T cell expressing chimeric antigen receptor

Abstract

To provide new approaches to treating B cell malignancies, including Mantle cell lymphoma (MCL).SOLUTION: Described herein are methods for producing and utilizing T cells comprising chimeric antigen receptors (CAR) comprising an extracellular domain that binds CD79b, or CD79b and CD19. Further, this invention is related to methods of treating cancer, plasma cell diseases or disorders, or autoimmune diseases or disorders.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The technologies described herein relate to immunotherapy. [Background technology]

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

[0003] Mantle cell lymphoma (MCL) is characterized by an invasive 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 modified to express chimeric antigen receptors (CARs) is a promising approach. + It has shown great promise as a treatment for B-cell malignancies. However, treatment failure due to antigen escape has been reported in patients receiving CD19 CAR therapy.

[0004] A new approach to treating B-cell malignancies, including MCL, would be advantageous. [Overview of the project]

[0005] CAR T cells represent a cutting-edge therapy with great potential in treating cancer. The technology has proven particularly effective against various non-solid tumors, such as leukemia, lymphoma, and myeloma. One challenge encountered in CAR T therapy design is tumor escape through deficiencies in targeted antigens or tumor-associated factors recognized by CARs. When a tumor is downregulated or otherwise loses cell surface expression of a targeted antigen or factor, the attack by CAR T cells designed to target that antigen or factor becomes inefficient. This has been observed, for example, in CAR T therapies targeting B-cell maturation antigens (BCMAs) expressed in B-cell malignancies, leukemia, lymphoma, and multiple myeloma. It has also been observed in connection with CD19-targeted CAR T therapy.

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

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

[0008] In various embodiments, the extracellular domain of the CAR polypeptide further includes a sequence that specifically binds to CD19, such as the antigen-binding region of an antibody against CD19. In certain embodiments, the sequence that binds to CD19 includes a single-chain antibody (scFv) against CD19. The scFv may optionally include a light chain and a heavy chain. The light chain may be N-terminal to the heavy chain, or the heavy chain may 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 NOs: 1, 2, 10, or 11, or a variant thereof, wherein the sequence optionally does not include the CD8 reader sequence of SEQ ID NO: 3.

[0010] In certain embodiments, the CAR polypeptide includes the CD8 reader sequence of SEQ ID NO: 3, or a variant thereof; the anti-CD79b light chain sequence of SEQ ID NO: 4, or a variant thereof; the anti-CD79b heavy chain sequence of SEQ ID NO: 6, or a variant thereof; the linker sequence of SEQ ID NO: 5, or a variant thereof; the CD8 transmembrane and hinge sequence of SEQ ID NO: 7, or a variant thereof; the 4-1BB ICD sequence of SEQ ID NO: 8, or a variant thereof; the CD3ζ ICD sequence of SEQ ID NO: 9, or a variant thereof; and / or the anti-CD19 scFv sequence of SEQ ID NO: 13, or a variant thereof. Any combination of these sequences is included in the present invention.

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

[0012] The present invention also provides a method for treating subjects who have or are at risk of developing cancer (e.g., B-cell malignancies) by administering a pharmaceutical composition as described herein to the subject. In various embodiments, the cancer is lymphoma (e.g., non-Hodgkin lymphoma, e.g., 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 a pharmaceutical composition as described herein in the treatment of a subject (e.g., a subject who has or is at risk of developing cancer as described herein).

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

[0014] The present invention further provides a method for producing CAR T cells expressing CD79b, or a CAR polypeptide specific to CD79b and CD19. The method comprises introducing a nucleic acid molecule or vector as described herein into T cells (e.g., primary T cells, e.g., human primary T cells, which may be autologous or allogeneic).

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

[0016] definition For convenience, the meanings of some terms and phrases used in this specification, the examples, and the attached claims are provided below. Unless otherwise noted, or as suggested by the context, the following terms and phrases include the meanings provided below. Since the scope of the art is not limited by the claims, the definitions are provided to aid in describing specific embodiments and are not intended to limit the claimed art. Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. If there is a clear conflict between the use of a term in the art and the definition provided herein, the definition provided herein shall prevail.

[0017] The definitions of general terms in immunology and molecular biology can be found 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 The contents of these can be found in 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 these are incorporated herein by reference in their entirety).

[0018] The terms “decrease,” “reduced,” “decrease,” or “inhibit” are all used herein to mean a statistically significant reduction. In some embodiments, “decrease,” “decrease,” “decrease,” or “inhibit” typically mean a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment or drug), and may include reductions 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, “decrease” or “inhibit” does not include complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level. Where applicable, the reduction may be a decrease to a level recognized as within the normal range in an individual without a given disorder.

[0019] The terms “increased,” “enhance,” “boost,” or “activate” are all used herein to mean an increase of a statistically significant amount. In some embodiments, the terms “increased,” “enhance,” or “activate” may mean an increase of at least 10% compared to a reference level, for example, 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 less than or equal to 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level, or any increase between 2 times and 10 times or more. In relation to a marker or symptom, “increase” is a statistically significant increase at such a level.

[0020] As used herein, "subject" means a human or an animal. Usually, the animal is a vertebrate such as a primate, a rodent, a domestic animal, or a game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, rhesus monkeys, and macaques, such as the rhesus macaque. Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic or game animals include, for example, cows, horses, pigs, deer, bison, water buffalo, felines, such as domestic cats, canines, such as dogs, foxes, wolves, avians, such as chickens, emus, ostriches, and fish, such as salmon, catfish, and trout. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual", "patient", and "subject" are used interchangeably herein.

[0021] In various embodiments, the subject is a mammal. The mammal can 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 representing animal models of diseases, such as cancer. The subject can be an adult, a child, or a neonate, and can be male or female.

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

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

[0024] A "disease" is a health condition in an animal, such as a human, where the animal is unable to maintain homeostasis and, if the disease is not resolved, 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 the animal's health is less desirable than what would be expected in the absence of the disorder. A disorder, if left untreated, does not necessarily lead to a further decline in the animal's health.

[0025] As used herein, the terms “tumor antigen” and “cancer antigen” are interchangeable to refer to antigens expressed separately by cancer cells and therefore can be used to target cancer cells. Cancer antigens are antigens that can potentially stimulate a tumor-specific immune response. Some of these antigens are encoded but not necessarily expressed by normal cells. These antigens can be characterized as those that are usually 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 mutant cell 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 may be encoded by viral genes, such as those carried on RNA and DNA oncoviruses. Numerous tumor antigens are defined in terms of several solid tumors: immune-defined MAGE1, 2, and 3; MART-1 / Melan-A, gp100, carcinoembryonic antigen (CEA), HER2, mucin (i.e., MUC-1), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). Furthermore, viral proteins such as those encoded by hepatitis B (HBV), Epstein-Barr (EBV), and human papillomavirus (HPV) have been shown to be important in the expression of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively.

[0026] As used herein, the term “chimera” refers to the product of the fusion of portions of at least two 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” may refer to a state of T cells that are sufficiently stimulated to induce detectable cell proliferation. In some embodiments, activation may refer to induced cytokine production. In other embodiments, activation may refer to detectable effector function. At a minimum, “activated T cells” as used herein are proliferative T cells.

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

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

[0030] When the term "stimulating molecule" is used herein, it refers to a molecule on a T cell that specifically binds to a homologous stimulating ligand present on an antigen-presenting cell.

[0031] When the term is used herein, “co-stimulating ligand” includes molecules on APCs that specifically bind to homologous co-stimulating molecules on T cells, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, and differentiation, in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to peptide-loaded MHC molecules. Co-stimulating ligands may include, but are not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible co-stimulating ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll-like receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands may also include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as 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 co-stimulating ligand that binds to the receptor 4-1BB (CD137) expressed on T cells. 4-1BBL is expressed on specialized 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 the 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 may refer to human 4-1BBL, including naturally occurring variants, molecules, and their alleles. In some embodiments of any aspect, for example in animal applications, 4-1BBL may refer to 4-1BBL in animals such as dogs, cats, cows, horses, and pigs. Homologs and / or orthologs of human 4-1BBL can be readily identified by those skilled in the art, for example, by using the NCBI ortholog search function or available sequence data for a given species for sequences similar to the reference 4-1BBL sequence.

[0033] A "co-stimulatory molecule" refers to a congenital binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by T cells, such as proliferation, but is not limited to. Co-stimulatory 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, may refer to one or more modifications of nucleic acids, for example, nucleic acids in the genome of an organism, that are designed for human use. In another embodiment, “modified” may refer to a change, addition, and / or deletion of a gene. “Modified cell” may refer to a cell having an added, deleted, and / or modified gene. The terms “cell” or “modified cell” and their grammatical equivalents, as used herein, may refer to a cell of human or non-human animal origin.

[0035] As used herein, the term “operably ligated” means that a first polynucleotide molecule, such as a promoter, is ligated to a second transcribable polynucleotide molecule, such as a gene of interest, where the polynucleotide molecules are arranged such that the first polynucleotide molecule influences the function of the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single adjacent polynucleotide molecule, and may or may not be adjacent. For example, if a promoter controls or mediates the transcription of a gene of interest within a cell, the promoter is operably ligated to the gene of interest.

