein the hinge domain and transmembrane domain are the CD8 hinge domain and transmembrane domain. 10. The CAR polypeptide of any one of paragraphs 7 to 9, wherein the costimulatory domain is a 4-1BB costimulatory domain. 11. The CAR polypeptide of any one of paragraphs 7 to 10, wherein the signaling domain is a CD3ζ signaling domain. 12. The CAR polypeptide of any one of paragraphs 1 to 11, comprising an anti-CD79b scFv, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. 13. The CAR polypeptide of any one of paragraphs 1 to 12, wherein the extracellular domain further comprises a sequence that specifically binds to CD19. 14. The CAR polypeptide of paragraph 13, wherein the sequence that specifically binds to CD19 comprises an antigen-binding region of an antibody to CD19. 15. The CAR polypeptide of paragraph 13 or 14, wherein the sequence that binds to CD19 comprises a single chain antibody (scFv) against CD19. 16. The CAR polypeptide of paragraph 15, wherein the scFv comprises a light chain and a heavy chain. 17. The CAR polypeptide of paragraph 16, wherein the light chain is N-terminal to the heavy chain. 18. The CAR polypeptide of paragraph 16, wherein the heavy chain is N-terminal to the light chain. 19. The CAR polypeptide of any one of paragraphs 13 to 18, wherein the sequence that binds to CD79b is N-terminal to the sequence that binds to CD19. 20. The CAR polypeptide of any one of paragraphs 13 to 18, wherein the sequence that binds to CD19 is N-terminal to the sequence that binds to CD79b. 21. The CAR polypeptide of any one of paragraphs 1 to 20, comprising the sequence of SEQ ID NO: 1, 2, 10, or 11, or a variant thereof, wherein the sequence optionally does not include the CD8 leader sequence of SEQ ID NO: 3. 22. The CAR polypeptide of any one of paragraphs 1 to 21, comprising a CD8 leader sequence of SEQ ID NO: 3, or a variant thereof. 23. The CAR polypeptide of any one of paragraphs 1 to 22, comprising the anti-CD79b light chain sequence of SEQ ID NO: 4, or a variant thereof. 24. The CAR polypeptide of any one of paragraphs 1 to 23, comprising an anti-CD79b heavy chain sequence of SEQ ID NO: 6, or a variant thereof. 25. The CAR polypeptide of any one of paragraphs 1 to 24, comprising a linker sequence of SEQ ID NO: 5, or a variant thereof. 26. The CAR polypeptide of any one of paragraphs 1 to 25, comprising a CD8 transmembrane and hinge sequence of SEQ ID NO: 7, or a variant thereof. 27. The CAR polypeptide of any one of paragraphs 1 to 26, comprising the 4-1BB ICD sequence of SEQ ID NO: 8, or a variant thereof. 28. The CAR polypeptide of any one of paragraphs 1 to 27, comprising the CD3ζ ICD sequence of SEQ ID NO: 9, or a variant thereof. 29. The CAR polypeptide of any one of paragraphs 13 to 20, comprising the anti-CD19 scFv sequence of SEQ ID NO: 13, or a variant thereof. 30. A nucleic acid molecule comprising a sequence encoding the CAR polypeptide of any one of paragraphs 1 to 29. 31. A vector comprising the nucleic acid molecule of paragraph 30. 32. A cell comprising the CAR polypeptide of any one of paragraphs 1 to 29, the nucleic acid molecule of paragraph 30, or the vector of paragraph 31. 33. The cell of paragraph 32, which is a human primary T cell. 34. A pharmaceutical composition comprising the CAR polypeptide of any one of paragraphs 1 to 29, the nucleic acid molecule of paragraph 30, the vector of paragraph 31, or the cell of paragraph 32 or 33. 35. A method of treating a subject having or at risk of developing cancer, comprising administering to the subject the pharmaceutical composition of paragraph 34. 36. The method of paragraph 35, wherein the cancer is lymphoma. 37. The method of paragraph 36, wherein the lymphoma is non-Hodgkin's lymphoma. 38. The method of paragraph 37, wherein the non-Hodgkin's lymphoma is selected from the group consisting of mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBCL), chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL). 39. A method of treating a subject experiencing a relapse of CD19-negative lymphoma after receiving CD19 CAR therapy, comprising administering to the subject the pharmaceutical composition of paragraph 34. 40. A method of generating a CAR T cell that expresses a CAR specific for CD79b, or CD79b and CD19, comprising introducing the nucleic acid molecule of paragraph 30 or the vector of paragraph 31 into a T cell. 41. The method of paragraph 40, wherein the T cells are human primary T cells.

