Chimeric antigen receptor targeting CD37 and CD19

Abstract

The present invention provides bispecific chimeric antigen receptors (CARs) that target CD37 and CD19, as well as related molecules, immune cells containing same, compositions thereof, and methods of using same. The present invention further provides methods for treating diseases or disorders, such as cancer. [Selection diagram] None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 688,775, filed June 22, 2018, and No. 62 / 757,562, filed November 8, 2018, both of which are incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on June 19, 2019, is named 51295-018WO3_Sequence_Listing_6.19.19_ST25, and is 56,545 bytes in size. [Background technology]

[0003] Immunotherapy harnesses a patient's immune system to treat diseases such as cancer, autoimmune diseases, or plasma cell disorders. Adoptive cell transfer uses antigen-specific immune cells, such as T cells, to treat such diseases. The immune cells used in this therapy can be modified to exhibit a desired specificity, for example, by expressing a chimeric antigen receptor (CAR). CARs provide a way to direct a cytotoxic T cell or NK cell response to target cells expressing a selected target antigen, most often a tumor antigen or tumor-associated antigen, and are an adaptation of a T cell receptor, where the antigen-binding domain is replaced by the antigen-binding domain of an antibody specific for the target antigen. Engagement of the target antigen on the surface of the target cell by the CAR expressed on the T cell promotes the killing of the target cell. The use of CAR-expressing T cells for the treatment of disease is known as CAR T cell immunotherapy.

[0004] So far, two CAR T cell products have been approved for the treatment of relapsed or refractory large cell lymphoma, both of which target CD19: axicabtagene ciloleucel with a CD28 costimulatory domain, and tisagenlecleucel with a 4-1BB costimulatory domain. Tisagenlecleucel is also approved for the treatment of relapsed or refractory acute B-cell lymphoblastic leukemia (ALL) in children and young adults. Anti-CD19 CAR T cell therapy has produced responses ranging from 60 to 80%, with approximately 40% of patients achieving long-term complete remission. However, disease relapse due to CD19 antigen target loss has been observed in all patient subsets, including both acute lymphoblastic (ALL) and non-Hodgkin lymphoma (NHL) patients (Maude et al., N. Engl. J. Med. 371:1507-17, 2014; Evans et al., Br. J. Haematol. 171(2):205-209, 2015; Schuster et al., N. Engl. J. Med. 377(26):2545-2554, 2017). There is an unmet clinical need for improved therapies.

[0005] Furthermore, patients with T-cell lymphomas are not candidates for anti-CD19 CAR T therapy. For example, peripheral T-cell lymphomas (PTCLs) are an aggressive, heterogeneous group of tumors that account for 12-15% of all non-Hodgkin lymphomas. Despite the recognition of their complex heterogeneity and the discovery of recurrent defects, PTCLs remain a clinical dilemma and are poorly treated. Although CAR T immunotherapy has shown excellent clinical results in ALL and B-cell NHL, success in treating T-cell malignancies has yet to be demonstrated.

[0006] Thus, new or improved treatments for B and T cell malignancies are needed. Summary of the Invention

[0007] The present invention provides, inter alia, a bispecific chimeric antigen receptor (CAR) targeting CD37 and CD19 for use in the treatment of a disease or disorder described herein, such as cancer.

[0008] In one aspect, the invention features a chimeric antigen receptor (CAR) that includes: (i) an extracellular domain that includes a CD37 binding domain and a CD19 binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0009] In some embodiments, the CD37 binding domain and / or the CD19 binding domain comprises an antibody, or an antigen-binding fragment thereof, such as a single chain variable fragment (scFv). In some embodiments, the CD19 binding domain is located N-terminal to the CD37 binding domain, or alternatively, the CD37 binding domain is located N-terminal to the CD19 binding domain.

[0010] In some embodiments, the CAR further comprises (iv) one or more costimulatory domains. In some embodiments, the transmembrane domain comprises a hinge / transmembrane domain, e.g., of CD8 or 4-1BB. In certain embodiments, the hinge / transmembrane domain comprises a hinge / transmembrane domain of CD8, which may comprise the amino acid sequence of SEQ ID NO:9.