[0036] It will be further considered that various embodiments described herein may include variants (naturally occurring or otherwise), alleles, homologs, conservedly modified variants, and / or conserved substitution variants of any of the specific polypeptides described herein. 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 low percentage of amino acids in the encoded sequence are “conservedly modified variants” if the modification results in the substitution of an amino acid with a chemically similar amino acid and preserves the desired activity of the polypeptide. Such conservedly modified variants do not, in addition, exclude polymorphic variants, interspecific homologs, and alleles consistent with this disclosure. Variants of sequences provided herein (see, for example, Examples 2 and 3) are included in the present invention.

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

[0038] Amino acids can be grouped according to their similarities in the properties of their side chains (as described in ALLehninger, 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) Non-charged: 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 affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging one member of one of these classes for another. Specific 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, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of the amino acid sequences described herein. As used herein, “functional fragment” is a fragment or segment of a peptide that is known in the art or that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described below herein. Functional fragments may include conservative substitutions of the sequences disclosed herein.

[0040] In some embodiments, the polypeptides described herein may be variants of polypeptides or molecules as described herein (see, for example, the sequences in Examples 2 and 3). In some embodiments, the variants are conservatively modified variants. Conservative substitution variants may be obtained, for example, by mutations in the native nucleotide sequence. When "variant" is used herein, it refers to a polypeptide that is substantially homologous to the native or reference polypeptide but has a different amino acid sequence from the native or reference polypeptide due to one or more deletions, insertions, or substitutions. DNA sequences encoding a variant polypeptide include sequences that encode a variant protein or fragment thereof that contains one or more nucleotide additions, deletions, or substitutions 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 mutagenicity methods are known in the art and can be applied by those skilled in the art.

[0041] The amino acid or DNA sequence of the mutant may 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 the natural or reference sequence (see, for example, the sequence in Example 2). The degree of homology (percent identity) between the natural and mutant sequences can be determined, for example, by comparing the two sequences using a freely available computer program commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0042] Modification of natural amino acid sequences can be achieved by any of several techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides having mutant sequences flanked by restriction sites that allow ligation to fragments of the natural sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, site-directed mutagenic techniques induced by oligonucleotides can be used to provide modified nucleotide sequences having specific codons altered according to the required substitution, deletion, or insertion. The 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. Patent Nos. 4,518,584 and 4,737,462 (both incorporated herein by reference). Any cysteine ​​residues not involved in maintaining the proper stereochemistry of the polypeptide can also be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, adding cysteine ​​bonds to a polypeptide can improve its stability and promote oligomer formation.

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

[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 having an amino acid length between about 2 and 60. Polypeptides as used herein typically have amino acids such as 20L amino acids, most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art may be used. One or more amino acids in a polypeptide may be modified by the addition of chemical entities, such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for conjugation, or functional differentiation. Polypeptides having a non-polypeptide moiety covalently or non-covalently associated with such entities are also considered “polypeptides.” Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, 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, may refer to the polypeptide material itself and / or sequence information (i.e., a series of letters or three-letter codes used as abbreviations for amino acid names) that biochemically characterize the polypeptide. Unless otherwise indicated, polypeptide sequences presented herein are shown in the N-terminal to C-terminal direction.

[0045] In some embodiments, nucleic acids encoding polypeptides as described herein (e.g., CAR polypeptides) are contained by a vector. In some embodiments described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably ligated to a vector. As used herein, the term “vector” refers to a nucleic acid construct designed for delivery to or introduction between different host cells. As used herein, a vector may be viral or nonviral. The term “vector” encompasses any genetic element that is capable of replication upon association with appropriate regulatory elements and can introduce a gene sequence into a cell. A vector may include, but is not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virions, and the like.

[0046] As used herein, the term “expression vector” refers to a vector that induces the expression of RNA or polypeptides from sequences ligated to transcriptional regulatory sequences on the vector. The expressed sequences are often heterogeneous to cells, though not necessarily. An expression vector may contain additional sequences, for example, an expression vector may have two replication systems, thereby enabling its maintenance in two organisms, such as human cells for expression and a prokaryotic host for cloning and amplification. The term “expression” refers to cellular processes involved in the production of RNA and proteins, and, if applicable, protein secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing. “Expression products” include RNA transcribed from genes and polypeptides obtained by translation of mRNA transcribed from genes. The term “gene” means a nucleic acid sequence that is transcribed (from DNA) to RNA in vitro or in vivo when operably ligated to an appropriate regulatory sequence. A gene may or may not include regions preceding and following the coding region, such as the 5' untranslated (5'UTR) or "leader" sequence and the 3'UTR or "trailer" sequence, 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 comprising at least one sequence of viral origin and having the ability to be packaged into a viral vector particle. Viral vectors may contain nucleic acids encoding polypeptides, as described herein, instead of non-essential viral genes. Vectors and / or particles may be used for the purpose of introducing nucleic acids into cells, either in vitro or in vivo. A great many forms of viral vectors are known in the art.

[0048] "Recombinant vector" means a vector containing a heterologous nucleic acid sequence or "transgene" capable of being expressed 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 an episome. The use of a suitable episomal vector provides a means of maintaining a target nucleotide in a subject with high copy number extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0049] As used herein, the terms “treat,” “treatment,” “treating,” or “remission” refer to therapeutic actions taken to reverse, reduce, remit, inhibit, slow, or halt the progression or severity of a condition associated with a disease or disorder, such as acute lymphoblastic leukemia or other cancer, disease, or disorder. The term “treat” includes reducing or mitigating 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 the progression of the disease is slowed or halted. That is, “treatment” includes not only improvement of symptoms or markers but also cessation, or at least slowing, of the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Favorable or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, a reduction in the severity of the disease, a stabilization (i.e., non-worsening) state of the disease, a delay or slowing of disease progression, remission or mitigation of the condition, remission (whether partial or complete), and / or a reduction in mortality (whether detectable or undetectable). The term “treatment” in relation to the 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, such as a carrier commonly used in the pharmaceutical industry. The term “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or dosage form suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other issues or complications, in proportion to a reasonable risk-benefit ratio within the bounds of sound medical judgment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticles, and / or ointment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be an artificial or modified carrier, such as a carrier in which the active ingredient is not found to exist naturally.

[0051] As used herein, the term “administer” means to place a therapeutic or pharmaceutical composition on a subject, such as those disclosed herein, by a method or route that results in at least partial delivery of the drug to the desired site. Pharmaceutical compositions containing drugs such as those disclosed herein may be administered by any suitable route that results in effective treatment on a subject.

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

[0053] Unless otherwise indicated in the experimental examples or elsewhere, all numbers used herein to express quantities of components or reaction conditions should be understood to be modified in all examples by the term “approximately.” When used in relation to percentages, the term “approximately” may mean ±1%.

[0054] As used herein, the term "including" means that, in addition to the defined element presented, other elements may also exist. The use of "including" indicates inclusion, not limitation.

[0055] The term "consisting of" refers to compositions, methods, and their respective components as described herein, excluding any elements not enumerated in the description of the embodiments.

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

[0057] The singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Similarly, the term “or” is intended to include “and” unless the context clearly indicates otherwise. Similar or equivalent methods and materials may be used in the practice or testing of the present disclosure as described herein, but preferred 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 aspect, the disclosures described herein do not relate to processes for cloning humans, processes for modifying the genetic identity of human germlines, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that are susceptible to disease and do not provide any substantial medical benefit to humans or animals, nor to animals resulting from such processes.

[0059] Other terms are defined within the scope of the following descriptions of various aspects and embodiments of the technology. [Brief explanation of the drawing]

[0060] [Figure 1] Surface expression of CD79b, CD79a, CD19, CD37, BCMA, TACI, Fas, CD38, and CD138 on the MCL cell line Jeko-1 is shown. [Figure 2] (i) shows the structure that codes for CD79b and (ii) the structure that codes for CD79b and CD19. [Figure 3] This graph shows the transduction efficiency of a specified CAR molecule in primary T cells (n=3). [Figure 4] These are growth curves for untransduced (UTD) and specified CAR-transduced cells. [Figure 5] This graph shows the level of activation in CAR-transduced cells. [Figure 6]This graph shows the in vitro cytotoxicity efficacy of CAR-transduced T cells on Jeko-1 cells (n=2). CD19(H / L)CAR - black circles; CD79b(L / H)CAR - pentagons; CD79b(H / L) - triangles; UTC - white circles. [Figure 7] This graph shows the levels of effector cytokines generated by CAR-transduced cells. [Figure 8A] This is the schedule for a xenograft model of mice that received Jeko-1 cells, followed by CAR T cells. [Figure 8B] This graph shows the cytotoxic efficacy of CAR T cells against Jeko-1 cells, as measured by FLUX. [Figure 8C] This graph shows the number of CAR T cells present in the blood 14 days after injection. [Figure 9A] This is the schedule for a mouse xenograft model that received MCL PDX cells, followed by CAR T cells. [Figure 9B] This graph shows the cytotoxic efficacy of CAR T cells against PDX tumors, as measured by FLUX. [Figure 10] This graph shows the percentage activation of bispecific CARs activated by cells expressing CD19 and CD79b (n=3). [Modes for carrying out the invention]

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

[0062] Furthermore, bispecific CARs specific to CD79b and CD19 are described. The bispecific CARs described herein are advantageous because they reduce the likelihood of tumor escape due to target antigen deficiency. In particular, CARs that bind to two different tumor-associated antigens or factors (such as CD79b and CD19) will not lose efficacy if one or the other antigen or factor is downregulated by the targeting cells. Similarly, CD79b CARs are advantageous because they can be used in the treatment of subjects who have been previously treated with CD19 CARs but are experiencing CD19-negative relapse.