Claims

1. 1. A chimeric antigen receptor (CAR) polypeptide comprising an extracellular domain comprising a sequence that specifically binds to CD79b, wherein the sequence that specifically binds to CD79b comprises an antigen-binding region of an antibody against CD79b, and the extracellular domain further comprises a sequence that specifically binds to CD19.

2. 2. The CAR polypeptide of claim 1, wherein the sequence that specifically binds to CD79b comprises a single chain antibody (scFv) against CD79b.

3. 3. The CAR polypeptide of claim 2, wherein the scFv comprises a light chain and a heavy chain.

4. The CAR polypeptide of claim 3, wherein the light chain is N-terminal to the heavy chain.

5. The CAR polypeptide of claim 3, wherein the heavy chain is N-terminal to the light chain.

6. 2. The CAR polypeptide of claim 1, further comprising one or more or all of a hinge domain, a transmembrane domain, a costimulatory domain, and a signaling domain.

7. The CAR polypeptide of claim 6, comprising all of the hinge domain, transmembrane domain, costimulatory domain, and signaling domain.

8. 7. The CAR polypeptide of claim 6, wherein the hinge domain and transmembrane domain are a CD8 hinge domain and transmembrane domain.

9. The CAR polypeptide of claim 6, wherein the costimulatory domain is a 4-1BB costimulatory domain.

10. The CAR polypeptide of claim 6, wherein the signaling domain is a CD3ζ signaling domain.

11. 2. The CAR polypeptide of claim 1, comprising an anti-CD79b scFv, a CD8 hinge domain and transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

12. The CAR polypeptide of claim 11, wherein the sequence that specifically binds to CD19 comprises an antigen-binding region of an antibody to CD19.

13. 12. The CAR polypeptide of claim 11, wherein the sequence that binds to CD19 comprises a single chain antibody (scFv) against CD19.

14. 14. The CAR polypeptide of claim 13, wherein the scFv comprises a light chain and a heavy chain.

15. 15. The CAR polypeptide of claim 14, wherein the light chain is N-terminal to the heavy chain.

16. 15. The CAR polypeptide of claim 14, wherein the heavy chain is N-terminal to the light chain.

17. 12. The CAR polypeptide of claim 11, wherein the sequence that binds to CD79b is N-terminal to the sequence that binds to CD19.

18. 12. The CAR polypeptide of claim 11, wherein the sequence that binds to CD19 is N-terminal to the sequence that binds to CD79b.

19. (i) the sequence of SEQ ID NO: 1, 2, 10, or 11; or (ii) a sequence at least 90% identical to the sequence of SEQ ID NO: 1, 2, 10, or 11, wherein the CDR sequences of said "sequence at least 90% identical to the sequence of SEQ ID NO: 1, 2, 10, or 11" are 100% identical to the CDR sequences of the sequence of SEQ ID NO: 1, 2, 10, or 11, and said "sequence at least 90% identical to the sequence of SEQ ID NO: 1, 2, 10, or 11" optionally does not include the CD8 leader sequence of SEQ ID NO: 3; The CAR polypeptide of claim 1, comprising:

20. 2. The CAR polypeptide of claim 1, comprising a CD8 leader sequence of SEQ ID NO: 3, or a sequence at least 90% identical to the sequence of SEQ ID NO:

3.