[0011] In further embodiments, the intracellular signaling domain comprises an intracellular signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the intracellular signaling domain comprises an intracellular signaling domain of CD3ζ, which may comprise the amino acid sequence of SEQ ID NO: 11. In some embodiments, the costimulatory domain comprises a costimulatory domain of 4-1BB, CD28, or OX-40. In certain embodiments, the costimulatory domain comprises a costimulatory domain of 4-1BB, which may comprise the amino acid sequence of SEQ ID NO: 10.

[0012] In some embodiments, the CAR includes an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 15, 16, 19, or 20, or an amino acid sequence that includes the sequence of the amino acid sequence of SEQ ID NO: 15, 16, 19, or 20.

[0013] In some embodiments, the CD37 binding domain comprises a heavy chain variable domain (VH) that comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1 or that comprises the sequence of the amino acid sequence of SEQ ID NO: 1, and a light chain variable domain (VL) that comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 2 or that comprises the sequence of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VH is positioned N-terminal to the VL, or alternatively, the VL is positioned N-terminal to the VH. In further embodiments, the CD37 binding domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:4 or 5, or an amino acid sequence including the amino acid sequence of SEQ ID NO:4 or 5.

[0014] In a further embodiment, the CD19 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 12 or comprising the sequence of the amino acid sequence of SEQ ID NO: 1, and a light chain variable domain (VL) comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 13 or comprising the sequence of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD19 binding domain includes an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:14, or an amino acid sequence including the amino acid sequence of SEQ ID NO:14.

[0015] In another aspect, the invention features a polynucleotide encoding a CAR of any of the previous aspects.

[0016] In some embodiments, the polynucleotide further comprises a suicide gene. In some embodiments, the polynucleotide further comprises a sequence encoding a signal sequence.

[0017] In another aspect, the invention features an immune cell comprising the CAR and / or polynucleotide of any of the previous aspects.

[0018] In some embodiments, the immune cell (e.g., a human cell) is a T cell or a natural killer (NK) cell.

[0019] In another aspect, the invention features a pharmaceutical composition including the immune cells of any of the previous aspects and a pharma- ceutically acceptable carrier.

[0020] In another aspect, the invention features a method of treating cancer in a subject in need thereof comprising administering to the subject an immune cell, or pharmaceutical composition thereof, of any one of the previous aspects.

[0021] In some embodiments, the cancer expresses CD37. In some embodiments, the cancer is a B cell non-Hodgkin's lymphoma (e.g., mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), or Burkitt's lymphoma), a T cell lymphoma (e.g., peripheral T cell lymphoma (PTCL), cutaneous T cell lymphoma (CTCL), angioimmunoblastic T cell lymphoma (AITL), or anaplastic large cell lymphoma (ALCL)), or a leukemia (e.g., chronic lymphocytic leukemia (CLL)).

[0022] In some embodiments, the subject is non-responsive to anti-CD19 therapy. In further embodiments, the subject is co-administered anti-CD19 therapy.

[0023] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise specified or implied from the context, the following terms and phrases have the meanings provided below. The definitions are provided to help explain certain embodiments and are not intended to limit the claimed technology, since the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0024] Definitions of common terms in immunology and molecular biology are given in The Merck Manual of Diagnosis and Therapy, 19 thEdition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al.(eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway’s Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 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) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the entire contents of each of which are incorporated herein by reference in their entirety.

[0025] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "reduce," "reduction," or "reduce" or "inhibit" typically means a decrease of at least 10% compared to a baseline level, e.g., 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 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 10 ... The term "reduction" or "inhibition" as used herein does not encompass complete inhibition or reduction as compared to a reference level. "Complete inhibition" is 100% inhibition as compared to a reference level. Where applicable, the reduction can be preferably down to a level that is recognized to be within the normal range for an individual without a given disorder.

[0026] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase of a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a base level, for example, 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% increase, or up to a 100% increase, or any increase between 10-100% compared to a gold pure level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a base level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0027] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, birds, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. 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.

[0028] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects that represent animal models of disease, such as cancer. The subject may be male or female.

[0029] The subject may have been previously diagnosed or identified as suffering from or having a condition in need of treatment (e.g., lymphoma, leukemia, or other types of cancer, among others) or one or more complications associated with such a condition, and may, optionally, have already received treatment for the condition or one or more complications associated with the condition. Alternatively, the subject may not have been previously diagnosed as having such a condition or associated complication. For example, the subject may exhibit one or more risk factors for the condition or one or more complications associated with the condition, or may exhibit no risk factors.