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

[0064] Accordingly, one aspect of the present invention described herein relates to a CAR polypeptide comprising (a) an extracellular domain including (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 a transmembrane domain, and (c) an intracellular signaling domain. Optionally, the CAR polypeptide also comprises a co-stimulation domain as described herein.

[0065] The following are considerations for constructing and utilizing these and other aspects of the technology.

[0066] Chimeric antigen receptor The technologies described herein provide improved CARs intended for use in immunotherapy. The CARs and various improvements are discussed below.

[0067] The terms “chimeric antigen receptor,” “CAR,” or “CARs,” as used herein, refer to modified T cell receptors that transfer ligand or antigen specificity to T cells (e.g., naive T cells, central memory T cells, effector memory T cells, or combinations thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0068] CARs are configured such that a chimeric extracellular target-binding domain, which specifically binds to a target expressed on the cell surface, such as a polypeptide, is targeted in the T cell response to a construct comprising the transmembrane domain and intracellular domain (including the signaling domain) of a T cell receptor molecule. In one embodiment, the chimeric extracellular target-binding domain includes an antigen-binding domain of an antibody that specifically binds to an antigen expressed on the cell to be targeted in the T cell response. In another embodiment, the chimeric extracellular target-binding domain includes an antigen-binding domain of a first antibody that specifically binds to a first antigen expressed on the cell that is the target of the T cell response, in addition to an antigen-binding domain of a second antibody that specifically binds to a second antigen expressed on the cell that is the target of the T cell response. The properties of the intracellular signaling domain of the CAR can vary as is known in the art and as disclosed herein, but the chimeric target / antigen-binding domain makes the receptor sensitive to signaling activation when the chimeric target / antigen-binding domain binds to the target / antigen on the surface of the target cell.

[0069] Regarding intracellular signaling domains, so-called "first-generation" CARs include those that provide a CD3 zeta (CD3ζ) signal alone upon antigen binding. So-called "second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ) domains, and so-called "third-generation" CARs include those that provide multiple co-stimulation (e.g., CD28 and CD137) domains and an activation domain (e.g., CD3ζ). In various embodiments, CARs are selected to have high affinity or binding activity to a target / antigen. For example, antibody-derived target or antigen-binding domains generally have higher affinity and / or binding activity to the target antigen than in the case of naturally occurring T cell receptors. This property, combined with high specificity that allows for antibody selection, results in more 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 CAR. When expressed within T cells, CAR has the ability to re-induce the specificity and responsiveness of T cells to selected targets in a non-MHC restriction manner and to utilize the antigen-binding properties of monoclonal antibodies. Non-MHC restriction antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape.

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

[0072] Antibody reagents In various embodiments, the CAR described herein includes an antibody reagent or its antigen-binding domain as an extracellular target-binding domain.

[0073] As used herein, the term “antibody reagent” refers to a polypeptide comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binding 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 embodiments, 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 (V as used herein). H (abbreviated as V in this specification), and the light (L) chain variable region. L(abbreviated as), may be included. In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" includes 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; which is incorporated herein by reference in its entirety)), as well as full antibodies. Antibodies may have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (as well as subtypes and combinations thereof). Antibodies are derived from any source including mice, rabbits, pigs, rats, and primates (human and non-human primates), and may be primatized antibodies. Antibodies also include, e.g., midibodies, humanized antibodies, chimeric antibodies, etc. A fully human antibody binding domain can be selected using methods known to those skilled in the art, e.g., from a phage display library.

[0074] V H and V L regions can be further subdivided into hypervariable regions termed "complementary determining regions" ("CDRs") interspersed with more conserved regions termed "framework regions" ("FRs"). The ranges of the framework regions and CDRs have been previously defined (see Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. 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 entirety). Each of the V H and V L is typically composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0075] In one embodiment, the antibody or antibody reagent is not a human antibody or antibody reagent (i.e., the antibody or antibody reagent is from a mouse), but is humanized. “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 method for humanizing an antibody involves transplanting a mouse or other non-human CDR into a human antibody framework.

[0076] In one embodiment, the extracellular target binding domain of CAR is linked to an antibody, generally a monoclonal antibody, via a flexible linker peptide. H and V L The molecule contains, or essentially consists of, a single-stranded Fv (scFv) fragment created by fusion of 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 these are well known to those skilled in the art.

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

[0079] Therefore, for example, in one embodiment, the extracellular domain of the CAR polypeptide consists essentially of, or comprises, the light chain sequence of SEQ ID NO: 4 and / or the heavy chain sequence of SEQ ID NO: 6, or consists essentially of, 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 with SEQ ID NO: 4 and / or SEQ ID NO: 6. The light chain and heavy chain sequences can be in any order; for example, the light chain sequence may be at the N-terminal end of the heavy chain sequence, or the heavy chain sequence may be at the N-terminal end of the light chain sequence. In various embodiments, the light chain 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 essentially consists of, or comprises, a sequence containing (i) a light chain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a heavy chain (SEQ ID NO: 6), a sequence containing an scFv for CD79b (SEQ ID NO: 12), (ii) an optional linker (SEQ ID NO: 5), and (iii) a sequence containing an scFv for CD19 (SEQ ID NO: 13). The extracellular domain of a CAR polypeptide may optionally contain a sequence with 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 with one or more of these sequences. Furthermore, the order of the light and heavy chains in, for example, the CD79b scFv may be reversed.

[0081] In one embodiment, a CAR polypeptide generates a mutant sequence as described herein by containing one or more mutations within its coding region. Those skilled in the art can 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 or PCR techniques. Site-directed mutagenesis kits are commercially available, for example, through New England Biolabs; Ipswich, MA. Non-limiting alternative methods for introducing point mutations into the nucleic acid sequence of a gene or gene product include cassette mutagenesis or whole plasmid mutagenesis.

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

[0083] target / antigen In general, any cell surface region can be targeted by a CAR. Most often, the target is a cell surface polypeptide that is differentially or preferentially expressed on cells that are desired to be targeted for T cell response. In this context, tumor antigens or tumor-associated antigens offer 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 to CD79b, or to both CD79b and CD19, are described herein. Further non-limiting examples of 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 for one or more of these antigens may be used in combination with CARs for CD79b, or CD79b and CD19, as described herein, if deemed appropriate by those skilled in the art.

[0084] Hinge and TM domain Each CAR described herein may 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 enables the binding site and the separation and flexibility of the T cell membrane. The length of the flexible hinge also allows for better binding to relatively isolated epitopes, and longer hinge regions, for example, allow for optimal binding. Those skilled in the art will be able to determine the 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 described herein includes 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 a generally hydrophobic region of a CAR that spans the cell’s plasma membrane. A TM domain may be a transmembrane region or fragment thereof of a transmembrane protein (e.g., type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Specific examples are presented herein and used in the examples, while other transmembrane domains will be obvious to those skilled in the art and may be used in connection with alternative embodiments of the art. A selected transmembrane region or fragment thereof will preferably not interfere with the intended function of the CAR. When used in connection with a transmembrane domain of a protein or polypeptide, “fragment thereof” refers to a portion of a transmembrane domain sufficient to anchor or adhere 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 CD8 or 4-1BB transmembrane domain. In an alternative embodiment of any embodiment, the transmembrane domain or fragment thereof of the CAR described herein comprises 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 (CD1a, 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 It includes 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 preferentially found 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 consists of α (CD8a) and β (CD8b) chains. CD8a sequences are known in several species, including, for example, human CD8a (NCBI Gene ID: 925), polypeptide (NCBI Ref Seq NP_001139345.1), and mRNA (e.g., NCBI Ref Seq NM_000002.12). CD8 may refer to human CD8, including naturally occurring variants, molecules, and their alleles. In some embodiments of any aspect, for example in veterinary applications, CD8 may refer to CD8 from, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human CD8 can be easily identified for such species by a person skilled in the art, for example, by using the NCBI ortholog search function, or by searching the available sequence data for a given species for sequences similar to the reference CD8 sequence.

[0090] In one embodiment, the CD8 hinge and the transmembrane sequence include the sequence of Sequence ID No. 7; or include 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 with respect to the sequence of Sequence ID No. 7.

[0091] Simultaneous stimulation domain Each CAR described herein may optionally contain one or more intracellular domains of a co-stimulating molecule, or co-stimulating domains. As used herein, the term “co-stimulating domain” refers to the intracellular signaling domain of a co-stimulating molecule. A co-stimulating molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for the efficient activation and function of T lymphocytes upon binding to an antigen. Exemplary examples of such co-stimulating 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] Therefore, in one embodiment, the CAR polypeptide further comprises an intracellular domain. As used herein, “intracellular domain” refers to a sequence that is entirely contained within a cell. In one embodiment, the intracellular domain refers to the intracellular domain of a receptor. The intracellular domain can interact with the inside of the cell. In relation to the intracellular domain of a receptor, the intracellular domain can function to relay signals being transmitted. The intracellular domain of a receptor may 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 contains the sequence of Sequence ID No. 8; or contains Sequence ID No. 8 with 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.