21. 2. The CAR polypeptide of claim 1, comprising an anti-CD79b light chain sequence of SEQ ID NO:

4.

22. 2. The CAR polypeptide of claim 1, comprising an anti-CD79b heavy chain sequence of SEQ ID NO:

6.

23. 2. The CAR polypeptide of claim 1, comprising a linker sequence of SEQ ID NO: 5, or a sequence at least 90% identical to the sequence of SEQ ID NO:

5.

24. 2. The CAR polypeptide of claim 1, comprising a CD8 transmembrane and hinge sequence of SEQ ID NO: 7, or a sequence at least 90% identical to the sequence of SEQ ID NO:

7.

25. 2. The CAR polypeptide of claim 1, comprising the 4-1BB ICD sequence of SEQ ID NO: 8, or a sequence that is at least 90% identical to the sequence of SEQ ID NO:

8.

26. 2. The CAR polypeptide of claim 1, comprising the CD3ζ ICD sequence of SEQ ID NO:9, or a sequence at least 90% identical to the sequence of SEQ ID NO:

9.

27. (i) the anti-CD19 scFv sequence of SEQ ID NO: 13, or (ii) a sequence at least 90% identical to the sequence of SEQ ID NO: 13, wherein the CDR sequences of said sequence at least 90% identical to the sequence of SEQ ID NO: 13 are 100% identical to the CDR sequences of the sequence of SEQ ID NO:

13. The CAR polypeptide of claim 11, comprising:

28. A nucleic acid molecule comprising a sequence encoding the CAR polypeptide of claim 1.

29. A vector comprising the nucleic acid molecule of claim 28.

30. A cell comprising the CAR polypeptide of claim 1.

31. 31. The cell of claim 30, which is a human primary T cell.

32. 10. A pharmaceutical composition comprising a T cell comprising the CAR polypeptide of claim 1.

33. 10. A medicament for treating a subject having or at risk of developing cancer, the medicament comprising a T cell comprising the CAR polypeptide of claim 1.

34. The pharmaceutical composition of claim 33, wherein the cancer is lymphoma.

35. The pharmaceutical composition of claim 34, wherein the lymphoma is non-Hodgkin's lymphoma.

36. 36. The pharmaceutical composition of claim 35, wherein the non-Hodgkin's lymphoma is selected from the group consisting of mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBCL), chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL).

37. A medicament for treating a subject experiencing relapse of CD19-negative lymphoma after receiving CD19 CAR therapy, the medicament comprising a T cell comprising the CAR polypeptide of claim 1.

38. 29. A method for generating CAR T cells expressing a CAR specific for CD79b, or CD79b and CD19, comprising introducing the nucleic acid molecule of claim 28.

39. 39. The method of claim 38, wherein the T cells are human primary T cells.

40. (a) an extracellular binding domain comprising a CD79b-binding domain and a CD19-binding domain; (b) CD8 hinge and transmembrane domain; (c) a 4-1BB costimulatory domain; and (d) CD3ζ signaling domain 1. A CAR polypeptide comprising:

41. (a) a CD79b-binding domain comprising: (i) a VL domain comprising the variable light chain (VL) CDR sequence of SEQ ID NO: 4 and a VH domain comprising the variable heavy chain (VH) CDR sequence of SEQ ID NO: 6; and (ii) an extracellular binding domain comprising a CD19-binding domain comprising the amino acid sequence of SEQ ID NO: 13; (b) a CD8 hinge and transmembrane domain comprising the amino acid sequence of SEQ ID NO: 7; (c) a 4-1BB costimulatory domain comprising the amino acid sequence of SEQ ID NO: 8; and (d) a CD3ζ signaling domain comprising the amino acid sequence of SEQ ID NO:

9.

1. A CAR polypeptide comprising:

42. 42. A T cell comprising the CAR polypeptide of claim 40 or 41.

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