[0030] A subject "in need" of treatment for a particular condition can be a subject who has the condition, a subject who has been diagnosed with the condition, or a subject who is at risk for developing the condition.

[0031] A "disease" is a health condition of an animal, e.g., a human, in which the animal is unable to maintain homeostasis and in which the animal's health continues to deteriorate if the disease is not ameliorated. In contrast, an animal's "disorder" is a health condition in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be in the absence of the disorder. A disorder, if left untreated, does not necessarily result in a further deterioration of the animal's health.

[0032] As used herein, the terms "tumor antigen" and "cancer antigen" are used interchangeably to refer to antigens that are differentially expressed by cancer cells and can thereby be utilized to target cancer cells. Cancer antigens are antigens that can stimulate apparently tumor-specific immune responses. Some of these antigens are encoded by normal cells, although not necessarily expressed. These antigens can be characterized as those that are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation, and those that are transiently expressed, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes, such as those carried by RNA and DNA tumor viruses. Many tumor antigens have been defined for a number of solid tumors: immunologically defined MAGE 1, 2, & 3; MART-1 / Melan-A, gp100, carcinoembryonic antigen (CEA), HER2, mucins (i.e., MUC-1), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP). In addition, viral proteins such as those encoded by Hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV) have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively.

[0033] As used herein, the term "chimera" refers to the product of the fusion of portions of at least two or more different polynucleotide molecules. In one embodiment, the term "chimera" refers to a gene expression element that is generated through the manipulation of known elements or other polynucleotide molecules.

[0034] In some embodiments, "activation" may refer to the state of an immune cell, such as a T cell or NK cell, that has been stimulated sufficiently 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, an activated T cell or NK cell as used herein is a proliferating T cell or NK cell.

[0035] As used herein, the terms "specific binding" and "specifically bind" refer to a physical interaction between two molecules, compounds, cells and / or particles, where a first entity binds to a second target, entity, with greater specificity and affinity than it binds to a third, non-target entity. In some embodiments, specific binding may refer to an affinity of the first entity for a second target, entity, that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more than the affinity of a third, non-target entity under the same conditions. A reagent specific for a given target is one that exhibits specific binding to that target under the conditions of the assay being used. Non-limiting examples include antibodies or ligands that recognize and bind to their cognate binding partner (e.g., stimulatory and / or costimulatory molecules present on T cells) proteins.

[0036] A "stimulatory ligand" as used herein refers to a ligand that, when present on an antigen-presenting cell (APC, e.g., macrophage, dendritic cell, B cell, artificial APC, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule" or "costimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, proliferation, activation, initiation of an immune response, etc. Stimulatory ligands are well known in the art and include, among others, peptide-loaded MHC Class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0037] As used herein, the term "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0038] As used herein, the term "costimulatory ligand" includes a molecule on an APC that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory ligands can include, but are not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll-like receptors, and ligands that specifically bind B7-H3. Costimulatory ligands can also include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0039] A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll-like receptors, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0040] In one embodiment, the term "engineered" and its grammatical equivalents as used herein may refer to a modification or modifications of a nucleic acid designed by human, e.g., a nucleic acid in the genome of an organism. In another embodiment, engineered may refer to a modification, addition, and / or deletion of a gene. An "engineered cell" may refer to a cell that has added, deleted, and / or modified genes. The terms "cell" or "engineered cell" and their grammatical equivalents as used herein may refer to a cell derived from a human or non-human animal.

[0041] As used herein, the term "operably linked" refers to a first polynucleotide molecule, e.g., a promoter, linked to a second transcribable polynucleotide molecule, e.g., a gene of interest, when the polynucleotide molecules are positioned such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single contiguous polynucleotide molecule, and may or may not be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter controls or mediates the transcription of the gene of interest in a cell.

[0042] It is further contemplated that the various embodiments described herein encompass any variant (naturally occurring or not), allele, homolog, conservatively modified variant, and / or conservatively substituted variant of the particular polypeptide described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that modify a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" in which the modification results in the replacement of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0043] A given amino acid may be replaced by a residue having similar physicochemical properties, for example, by substituting one aliphatic residue for another (e.g., substituting Ile, Val, Leu, or Ala for one another), or by substituting one polar residue for another (e.g., between Lys and Arg, between Glu and Asp, or between Gln and Asn). Other such conservative substitutions, for example, the substitution of entire regions with similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, for example, ligand-mediated receptor activity and specificity of the native or reference polypeptide, is retained.