[0094] Intracellular signal transduction domains CARs as described herein include an intracellular signaling domain. The “intracellular signaling domain” refers to a portion of a CAR polypeptide that is involved in transducing the message of effective CAR binding to a target antigen into the interior of immune effector cells and inducing effector cell function, such as activation, cytokine production, proliferation and cytotoxic activity, such as the release of cytotoxic factors to CAR-bound target cells, or other cellular responses induced after antigen binding to an extracellular CAR domain.

[0095] CD3 is a T-cell co-receptor that promotes T-lymphocyte activation when it associates with appropriate co-stimuli (e.g., binding of co-stimulatory molecules). The CD3 complex consists of four distinct chains; mammalian CD3 consists of a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with molecules known as the T-cell receptor (TCR) and CD3ζ to generate activation signals in T lymphocytes. The complete TCR complex includes the TCR, CD3ζ, and the complete CD3 complex.

[0096] In some embodiments of any aspect, the CAR polypeptide described herein comprises an intracellular signaling domain including an activation motif based on the immune receptor tyrosine from CD3 zeta (CD3ζ), namely ITAM. In some embodiments of any aspect, ITAM comprises three motifs of ITAM from CD3ζ (ITAM3). In some embodiments of any aspect, the three motifs of ITAM from CD3ζ are mutated.

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

[0098] Those skilled in the art can introduce mutations into the nucleic acid sequence of a gene or gene product, such as an ITAM, using standard techniques. For example, point mutations can be introduced via site-directed mutagenesis or PCR techniques. Site-directed mutagenesis kits are commercially available, for example, through New England Biolabs; Ipswich, MA. Non-limiting alternative methods for introducing point mutations into the nucleic acid sequence of a gene or gene product include cassette mutagenesis or whole plasmid mutagenesis.

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

[0100] In one embodiment, the CD3ζ intracellular signaling sequence corresponds to the amino acid sequence of SEQ ID NO: 9; or contains the sequence of SEQ ID NO: 9; or contains 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 with respect to the sequence of SEQ ID NO: 9.

[0101] More detailed descriptions 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 these is incorporated herein by reference in its entirety).

[0102] In one embodiment, the CAR polypeptide further comprises a CD8 leader sequence. As used herein, “leader sequence” refers to the region of mRNA immediately upstream of the start codon, which is also known as leader RNA. The leader sequence may be important for the regulation of transcript translation.

[0103] In one embodiment, the CD8 reader sequence corresponds to the amino acid sequence of SEQ ID NO: 3; or includes SEQ ID NO: 3; or includes 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 with respect 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 of approximately 2 to 100 amino acids in length that links together any of the domains / regions of the CAR as described herein. In some embodiments, the linker may include, or be composed of, mobile residues such as glycine and serine, so that the adjacent protein domains move freely in relation to each other. Longer linkers may be used when it is desirable to ensure that the two adjacent domains do not sterically interfere with each other. The linker may be cleavable or incleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or their functional equivalents and combinations thereof. In one embodiment, the linker region is T2A derived from the Zosea asigna virus. Non-limiting examples of linkers include linkers derived from the Zosea asigna virus and linkers derived from internal ribosome entry sites (IRES) sequences.

[0105] In one embodiment, the CAR described herein further comprises a reporter molecule to enable, for example, non-invasive imaging (e.g., positron emission tomography PET scans). In a bispecific CAR comprising a reporter molecule, the first extracellular binding domain and the second extracellular binding domain may comprise different or the same reporter molecule. In bispecific CAR T cells, the first CAR and the second CAR may express different or the same reporter molecule. In another embodiment, the CAR described herein may be alone or with a substrate or chemical (e.g., 9-[4-[ 18 F]Fluoro-3-(hydroxymethyl)butyl]guanine([ 18 The CAR further comprises a reporter molecule (e.g., hygromycin phosphotransferase (hph)) that can be imaged in combination with F[FHBG). In another embodiment, the CAR as described herein is a nanoparticle (e.g.,) that can be easily imaged using non-invasive techniques. 64 Cu 2+The solution further comprises gold nanoparticles (GNPs) functionalized with [unspecified material]. Labeling of CAR T cells for non-invasive imaging has been 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 (both of which are incorporated herein by reference).

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

[0107] Another aspect of the present 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 with a sequence selected from sequence numbers 1, 2, 10, and 11 (optionally excluding the CD8 reader sequence of sequence number 3). Another aspect of the present invention relates to a CAR polypeptide comprising a sequence selected from sequence numbers 1, 2, 10, and 11 (optionally excluding the CD8 reader sequence of sequence number 3).

[0108] Another aspect of the present invention described herein relates to a polypeptide complex comprising two or more of the CAR polypeptides described herein (for example, 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). In one embodiment, the polypeptide complex comprises three of the CAR polypeptides described herein.

[0109] Another aspect of the present invention relates to mammalian cells comprising any of the CAR polypeptides described herein; or nucleic acids encoding any of the CAR polypeptides described herein. In one embodiment, the mammalian cells comprise an antibody, an antibody reagent, its antigen-binding moiety, or any of the CAR polypeptides described herein, or nucleic acids encoding such an antibody, an antibody reagent, its antigen-binding moiety, or any of the CAR polypeptides described herein. The mammalian cells or tissues may be derived from humans, primates, hamsters, rabbits, rodents, cows, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells may be used. In a preferred embodiment of any aspect, the mammalian cells are human.

[0110] In one embodiment, the cells are T cells. In alternative embodiments of any aspect, the cells are immune cells. As used herein, “immune cells” refers to cells that play a role in the immune response. Immune cells are hematopoietic in origin and include lymphocytes, e.g., B cells and T cells; natural killer cells; myeloid cells, e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cells are T cells; NK cells; NKT cells; lymphocytes, e.g., B cells and T cells; and myeloid cells, e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

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

[0112] When used herein, "cancer" may refer to excessive cell proliferation where a reduction in characteristic traits (normal cellular control) results in uncontrolled growth, lack of differentiation, local tissue invasion, and metastasis, and may include, for example, lymphoma, leukemia, multiple myeloma, or solid tumors. In specific cases, 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, and mucosa-associated lymphoid tissue lymphoma (MALT). Examples include lymphoplasmacytic lymphoma, nodular marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granuloma, primary central nervous system lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, and unclassifiable B-cell lymphoma.

[0113] Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In one embodiment, cancer is ALL or CLL. Non-limiting examples of solid tumors include adrenocortical tumors, hydatidiform soft tissue sarcomas, carcinomas, chondrosarcomas, colorectal cancers, tendonoid tumors, fibrous round cell tumors, endocrine tumors, yolk sac tumors, epithelioid hemangioendotheliomas, Ewing's sarcoma, germ cell tumors (solid tumors), giant cell tumors of bone and soft tissue, hepatoblastoma, hepatocellular carcinoma, melanoma, renal tumors, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, paravertebral sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms' tumor. Solid tumors can be found in bone, muscle, or organs and may be sarcomas or carcinomas. Any aspect of the invention described herein is considered to be usable for treating any type of cancer, including cancers not enumerated herein. As used herein, the term “tumor” refers to, for example, the abnormal proliferation of cells or tissues of malignant or benign type.

[0114] As used herein, “autoimmune disease or disorder” is characterized by the inability of the body’s own immune system to distinguish between foreign cells and healthy cells. As a result, the body’s own immune system targets its own healthy cells as 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, e.g., allergic asthma, atopic dermatitis, and contact hypersensitivity, rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, Graves’ disease (hyperactive thyroid), Hashimoto’s thyroiditis (hypoactive thyroid), chronic graft-versus-host disease, hemophilia with antibodies against coagulation factors, celiac disease, Crohn’s disease, and ulcerative colitis, Guillain-Barré syndrome, primary biliary sclerosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, and Raynos' disease. Examples include: scleroderma, Sjögren's syndrome, Goodpasteur'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, ocular clonus-myoclonus syndrome, optic neuritis, Oud's thyroiditis, pemphigus, pernicious anemia, polyarthritis in dogs, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, and fibromyalgia (FM).

[0115] In one embodiment, mammalian cells are obtained in patients with an immune system disorder resulting in abnormally low activity in the immune system or immunodeficiency, which inhibits their own ability to fight foreign cells (i.e., viral or bacterial cells).

[0116] Plasma cells are white blood cells produced from B lymphocytes that function to produce and release antibodies necessary to fight infection. As used herein, “plasmacytotoxicity or disease” is characterized by the abnormal proliferation of plasma cells. Abnormal plasma cells have the ability to “push out” healthy plasma cells, thereby reducing their ability to fight exogenous targets, such as viruses or bacterial cells. Non-limiting examples of plasmacytotoxicity include amyloidosis, Waldenström macroglobulinemia, sclerosing myeloma (POEMS syndrome), monoclonal immunoglobulinemia of unknown significance (MGUS), and plasma cell myeloma.

[0117] T cells can be obtained from subjects using standard techniques known in the art; for example, T cells are isolated from peripheral blood collected from patients.

[0118] Cells, such as T cells, may be modified to contain any of the CAR polypeptides described herein; or nucleic acids 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 cells using standard infection techniques.

[0119] Retroviruses, such as lentiviruses, provide a convenient platform for delivering genes, i.e., nucleic acid sequences encoding a target chimeric gene. Selected nucleic acid sequences can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant viruses can then be isolated, for example, in vitro or in vitro, and delivered to cells. Retroviral systems are well known in the art and are described, for example, in U.S. Patent 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 (both of which are incorporated herein by reference). Lentiviral systems for efficient DNA delivery can be purchased from OriGene; Rockville, MD. In other embodiments, any CAR polypeptide 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 of transfection or electroporation are known in the art.

[0120] The efficient expression of any of the CAR polypeptides described herein can be evaluated using standard assays that detect the gene product of mRNA, DNA, or nucleic acid encoding the CAR. Examples include RT-PCR, FACS, Northern blotting, Western blotting, ELISA, or immunohistochemistry.

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

[0122] One aspect of the present invention described herein is a method for treating cancer, plasma cell dysfunction, amyloidosis, or autoimmune disease in a subject, comprising: modifying T cells to contain one of the CAR polypeptides described herein on the surface of the T cells; and administering the modified T cells to the subject.

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

[0124] In one embodiment, the method further includes activating or stimulating CAR-T cells before administering them to the target, for example, according to a method described elsewhere in this specification.

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

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

[0127] The compositions described herein may be administered to subjects having or diagnosed with a condition. In some embodiments, the methods described herein include administering an effective dose of activated CAR T cells described herein to a subject to alleviate the symptoms of the condition. As used herein, “alleviating the symptoms of the condition” means achieving remission of any condition or symptom associated with the condition. When compared to a corresponding untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or higher, 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 topically. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered to a tumor site.

[0128] As used herein, the term “effective dose” refers to the amount of activated CAR T cells necessary to alleviate at least one symptom of a disease or disorder, and relates to an amount sufficient to produce the desired effect of a cell preparation or composition. Thus, the term “therapeutic effective dose” refers to an amount of activated CAR T cells sufficient to produce a specific anti-condition effect when administered to a typical subject. As used herein, an effective dose also includes, under various circumstances, an amount sufficient to delay the onset of disease symptoms, alter the course of symptoms / disease (e.g., slow the progression of the condition), or reverse the symptoms of a condition. Therefore, it is generally not feasible to specify an exact “effective dose.” However, in certain specific cases, an appropriate “effective dose” can be determined by those skilled in the art, solely through conventional experimentation.

[0129] The effective dose, toxicity, and therapeutic effect can be evaluated in cell cultures or experimental animals using standard medical procedures. The dose may vary depending on the dosage form used and the route of administration utilized. The dose-to-toxicity ratio is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compositions and methods exhibiting a large therapeutic index are preferred. The therapeutic effective dose may first be evaluated from a cell culture assay. The dose may also be formulated in an animal model to achieve a circulating plasma concentration range including IC50 (i.e., the concentration of activated CAR T cells that achieve maximum half-dose inhibition of symptoms), as determined in cell cultures or appropriate animal models. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dose can be monitored, in particular, by suitable bioassays, such as those for bone marrow examination. The dose is determined by the physician and may be adjusted as needed to suit the observed therapeutic effect.

[0130] In one aspect of the present invention, the techniques described herein relate to a pharmaceutical composition comprising activated CAR T cells as described herein and, optionally, a pharmaceutically acceptable carrier. The active component of the pharmaceutical composition comprises at least activated CAR T cells as described herein. In some embodiments, the active component of the pharmaceutical composition consists essentially of activated CAR T cells as described herein. In some embodiments, the active component of the pharmaceutical composition consists of activated CAR T cells as described herein. A pharmaceutically acceptable carrier for cell-based therapeutic formulations includes physiological 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, the pharmaceutical composition containing activated CAR T cells as described herein may be in parenteral administration form. Since parenteral administration typically bypasses the patient's natural defenses against contaminants, components other than the CAR T cells themselves may be sterile or sterilized before administration to the patient. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, dried products readily soluble or suspended in pharmaceutically acceptable media for injection, injectable suspensions, and emulsions. Any of these may be added to the activated CAR T cell preparation before administration.

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

[0133] dose When the term is used herein, “unit dosage form” refers to a suitable single dose. For example, a unit dosage form may be the amount of therapeutic agent administered by a delivery device, such as a syringe or intravenous infusion bag. In one embodiment, a unit dosage form is administered as 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 monotherapy, i.e., no other therapeutic agents for the condition are administered to the subject simultaneously.

[0135] The pharmaceutical compositions containing T cells described herein generally have 10 4 ~10 9Cells / kg body weight, in some cases 10 5 ~10 6 The cells may be administered in doses of cells / kg body weight (including all integer values ​​within that range). If necessary, the T cell composition may also be administered multiple times at these doses. The cells may be administered using infusion techniques commonly known in immunotherapy (see, for example, 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 target, then subsequently collect blood again (or perform apheresis), activate the T cells therefrom as described herein, and reinject these activated and expanded T cells into the patient. This process may be performed multiple times at intervals of several weeks. In certain embodiments, T cells may be activated from 10cc to 400cc of blood sample. In certain embodiments, T cells may be activated from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc of blood sample.

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

[0138] In certain exemplary embodiments, the subject may undergo leukocyte apheresis, where leukocytes are collected, concentrated, or ex vivo depleted, and the target cells, such as T cells, are selected and / or isolated. These T cell isolates may be expanded by contact with artificial antigen-presenting cells (aAPCs), such as aAPCs expressing anti-CD28 and anti-CD3 CDR, and processed to produce CAR T cells by introducing one or more CAR constructs of the present invention. Subjects requiring this may then undergo standard treatment with high-dose chemotherapy, followed by peripheral blood stem cell transplantation. Post-transplantation or concurrently with transplantation, the subject may receive infusion of the expanded CAR T cells. In one embodiment, the expanded cells are administered pre- or post-operatively.

[0139] In some embodiments, lymphocyte depletion is performed on a subject before administering one or more CAR T cells as described herein. In such embodiments, lymphocyte depletion may include administering one or more of melphalan, cytoxane, cyclophosphamide, and fludarabine.

[0140] The dosage of the above-mentioned therapeutic agents to be administered to the patient will vary depending on the patient's condition during treatment and the specific characteristics of the treatment recipient. Scaling of human doses may be carried out in accordance with established practices in the art.

[0141] In some embodiments, a single treatment plan is required. In others, one or more subsequent doses or treatment plans may be implemented. For example, after treatment every two weeks for three months, treatment may be repeated once every month, six months, or year or longer. In some embodiments, no additional treatment is administered after the initial treatment.

[0142] The dosage of compositions as described herein is determined by a physician and may be adjusted as needed to suit the observed effects of the treatment. It is typical for an experienced clinician to monitor the patient to determine when the treatment is producing a therapeutic effect, in relation to the duration and frequency of treatment, and to determine whether further cells should be administered, whether treatment should be interrupted, whether treatment should be resumed, or whether any changes should be made outside of the treatment plan. 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 patient's age, condition, and sex and may be determined by those skilled in the art. The dosage may also be adjusted by the individual physician if any complications arise.

[0143] Combination therapy The activated CAR T cells described herein may be used in combination with other known drugs and therapies. In one embodiment, the subject is subjected to anti-CD19 therapy and anti-CD79b therapy. In another embodiment, the subject is further subjected to anti-BCMA therapy. When used herein, “combination” administration means that two (or more) different therapeutic agents are delivered to the subject during the course of the subject’s suffering due to the disorder, for example, two or more therapeutic agents are delivered from the time the subject is diagnosed with the disorder and before the disorder is cured or eliminated, or before treatment is completed for other reasons. In some embodiments, there is an overlap in administration, with the delivery of one therapeutic agent still taking place when the second delivery begins. This is sometimes referred to herein as “simultaneous” or “combination delivery.” In other embodiments, the delivery of one therapeutic agent is completed before the delivery of the other therapeutic agent begins. In some embodiments of either case, the therapeutic agents are more effective due to combination administration. For example, the second therapeutic agent may be more effective, for instance, an equivalent effect may be observed with the reduction of the second therapeutic agent, or the second therapeutic agent may reduce symptoms more broadly than would be observed if the second therapeutic agent were administered in the absence of the first therapeutic agent, or a similar situation may be observed in the presence of the first therapeutic agent. In some embodiments, delivery is made such that the degree of reduction in symptoms, or other parameters relating to impairment, is greater than that observed if 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 more than additive. Delivery may be made such that the effect of the first therapeutic agent being delivered is still detectable when the second therapeutic agent is delivered. The activated CAR T cells and at least one additional therapeutic agent described herein may be administered simultaneously, in the same composition, in separate compositions, or sequentially. In sequential administration, the cells expressing the CAR described herein may be administered first, the additional agent second, or the order of administration may be reversed. CAR T therapy and / or other therapeutic drugs, procedures, or forms of treatment may be administered during periods of active disorder or during periods of remission or less active disorder. CAR T therapy may be administered before, concurrently with, after, or during remission of another treatment.