[0044] Amino acids can be grouped according to similarities in the properties of their side chains (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):(1) nonpolar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M);(2) uncharged polar: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q);(3) acidic: Asp(D), Glu(E);(4) basic: Lys(K), Arg(R), His(H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions entail exchanging a member of one of these classes for another. Particular conservative substitutions include, for example, the following: 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.

[0045] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a peptide fragment or segment that retains at least 50% of the activity of a wild-type reference polypeptide according to assays known in the art or described herein below. Functional fragments may include conservative substitutions of the sequences disclosed herein.

[0046] In some embodiments, the polypeptides described herein may be variants of the polypeptides or molecules described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of a natural nucleotide sequence. A "variant" as referred to herein is a polypeptide having an amino acid sequence that is substantially homologous to a natural or reference polypeptide, but differs from the amino acid sequence of the natural or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide encompasses sequences encoding variant proteins or fragments thereof that contain one or more additions, deletions, or substitutions of nucleotides when compared to a natural or reference DNA sequence, but retain the activity of the non-variant polypeptide. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the skilled artisan.

[0047] A variant amino acid or DNA sequence is 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 more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0048] Modification of the native amino acid sequence can be accomplished by any of a number of techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutated sequence, flanked by restriction sites that allow ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis approach can be used to provide modified nucleotide sequences with specific codons modified according to the required substitution, deletion, or insertion. The techniques for making such modifications are well established and include, for example, those disclosed by 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, which are incorporated herein by reference in their entirety. Cysteine ​​residues that are not involved in maintaining the proper conformation of the polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve its stability or promote oligomerization.

[0049] As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term "polynucleotide" is used interchangeably with "nucleic acid" herein and refers to a polymer of nucleosides. Typically, polynucleotides are composed of nucleosides found naturally in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term encompasses molecules that contain nucleosides or nucleoside analogs that contain chemically or biologically modified bases, modified backbones, and the like, whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to polynucleotides, it is understood that both DNA, RNA, and in each case both single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and / or the sequence information (i.e., the series of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise indicated.

[0050] The term "polypeptide" as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. Peptides are relatively short polypeptides, usually between about 2 and 60 amino acids in length. A polypeptide as used herein typically contains amino acids, such as the 20 L-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 of the amino acids in a polypeptide may be modified, for example, by the addition of chemicals such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for conjugation, functionalization, and the like. Polypeptides having non-polypeptide moieties covalently or non-covalently bound to the polypeptide are also considered "polypeptides". Exemplary modifications include glycosylation and palmitoylation. Polypeptides may be purified from natural sources, produced using recombinant DNA technology, or synthesized through chemical means such as conventional solid phase peptide synthesis, and the like. The terms "polypeptide sequence" or "amino acid sequence" as used herein may refer to the polypeptide material itself and / or to sequence information that biochemically characterizes the polypeptide (i.e., the series of letters or three-letter codes used as abbreviations for amino acid names). Polypeptide sequences presented herein are presented in an N-terminal to C-terminal direction unless otherwise indicated.

[0051] In some embodiments, the nucleic acid encoding the polypeptides described herein (e.g., bispecific CAR polypeptides) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide described herein, or a module thereof, is operably linked to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that is replicable when linked to the appropriate control elements and can transfer genetic sequences into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virions, and the like.

[0052] As used herein, the term "expression vector" refers to a vector that expresses an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is often, but not necessarily, heterologous to the cell. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, allowing it to be maintained in two organisms, for example, a human cell for expression and a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and optionally secretion of proteins, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable. The term "expression product" includes RNA transcribed from a gene and a polypeptide resulting from translation of mRNA transcribed from a gene. The term "gene" refers to a (DNA) nucleic acid sequence that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0053] As used herein, "signal peptide" or "signal sequence" refers to a peptide at the N-terminus of a newly synthesized protein that functions to target the nascent protein to the endoplasmic reticulum. In some embodiments, the signal peptide is the CD8 signal peptide.