[0144] When administered in combination, activated CAR T cells and additional agents (e.g., a second or third agent), or all of them, may be administered in amounts higher, lower, or the same as the amounts or doses of each agent used individually, for example, as monotherapy. In certain embodiments, the amount or dose of activated CAR T cells, additional agents (e.g., a second or third agent), or all of them is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose of each agent used individually. In other embodiments, the amount or dose of activated CAR T cells, additional agents (e.g., a second or third agent), or all of them that produce the desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose 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 plan that combines 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, mycophenolate, steroids, FR901228, cytokines, or peptide vaccines, for example, as 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 used in combination with chemotherapeutic agents. 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), and antimetabolites. Examples include antiprosthetics (e.g., folate antagonists, 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., acrasinomycin A, gliotoxin, or bortezomib), and immunomodulators, such as thalidomide or thalidomide derivatives (e.g., lenalidomide). Common chemotherapeutic agents 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®), and cisplatin (Platin®). nol(registered trademark), cladribine (Leustatin(registered trademark)), cyclophosphamide (Cytoxan(registered trademark) or Neosar(registered trademark)), cytarabine, cytosine arabinoside (Cytosar-U(registered trademark)), cytarabine liposomal injection (DepoCyt(registered trademark)), dacarbazine (DTIC-Dome(registered trademark)), dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine(registered trademark)), daunorubicin tritate liposomal injection (DaunoXome(registered trademark)), dexamethasone, docetaxel (Taxotere(registered trademark)),Doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacytibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol) Includes registered trademarks ()), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), Phoenix (Yttrium90 / MX-DTPA), pentostatin, polyfeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). Exemplary alkylating agents include, but are not limited to, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®)Uramustin (registered trademark), Uramustine (registered trademark), Chlormethine (Mustargen (registered trademark)), Cyclophosphamide (Cytoxan (registered trademark), Neosar (registered trademark), Clafen (registered trademark), Endoxan (registered trademark), Procytox (registered trademark), Revimmune (trademark)), Ifosfamide (Mitoxana (registered trademark)), Melphalan (Alkeran (registered trademark)), Chlorambucil (Leukeran (registered trademark)), Pipobroman (Amedel (registered trademark), Vercyte (registered trademark)), Triethylenemel Examples include methyl (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozosin (Zanosar®), and dacarbazine (DTIC-Dome®). As additional exemplary alkylating agents, but not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (actinomycin-D, also known as Cosmegen®); melphalan (L-PAM, L-sarcolicin, and phenylalanine mustard, also known as Alkeran®); altoretamine (hexamethylmelamine (HMM), also known as Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (CCNU, also known as CeeNU®); cisplatin (CDDP,Platinol® and Platinol®-AQ (also known as 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 Examples include ane(registered trademark); mechloretamine (also known as nitrogen mustard, mustine, and mechloretamine hydrochloride, Mustargen(registered trademark)); streptozocin (Zanosar(registered trademark)); thiotepa (also known as thiophosphoamide, TESPA, and TSPA, Thioplex(registered trademark)); cyclophosphamide (Endoxan(registered trademark), Cytoxan(registered trademark), Neosar(registered trademark), Procytox(registered trademark), Revimmune(registered trademark)); and bendamustine HC1 (Treanda(registered trademark)). Examples of mTOR inhibitors include, for example, temsirolimus; ridafololimus (formerly known as deferolimus), (lR,2R,45)-4-[(2R)-2[(1R,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-II, 36-Dioxa-4-Azatricyclo[30.3.1.04'9]Hexatriaconta-16,24,26,28-Tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate is known, also known as AP23573 and MK8669, and is described in the PCT International Publication No. 03 / 064383); everolimus (Afinitor® or RADOOl); rapamycin (AY22989,Sirolimus®; simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-bis[(35,)-3-methylmorpholine-4-yl]pyrido[2,3-(i)]pyrimidine-7-yl}-2-methoxyphenyl)methanol (AZD 8055); 2-amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methylpyrido[2,3-JJpyrimidine-7(8H)-one (PF 04691502, CAS 1013101-36-4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-l-ben Examples of immunomodulatory agents include zopyran-2-yl)morpholinium-4-yl]methoxybutyl]-L-arginylglycyl-La-aspartyl L-serine, intramolecular salt (SF1126, CAS936487-67-1), and XL765. Exemplary immunomodulatory agents include, for example, aftuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimide (CC4047); and IRX-2 (a mixture of human cytokines including interleukin-1, interleukin-2, and interferon-γ, CAS951209-71-5, IRX Examples of anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposome (daunorubicin crate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence®); idarubicin (Idamycin®)Examples of vinca alkaloids include Idamycin PFS®, Mutamycin®, Geldanamycin, Herbimycin, Rabidomycin, and deacetylravidomycin. Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine®); vinblastine (also known as vinblastine sulfate, vincaloicoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). Exemplary proteosome inhibitors include bortezomib (Velcade®), carfilzomib (PX-1, 71-007, (5)-4-methyl-N-((5)-l-(((5)-4-methyl-l-((R)-2-methyloxiran-2-yl)-l-oxopentan-2-yl)amino)-l-oxo-3-phenylpropane-2-yl)-2-((5,)-2-(2-morpholinoacetamide)-4-phenylbutanamide)-pentanamide); marizomib (NPT0052) Examples include ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(llS')-2-[(2R)-2-methyl-2-oxyranyl]-2-oxo-l-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0147] Those skilled in the art can easily identify the chemotherapeutic agents to be used (e.g., 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 molecules that reduce the activity and / or levels of molecules that target GITR and / or modulate GITR function, molecules that reduce the Treg cell population, mTOR inhibitors, GITR agonists, kinase inhibitors, non-receptor tyrosine kinase inhibitors, CDK4 inhibitors, and / or BTK inhibitors.

[0149] Effectiveness For example, the effectiveness of activated CAR T cells in treating the conditions described herein, or in inducing a response such as those described herein (e.g., a reduction in cancer cells), can be determined by a skilled clinician. However, a treatment is considered "effective" when, if the terminology is used herein and one or more signs or symptoms of the conditions described herein are modified in a favorable manner, other clinically recognized symptoms are improved or further reduced, or a desired response is induced (e.g., at least 10% after treatment according to the method herein). Effectiveness can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence of the condition being treated according to the method herein, or any other measurable appropriate parameter. Treatment according to the method herein can reduce the level of markers or symptoms of the condition by, for example, 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] Effectiveness can also be assessed by whether the individual's condition does not worsen as a result of hospitalization, or by whether there is a need for further medical intervention (i.e., cessation of disease progression). Methods for measuring these indicators are known to those skilled in the art and / or are described herein.

[0151] Treatment includes any treatment of a disease in an individual or animal (including, in some non-limiting examples, humans or animals), and includes (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 recovery of symptoms. An effective dose in the treatment of a disease means an amount sufficient to produce an effective treatment of the disease, as defined herein, when administered to a subject in need of it. The effectiveness of a drug may be determined by evaluating physical indicators of the condition or the desired response. Monitoring the effectiveness of administration and / or treatment by measuring any one of such parameters or any combination of parameters is well within the capabilities of those skilled in the art. The effectiveness of a given method may be evaluated in animal models of the conditions described herein, e.g., in the treatment of lymphoma as described herein. When using experimental animal models, the effectiveness of a treatment is demonstrated when a statistically significant change in a marker is observed.

[0152] All patents and other publications cited throughout this application, such as references, granted patents, published patent applications, and concurrently pending patent applications, are explicitly incorporated herein by reference for the purpose of explaining and disclosing methods described in such publications, for example, which may be used in connection with the technology described herein. These publications are provided solely to be prior to the filing date of this application. In this regard, the inventors should not be construed as acknowledging that they are not authorized to precede such disclosures for the reasons of prior invention or for any other reason. All statements regarding dates or expressions relating to the contents of these documents are based on information available to the applicant and do not in any way constitute an acknowledgment of the accuracy of the dates or contents of these documents.

[0153] The descriptions of embodiments in this disclosure are not intended to be strict or limit to the exact form in which the disclosure is disclosed. While specific embodiments and examples in this disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be understood by those skilled in the art. For example, while method steps or functions are presented in a given order, in alternative embodiments the functions may be performed in a different order or substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods as needed. Further embodiments can be presented by combining the various embodiments described herein. The aspects of this disclosure can be modified as needed, and further embodiments can be presented by utilizing the above-mentioned references and compositions, functions and concepts of the application. Furthermore, by considering biological and functional equivalence, some modifications can be made to the protein structure without affecting the biological or chemical action in terms of type or quantity. Thanks to the detailed description, these and other modifications can be made to this disclosure. Not all such modifications are intended to be included within the scope of the appended claims.

[0154] Specific elements of any of the embodiments described above may be combined with or substituted for elements in other embodiments. Furthermore, while advantages related to specific embodiments of this disclosure are described in relation to those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments are required to demonstrate that such advantages are within the scope of this disclosure.

[0155] The techniques described herein are further illustrated by the following examples, but should not be construed as further limiting them. [Examples]

[0156] Example 1 We designed a carcinogenic agent (CAR) that targets CD79b as part of a B-cell receptor (BCR) complex. Using this approach, we are expanding treatment options for lymphoma patients, such as those with relapsed CD19-negative disease after CD19 CAR therapy. We also designed a bispecific CAR that targets both CD79b and CD19.

[0157] Materials and methods We constructed scFv-based anti-CD79b CAR constructs fused to 4-1BB and CD3ζ via the CD8 hinge and transmembrane domain. Human primary T cells were transduced with either the CD79b or CD19 CAR via lentiviral. Cytotoxicity, T cell activation, and cytokine production against the MCL cell line Jeko-1 were evaluated. Furthermore, the cytotoxic effect of the CD79b CAR was compared to that of the CD19 CAR in xenograft experiments in mice with Jeko-1 tumors and mice with MCL PDX tumors.

[0158] result Figure 1 shows the results of characterizing the cell surface expression of CD79b and CD19, as well as CD79a, CD37, BCMA, TACI, Fas, CD38, and CD138 in the MCL cell line Jeko-1. Human primary T cells were successfully 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 CD79b CAR after overnight incubation with designated target cells expressing CD19 or CD79b (n=3).

[0159] In vitro studies demonstrated the cytotoxic effects of CAR-transduced T cells incubated overnight with luciferase-expressing Jeko-1 cells (Figure 6). CD19(H / L)CAR and CD79b(L / H)CAR showed relatively high levels of cytotoxicity. Figure 7 shows 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).

[0160] Next, we tested CAR T cells in two in vivo animal models. Figure 8A shows 10 6 One Jeko-1-Luc+ cell, then 7 days later, 2 × 10 6 The schedule for a xenograft model using mice that received intravenous injection of CAR T cells is shown. Figure 8B shows the cytotoxic effect of CAR T cells (CD79b(L / H) and CD19 CAR) compared to untransduced cells, measured by FLUX, while Figure 8C shows the number of CAR T cells present in the blood 14 days after injection using TrueCount beads. Figure 9A shows 10 cells 39 days after tumor injection. 6 Individual MCL PDX cells and 3 × 10⁶ cells 6The schedule for a xenograft model using mice that received individual CAR T cells is shown. Figure 9B shows the cytotoxic effect of CAR T cells on PDX tumor cells, as measured by FLUX.

[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. Tumor clearance evaluation in a xenograft model of MCL showed that CD79b CARs achieved complete tumor clearance comparable to CD19 CARs through multiple healthy T-cell donors. Furthermore, it was shown that bispecific CARs are activated by cells expressing both CD19 and CD79b.

[0163] Example 2 The sequences of the two CAR polypeptides of the present invention, which are specific to CD79b, are provided and described as follows:

[0164] pMGH73 contains the following domains: CD8L, anti-CD79b L / H (separated by a linker), CD8™ and hinge, 4-1BB, and CD3ζ, and its sequence is as shown below. TIFF2023123452000002.tif63161

[0165] The sequence of the CD8 reader is MALPVTALLLPLALLLHAARP (sequence number 3).

[0166] The arrangement of the light chains is, The filename is TIFF2023123452000003.tif15161.

[0167] The linker sequence is GGGGSGGGGSGGGGSGGGGS (sequence number 5).

[0168] The arrangement of the heavy chains is, The filename is TIFF2023123452000004.tif20161.

[0169] The sequences of the CD8 transmembrane and hinge domains are: The filename is TIFF2023123452000005.tif14161.

[0170] The sequence of 4-1BB ICD is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (sequence number 8).

[0171] The arrangement of CD3ζ ICD is, The filename is TIFF2023123452000006.tif20161.

[0172] pMGH74 contains the following domains: CD8L, anti-CD79b H / L (separated by a linker), CD8™ and hinge, 4-1BB, and CD3ζ, and its sequence is as shown below. TIFF2023123452000007.tif62161

[0173] The sequence of the CD8 reader is MALPVTALLLPLALLLHAARP (sequence number 3).

[0174] The arrangement of the heavy chains is, The filename is TIFF2023123452000008.tif20161.

[0175] The linker sequence is GGGGSGGGGSGGGGSGGGGS (sequence number 5).

[0176] The arrangement of the light chains is, The filename is TIFF2023123452000009.tif14161.

[0177] The sequences of the CD8 transmembrane and hinge domains are: The filename is TIFF2023123452000010.tif17161.

[0178] The sequence of 4-1BB ICD is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (sequence number 8).

[0179] The arrangement of CD3ζ ICD is, The filename is TIFF2023123452000011.tif21161.

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

[0181] The first CAR comprises the following domains: CD8L, anti-CD79b L / H (having L and H separated by a linker), linker, anti-CD19 scFv (containing a glycine-rich linker between the heavy and light chains), CD8™ and hinge, 4-1BB, and CD3ζ, and its sequence is as shown below. TIFF2023123452000012.tif96161

[0182] The sequence of the CD8 reader is MALPVTALLLPLALLLHAARP (sequence number 3).

[0183] The sequence of anti-CD79b(L / H)scFv is: The filename is TIFF2023123452000013.tif34161.

[0184] The linker sequence is GGGGSGGGGSGGGGSGGGGS (sequence number 5).

[0185] The sequence of anti-CD19 scFv (which includes a glycine-rich linker separating the heavy and light chains) is: The filename is TIFF2023123452000014.tif35161.

[0186] The sequences of the CD8 transmembrane and hinge domains are: The filename is TIFF2023123452000015.tif16161.

[0187] The sequence of 4-1BB ICD is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (sequence number 8).

[0188] The arrangement of CD3ζ ICD is, The filename is TIFF2023123452000016.tif20161.

[0189] The second CAR comprises the following domains: CD8L, anti-CD19 scFv (containing a glycine-rich linker separating the heavy and light chains), linker, anti-CD79b L / H (having L and H separated by the linker), CD8 TM and hinge, 4-1BB, and CD3ζ, and its sequence is as shown below. TIFF2023123452000017.tif97161

[0190] The sequence of the CD8 reader is MALPVTALLLPLALLLHAARP (sequence number 3).

[0191] The sequence of anti-CD19 scFv (which includes a glycine-rich linker separating the heavy and light chains) is: The filename is TIFF2023123452000018.tif35161.

[0192] The linker sequence is GGGGSGGGGSGGGGSGGGGS (sequence number 5).

[0193] The sequence of anti-CD79b(L / H)scFv is: The filename is TIFF2023123452000019.tif36161.

[0194] The sequences of the CD8 transmembrane and hinge domains are: The filename is TIFF2023123452000020.tif14161.

[0195] The sequence of 4-1BB ICD is KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (sequence number 8).

[0196] The arrangement of CD3ζ ICD is, The filename is TIFF2023123452000021.tif21161.

[0197] Other embodiments fall within the scope of the following numbered paragraphs. 1. A chimeric antigen receptor (CAR) polypeptide containing an extracellular domain that includes a sequence that specifically binds to CD79b. 2. The CAR polypeptide from paragraph 1, wherein the sequence that specifically binds to CD79b includes the antigen-binding region of the antibody against CD79b. 3. A CAR polypeptide from paragraph 1 or 2, wherein the sequence that specifically binds to CD79b contains a single-chain antibody (scFv) against CD79b. 4.scFv is the CAR polypeptide from paragraph 3, which includes a light chain and a heavy chain. 5. The CAR polypeptide from paragraph 4, in which the light chain is at the N-terminus relative to the heavy chain. 6. The CAR polypeptide from paragraph 4, in which the heavy chain is at the N-terminus relative to the light chain. 7. A CAR polypeptide from any one of paragraphs 1 to 6, further comprising one or more, or all, of the hinge domain, transmembrane domain, co-stimulation domain, and signal transduction domain. 8. The CAR polypeptide of paragraph 7, comprising all of the hinge domain, transmembrane domain, co-stimulation domain, and signal transduction domain. 9. The CAR polypeptide of paragraph 7 or 8, wherein the hinge domain and transmembrane domain are CD8 hinge domain and transmembrane domain. 10. One of the CAR polypeptides from paragraphs 7-9, whose co-stimulation domain is a 4-1BB co-stimulation domain. 11. One of the CAR polypeptides from paragraphs 7-10, whose signaling domain is a CD3ζ signaling domain. 12. One CAR polypeptide from any of paragraphs 1-11, comprising an anti-CD79b scFv, a CD8 hinge domain and transmembrane domain, a 4-1BB co-stimulation domain, and a CD3ζ signaling domain. 13. A CAR polypeptide from any one of paragraphs 1-12, wherein the extracellular domain further contains a sequence that specifically binds to CD19. 14. The CAR polypeptide from paragraph 13, wherein the sequence that specifically binds to CD19 includes the antigen-binding region of the antibody against CD19. 15. A CAR polypeptide from paragraph 13 or 14, wherein the sequence that binds to CD19 contains a single-chain antibody (scFv) against CD19. 16.scFv is the CAR polypeptide from paragraph 15, which includes a light chain and a heavy chain. 17. The CAR polypeptide from paragraph 16, in which the light chain is at the N-terminus relative to the heavy chain. 18. The CAR polypeptide from paragraph 16, in which the heavy chain is at the N-terminus relative to the light chain. 19. One of the CAR polypeptides from paragraphs 13-18, wherein the sequence that binds to CD79b is at the N-terminus relative to the sequence that binds to CD19. 20. One of the CAR polypeptides from paragraphs 13-18, wherein the sequence that binds to CD19 is at the N-terminus of the sequence that binds to CD79b. 21. A CAR polypeptide from any one of paragraphs 1 to 20, comprising the sequence of sequence number 1, 2, 10, or 11, or a variant thereof, wherein the sequence optionally does not include the CD8 reader sequence of sequence number 3. 22. One CAR polypeptide from any of paragraphs 1-21, containing the CD8 reader sequence of sequence number 3, or a variant thereof. 23. A CAR polypeptide from any one of paragraphs 1 to 22, containing the anti-CD79b light chain sequence of SEQ ID NO: 4, or a variant thereof. 24. A CAR polypeptide from any one of paragraphs 1-23, containing the anti-CD79b heavy chain sequence of SEQ ID NO: 6, or a variant thereof. 25. A CAR polypeptide from any one of paragraphs 1 to 24, containing the linker sequence of SEQ ID NO: 5, or a variant thereof. 26. One CAR polypeptide from any of paragraphs 1 to 25, comprising the CD8 transmembrane and hinge sequence of SEQ ID NO: 7, or a variant thereof. 27. One CAR polypeptide from any of paragraphs 1-26, containing the 4-1BB ICD sequence of SEQ ID NO: 8, or a variant thereof. 28. One CAR polypeptide from any of paragraphs 1-27, containing the CD3ζ ICD sequence of sequence number 9, or a variant thereof. 29. One CAR polypeptide from any of paragraphs 13-20, containing the anti-CD19 scFv sequence of sequence number 13, or a variant thereof. 30. A nucleic acid molecule containing a sequence encoding one of the CAR polypeptides described in paragraphs 1-29. 31. A vector containing the nucleic acid molecule from paragraph 30. 32. A cell containing one of the CAR polypeptides from paragraphs 1-29, a nucleic acid molecule from paragraph 30, or a vector from paragraph 31. 33. Human primary T cells, as described in paragraph 32. 34. A pharmaceutical composition comprising one CAR polypeptide from any of paragraphs 1 to 29, a nucleic acid molecule from paragraph 30, a vector from paragraph 31, or a cell from paragraph 32 or 33. 35. A method for treating a subject who has cancer or is at risk of developing cancer, comprising administering the pharmaceutical composition of paragraph 34 to the subject. 36. The method for determining whether cancer is lymphoma, as described in paragraph 35. 37. The method described in paragraph 36 for lymphoma being non-Hodgkin lymphoma. 38. The method of paragraph 37, in which non-Hodgkin 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 for treating a subject whose CD19-negative lymphoma has relapsed after receiving CD19 CAR therapy, comprising administering the pharmaceutical composition of paragraph 34 to the subject. 40. A method for producing CAR T cells expressing CD79b, or a CAR specific to CD79b and CD19, comprising introducing the nucleic acid molecule of paragraph 30 or the vector of paragraph 31 into T cells. 41. The method described in paragraph 40, which posits that T cells are human primary T cells.