[0054] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector may contain a nucleic acid encoding a polypeptide as described herein in place of a non-essential viral gene. The vector and / or particle may be used to transfer the nucleic acid into cells in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0055] "Recombinant vector" refers to a vector that contains a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can be combined with other suitable compositions and therapies in some embodiments. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means to maintain the nucleotide of interest in a high copy number of extrachromosomal DNA in a subject, thereby eliminating the potential effects of chromosomal integration.

[0056] As used herein, the terms "treat", "treatment", "treating" or "amelioration" refer to therapeutic treatments whose purpose is to reverse, alleviate, ameliorate, inhibit, slow or halt the progression or severity of a condition associated with a disease or disorder, e.g., acute lymphoblastic leukemia or other cancer, disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is typically "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the halting or at least slowing of the progression or worsening of symptoms compared to that expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., does not worsen), delay or slowing of disease progression, amelioration or palliation of the pathology, remission (partial or complete), and / or reduction in mortality. The term "treatment" of a disease also includes providing relief (including palliative treatment) from the symptoms and side effects of the disease.

[0057] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharma- ceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio. In certain embodiments of any of the aspects of the invention, the pharma- ceutically acceptable carrier can be a carrier other than water. In certain embodiments of any of the aspects of the invention, the pharma- ceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In certain embodiments of the aspects of the invention, the pharma- ceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier in which the active ingredient is not found to occur in nature.

[0058] As used herein, the term "administering" refers to placing a therapeutic or pharmaceutical composition disclosed herein into a subject by a method or route that results in at least partial delivery of the agent to a desired site. Pharmaceutical compositions containing the agents disclosed herein can be administered by any suitable route that results in an effective treatment in the subject.

[0059] The terms "statistically significant" or "significantly" refer to statistical significance, usually meaning a difference of 2 standard deviations (2 SD) or greater.

[0060] Except in the working examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about," which when used in connection with percentages can mean ±1%.

[0061] As used herein, the term "comprising" means that other elements may be present in addition to the defined elements set forth. The use of "comprising" indicates inclusion rather than limitation.

[0062] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any elements not recited in that description of an embodiment.

[0063] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the technology.

[0064] The singular forms "a," "an," "the," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." In certain embodiments of any of the aspects of the invention, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the genetic identity of the germ line of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that may suffer without substantial medical benefit to humans or animals and that result from such processes.

[0065] Other terms are defined within the description of various aspects and embodiments of the technology below. [Brief description of the drawings]