Claims

1. (a) Extracellular binding domains including CD79b-binding domains and CD19-binding domains; (b) CD8 hinge and transmembrane domain; (c) 4-1BB costimulatory domain; and (d) CD3ζ signaling domain A polynucleotide encoding a CAR polypeptide, including [the specified character].

2. The polynucleotide according to claim 1, wherein the CD8 hinge and the transmembrane domain include the amino acid sequence of SEQ ID NO:

7.

3. The polynucleotide according to claim 1, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:

8.

4. The polynucleotide according to claim 1, wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:

9.

5. (a) The CD79b binding domain comprises a VL domain containing the variable light chain (VL) CDR sequence of SEQ ID NO: 4 and a VH domain containing the variable heavy chain (VH) CDR sequence of SEQ ID NO: 6; and (b) The CD19 binding domain contains the amino acid sequence of SEQ ID NO: 13, The polynucleotide according to claim 1.

6. The CD79b binding domain is (a) A VL domain comprising the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and an amino acid sequence having at least 90% identity with SEQ ID NO: 4; and (b) A VH domain comprising the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6 and an amino acid sequence having at least 90% identity with SEQ ID NO:

6. The polynucleotide according to claim 1, comprising:

7. (a) (i) A CD79b binding domain comprising a VL domain containing the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and a VH domain containing the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6; and (ii) CD19 binding domain containing the amino acid sequence of SEQ ID NO: 13 Extracellular binding domains, including; (b) CD8 hinge and transmembrane domain containing the amino acid sequence of Sequence ID No. 7; (c) A 4-1BB costimulatory domain containing the amino acid sequence of SEQ ID NO: 8; and (d) CD3ζ signaling domain containing the amino acid sequence of SEQ ID NO: 9 A polynucleotide encoding a CAR polypeptide, including [the specified character].

8. The CD79b binding domain is (a) A VL domain comprising the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and an amino acid sequence having at least 90% identity with SEQ ID NO: 4; and (b) A VH domain comprising the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6 and an amino acid sequence having at least 90% identity with SEQ ID NO:

6. The polynucleotide according to claim 7, comprising:

9. The polynucleotide according to any one of claims 1 to 8, wherein the CAR polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 10 or 11.

10. (a) Extracellular binding domains including CD79b-binding domains and CD19-binding domains; (b) CD8 hinge and transmembrane domain; (c) 4-1BB costimulatory domain; and (d) CD3ζ signaling domain A pharmaceutical product for treating subjects with cancer or at risk of developing cancer, comprising T cells containing CAR polypeptides.

11. The pharmaceutical product according to claim 10, wherein the CD8 hinge and transmembrane domain contain the amino acid sequence of SEQ ID NO:

7.

12. The pharmaceutical product according to claim 10, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:

8.

13. The pharmaceutical product according to claim 10, wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:

9.

14. (a) The CD79b binding domain comprises a VL domain containing the variable light chain (VL) CDR sequence of SEQ ID NO: 4 and a VH domain containing the variable heavy chain (VH) CDR sequence of SEQ ID NO: 6; and (b) The CD19 binding domain contains the amino acid sequence of SEQ ID NO: 13, The pharmaceutical product according to claim 10.

15. The CD79b binding domain is (a) A VL domain comprising the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and an amino acid sequence having at least 90% identity with SEQ ID NO: 4; and (b) A VH domain comprising the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6 and an amino acid sequence having at least 90% identity with SEQ ID NO:

6. The pharmaceutical product according to claim 10, including

16. (a) (i) A CD79b binding domain comprising a VL domain containing the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and a VH domain containing the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6; and (ii) CD19 binding domain containing the amino acid sequence of SEQ ID NO: 13 Extracellular binding domains, including; (b) CD8 hinge and transmembrane domain containing the amino acid sequence of Sequence ID No. 7; (c) A 4-1BB costimulatory domain containing the amino acid sequence of SEQ ID NO: 8; and (d) CD3ζ signaling domain containing the amino acid sequence of SEQ ID NO: 9 A pharmaceutical product containing a CAR polypeptide, for the treatment of subjects who have cancer or are at risk of developing cancer.

17. The CD79b binding domain is (a) A VL domain comprising the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and an amino acid sequence having at least 90% identity with SEQ ID NO: 4; and (b) A VH domain comprising the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6 and an amino acid sequence having at least 90% identity with SEQ ID NO:

6. The pharmaceutical product according to claim 16, including the above.

18. (a) Extracellular binding domains including CD79b-binding domains and CD19-binding domains; (b) CD8 hinge and transmembrane domain; (c) 4-1BB costimulatory domain; and (d) CD3ζ signaling domain A pharmaceutical product for treating subjects with cancer or at risk of developing cancer, comprising nucleic acids encoding a CAR polypeptide.

19. The pharmaceutical product according to claim 18, wherein the CD8 hinge and transmembrane domain contain the amino acid sequence of SEQ ID NO:

7.

20. The pharmaceutical product according to claim 18, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO:

8.

21. The pharmaceutical product according to claim 18, wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:

9.

22. (a) The CD79b binding domain comprises a VL domain containing the variable light chain (VL) CDR sequence of SEQ ID NO: 4 and a VH domain containing the variable heavy chain (VH) CDR sequence of SEQ ID NO: 6; and (b) The CD19 binding domain contains the amino acid sequence of SEQ ID NO: 13, The pharmaceutical product according to claim 18.

23. The CD79b binding domain is (a) A VL domain comprising the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and an amino acid sequence having at least 90% identity with SEQ ID NO: 4; and (b) A VH domain comprising the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6 and an amino acid sequence having at least 90% identity with SEQ ID NO:

6. The pharmaceutical product according to claim 18, including the following:

24. (a) (i) A CD79b binding domain comprising a VL domain containing the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and a VH domain containing the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6; and (ii) CD19 binding domain containing the amino acid sequence of SEQ ID NO: 13 Extracellular binding domains, including; (b) CD8 hinge and transmembrane domain containing the amino acid sequence of Sequence ID No. 7; (c) A 4-1BB costimulatory domain containing the amino acid sequence of SEQ ID NO: 8; and (d) CD3ζ signaling domain containing the amino acid sequence of SEQ ID NO: 9 A pharmaceutical product for treating subjects with cancer or at risk of developing cancer, comprising nucleic acids encoding a CAR polypeptide.

25. The CD79b binding domain is (a) A VL domain comprising the CDR sequence of the variable light chain (VL) of SEQ ID NO: 4 and an amino acid sequence having at least 90% identity with SEQ ID NO: 4; and (b) A VH domain comprising the CDR sequence of the variable heavy chain (VH) of SEQ ID NO: 6 and an amino acid sequence having at least 90% identity with SEQ ID NO:

6. The pharmaceutical product according to claim 24, including the above.

26. The pharmaceutical product according to any one of claims 10 to 25, wherein the CAR polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 10 or 11.

27. Cancer, CD79b ＋ and / or CD19 ＋ A pharmaceutical product according to any one of claims 10 to 25, comprising cells.

28. The pharmaceutical product according to any one of claims 10 to 25, wherein the cancer is lymphoma.

29. The pharmaceutical product according to claim 28, wherein the lymphoma is mantle cell lymphoma, diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, chronic lymphocytic leukemia, or small lymphocytic lymphoma.

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