[0066] [Figure 1A] FACS plots of tumor cell lines stained with CD37 and CD19 antibodies are shown. NALM6: acute lymphoblastic leukemia (ALL) cell line; K562 expressing CD19 and CD37: positive control; JEKO-1: mantle cell lymphoma (MCL) cell line; RAJI: Burkitt's lymphoma cell line. [Figure 1B] FACS plots of samples from three MCL patients are shown. [Figure 1C] The mean fluorescence intensity (MFI) of CD19 and CD37 on cells from MCL patients is shown. Each dot represents a separate xenograft sample. (n=3; medians shown). [Figure 1D] Expression of CD19 and CD37 on peripheral blood mononuclear cells (PBMCs) of CLL patients gated on CD3 lymphocytes is shown (n=20; mean±SD shown; **** indicates p<0.0001 by t-test). [Figure 1E] Figure 1 shows CD19 and CD37 on CLL samples. Expression levels of CD19 and CD37 in 21 patients with CLL by flow cytometry gated on CD3-CD20+ B cells. Mean ± SD shown. [Figure 2A] Distribution of CD19 and CD37 antigens on CLL PBMCs. Antibody binding per cell (ABC) gated on CD3- cells from each patient sample is shown. [Figure 2B] CD19 and CD37 antigen densities are shown. CD19 range: 24396-70952, mean: 43238; CD37 range: 8992-46550, mean: 23989. [Figure 2C] CD37 immunohistochemistry in primary ALK-negative (left) and ALK-positive (right) ALCL specimens from tissue microarrays is shown. [Figure 3A]Schematic diagram of two anti-CD37 second generation CAR constructs with different orientations of the single-chain variable fragment derived from a humanized murine antibody: light chain to heavy chain orientation (CAR-37 LH, top) and heavy chain to light chain (CAR-37 HL, bottom). [Figure 3B] Representative flow plots of transduction efficiency of primary human T cells 10 days after activation with CD3 / CD28 beads are shown. [Figure 3C] Shown are expanded T cells from three healthy donors contained variable CAR-37 expression, an average of 38% (LH) and 75% (HL). [Figure 3D] Figure 1 shows ex vivo expansion of CD3 / CD28 bead-activated and target-stimulated T cells using static culture conditions in three healthy donors over a period of 38 days. Each arrow represents antigen stimulation with K562 cells transduced to express CD37 and CD19. [Figure 3E] Activation of Jurkat reporter (NFAT-Luc) T cells transfected with different CAR constructs and co-cultured with tumor cells. Luciferase activity was measured after 16 hours. (CD3-CD28 beads: positive control). [Figure 3F] Whole blood from six normal donors stained with CD45, CD3, CD19, CD16, CD14, CD56, and CD37 antibodies or isotype controls are shown. 3F shows a histogram depicting CD37 expression gated on CD45+CD19+ B cells. [Figure 3G] Whole blood from six normal donors stained with CD45, CD3, CD19, CD16, CD14, CD56, and CD37 antibodies or isotype controls are shown. 3G shows a histogram depicting CD37 expression gated on the CD45+CD16+CD14+ monocyte population. [Figure 3H] Whole blood from six normal donors stained with CD45, CD3, CD19, CD16, CD14, CD56, and CD37 antibodies or isotype controls are shown. 3H shows a histogram depicting CD37 expression gated on T cells (CD45+CD3+). [Figure 3I]Whole blood from six normal donors stained with CD45, CD3, CD19, CD16, CD14, CD56, and CD37 antibodies or isotype controls are shown. 3I shows a histogram representing CD37 expression gated on CD45+CD16+CD56+ NK cells. [Figure 4A] Shown are the numbers of CSFE-labeled, unstimulated target T cells measured by flow cytometry 24 hours after co-culture with CAR-37 HL, CAR-19, or non-transduced T cells at the indicated E:T ratios. [Figure 4B] Shown are the numbers of CSFE-labeled T cells measured by flow cytometry 24 hours after stimulation with PMA / ionomycin for 6 hours followed by co-culture with CAR-37 HL, CAR-19, or non-transduced T cells at the indicated E:T ratios. [Figure 4C] Shown are the numbers of Jeko-1 CBG-GFP cells measured by flow cytometry 24 hours after co-culture with CAR-37 HL, CAR-19, or non-transduced T cells at the indicated E:T ratios. Bars show the mean ± SEM counts of triplicates from one normal donor representative of three normal donors. [Figure 4D] Figure 1 shows the production of CD107a and IFNγ to medium by CAR-37 HL and CAR-19 T cells incubated with primary immune cells at an E:T ratio of 1:1 for 6 hours, analyzed by flow cytometry. Bars represent the mean ± SEM percentages of three normal donors analyzed. [Diagram 5] Figure 1 shows the cytotoxic performance of CAR-37 T cells measured after overnight co-culture with targets. CAR T cells were co-cultured with the indicated tumor cell lines at the indicated E:T ratios. Increasing concentrations of CAR-37 and CAR-19 T cells led to specific killing, whereas no killing was observed in the control group (UTD). Cytotoxicity assays are representative of three independent experiments performed in different healthy donors. [Figure 6A]Figure 1 shows cytokine production by CAR-37 HL, CAR-19, or UTD T cells incubated with primary CLL tumor samples. CAR T cells were incubated with target cells at a 1:1 E:T ratio for 24 hours and culture supernatants were analyzed by Luminex assay. Data are plotted as the mean ± SEM of three donors. [Figure 6B] Figure 1 shows cytokine production by CAR-37 HL, CAR-19, or UTD T cells incubated with MCL PDX tumor samples. CAR T cells were incubated with target cells at a 1:1 E:T ratio for 24 hours and culture supernatants were analyzed by Luminex assay. Data are plotted as the mean ± SEM of three donors. [Figure 6C] Cytokine production by CAR-37, CAR-19, or UTD T cells incubated with the indicated tumor cell lines at a 1:1 E:T ratio for 24 hours was shown, and culture supernatants were analyzed by Luminex assay. Significant production of multiple cytokines was observed in the CAR-37 and CAR-19 groups, but not UTD. Three normal donors were analyzed, and the mean ± SEM is shown. [Figure 6D] IL-6 production by CAR-37, CAR-19, or UTD T cells incubated with the indicated tumor cell lines at a 1:1 E:T ratio for 24 hours and culture supernatants were analyzed by Luminex assay. Three healthy donors were analyzed and the mean ± SEM is shown. [Figure 7A-C]7A shows a direct comparison of anti-MCL activity of CAR-37 T cells in an MCL tumor model. 7A shows an experimental schematic. NSG mice were injected IV with 1×106 JEKO-1 (CBG-GFP) cells and monitored by BLI for tumor burden at different time points. On day 0, mice were randomized based on tumor burden (BLI) to receive 1×106 control T cells (UTD), CAR-37 LH, or CAR-37 HL. 7B shows representative bioluminescence images of JEKO-1 growth over time. 7C shows the mean radiance (p / s / cm2 / sr) across three groups of mice at different time points. The graph represents one experiment with five mice per group. Mean ± SD is shown. [Figure 7D] A schematic diagram of the experimental design is shown. NSG mice were injected IV with 1×10 JEKO-1 (CBG-GFP) cells and monitored by BLI for tumor burden at different time points. On day 0, mice were randomized (BLI) based on tumor burden to receive 2×10 control T cells (UTD), CAR-37, or CAR-37 HL. [Figure 7E] Representative bioluminescence images of JEKO-1 growth over time are shown. [Figure 7F] The average flux (photons / sec) across the three groups of mice at different time points is shown. The graph represents two experiments with 5 mice per group performed with CAR T cells obtained from two different healthy donors. Mean ± SD shown. *** indicates p<0.001 by two-way ANOVA. [Figure 7G] Absolute numbers of CAR T cells monitored by bleeding and flow cytometry detection are shown. Absolute numbers of CAR T cells in peripheral blood 14 days after CAR injection are shown. * indicates p<0.05 by t-test. [Figure 8A]A schematic of the experimental design is shown. NSG mice were injected IV with 1×106 MCL patient-derived cells and monitored over time for tumor burden by bioluminescence imaging (BLI). On day 0, mice were randomized based on tumor burden to receive 3×106 control T cells (UTD), CAR-37, or CAR-37 HL. [Figure 8B] Representative BLI of MCL xenografts over time. [Figure 8C] The average flux (photons / sec) across three groups of mice at different time points is shown. The graph represents two experiments with 5 mice per group performed with CAR T cells obtained from two different healthy donors and the data pooled. Mean ± SD shown (t-test, p<0.05). [Figure 8D] 1 shows that the absolute number of CAR T cells was monitored in peripheral blood using flow cytometry. The absolute number of CAR T cells is plotted at day 14. [Figure 9A] 1 shows CD37 expression on PTCL tumor cell lines. [Figure 9B] A typical FACS plot from a patient-derived sample is shown. [Figure 10A] Shown is CD69 expression of CAR T cells assessed by flow cytometry 6 hours after co-culture with the indicated tumor cells at an E:T ratio of 1:1. [Figure 10B] Shown is CD107a degranulation of CAR T cells assessed by flow cytometry 6 hours after co-culture with the indicated tumor cells at a 1:1 E:T ratio. Degranulation is relative to the PMA positive control and representative normal donor is shown. [Figure 10C] Figure 1 shows the cytotoxic performance of CAR-37 T cells, measured after overnight co-culture with Hut78 target cells at different E:T ratios. Cytotoxicity assays are representative of three independent experiments performed in different healthy donors. Mean ± SEM shown. [Figure 10D]Figure 1 shows the cytotoxic performance of CAR-37 T cells, measured after overnight co-culture with FEPD target cells at different E:T ratios. Cytotoxicity assays represent three independent experiments performed in different healthy donors. Mean ± SEM shown. [Figure 11A] Schematic diagram of two bispecific second generation CAR constructs with different orders of scFvs: CAR-19-37 (top) and CAR-37-19 (bottom). [Figure 11B] Representative flow plots of transduction efficiency of primary human T cells 10 days after activation with CD3 / CD28 beads are shown. [Figure 11C] We demonstrated the expansion of T cells from two healthy donors, which showed variable CAR expression, averaging 19% (CAR-19-37) and 48% (CAR-37-19). [Figure 12A] Activation of Jurkat reporter (NFAT-Luc) T cells transfected with different CAR constructs and co-cultured with tumor cells. Luciferase activity was measured after 16 hours. (CD3-CD28 beads: positive control). [Figure 12B] Figure 1 shows ex vivo expansion of CD3 / CD28 bead-activated and target-stimulated T cells in two healthy donors over a 30 day period. [Figure 12C] Figure 1 shows the cytotoxic potential of bispecific CAR T cells, measured after overnight co-culture with K562 targets transduced with CD37, CD19, or both, at the indicated E:T ratios. Cytotoxicity assays represent two independent experiments performed in different healthy donors. [Figure 12D]Tumor burden in NSG mice over time. NSG mice were injected IV with 1x106 JEKO-1 (CBG-GFP) cells and monitored by BLI for tumor burden. On day 0, mice were randomized based on tumor burden (BLI) to receive 2x106 control T cells (UTD), CAR-37, CAR-19, CAR-19-37 or CAR-37-19. All CAR T cell groups were normalized to have the same percentage of CAR+ and non-transduced cells. The average flux (photons / sec) across mice at different time points is shown. The graph shows one experiment with 6 mice per group. [Figure 12E] Absolute numbers of CAR T cells are shown, which were enumerated in peripheral blood by flow cytometry at the indicated time points. Absolute numbers of CAR T cells are shown as mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The present invention provides bispecific chimeric antigen receptor (CAR) polypeptides that target CD37 and CD19. Also described are nucleic acid molecules encoding the bispecific CARs, vectors containing said nucleic acid molecules, and methods of making and using the same. Additionally, methods of treating diseases or disorders as described herein, such as cancer, using the bispecific CARs and associated molecules as described herein are provided.

[0068] Chimeric Antigen Receptor The technology described herein provides bispecific CARs that target CD37 and CD19 for use in immunotherapy, e.g., to treat cancer.

[0069] The term "chimeric antigen receptor" or "CAR" as used herein refers to an engineered T cell receptor that transfers ligand or antigen specificity into a T cell (e.g., a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof) or a NK cell. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. Additionally, the term "CAR" includes bispecific CARs as described herein.

[0070] CARs place a chimeric extracellular target binding domain that specifically binds to a target, e.g., a polypeptide, expressed on the surface of a cell targeted by a T cell response on a construct that includes a transmembrane domain and an intracellular domain of a T cell receptor molecule. In one embodiment, the chimeric extracellular target binding domain comprises an antigen binding domain of an antibody that specifically binds to an antigen expressed on a cell targeted by a T cell response. The properties of the intracellular signaling domain of a CAR can vary, as known in the art and as disclosed herein, but the chimeric target / antigen binding domain renders the receptor susceptible to signaling activation when the chimeric target / antigen binding domain binds to a target / antigen on the surface of a target cell.

[0071] In terms of intracellular signaling domains, so-called "first generation", CARs include those that provide only a CD3 zeta (CD3ζ) signal upon antigen binding. So-called "second generation" CARs include those that provide both a costimulatory domain (e.g., CD28 or CD137) and an activation (CD3ζ) domain, and so-called "third generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) and activation domains (e.g., CD3ζ). In various embodiments, CARs are selected to have high affinity or affinity for the target / antigen, e.g., antibody-derived target or antigen binding domains typically have higher affinity and / or affinity for target antigens than naturally occurring T cell receptors. This property, combined with the high specificity that can be selected for antibodies, provides highly specific targeting of T cells by CAR T cells.

[0072] As used herein, "CAR T cells" or "CAR-T" refers to T cells expressing a CAR. Similarly, "CAR NK cells" refers to NK cells expressing a CAR. When expressed in a T cell or NK cell, a CAR can utilize the antigen-binding properties of a monoclonal antibody to redirect the specificity and reactivity of the T cell or NK cell to a selected target in an MHC-unrestricted manner. MHC-unrestricted antigen recognition allows a CAR-expressing T cell or NK cell to recognize antigens independent of antigen processing, thereby circumventing a major mechanism of tumor escape.

[0073] As used herein, the term "extracellular target binding domain" refers to a polypeptide found outside a cell sufficient to facilitate binding to a target. An extracellular target binding domain specifically binds to its binding partner, i.e., the target. As a non-limiting example, an extracellular target binding domain may include the antigen binding domain of an antibody, or a ligand that recognizes and binds to a cognate binding partner (e.g., CD37 or CD19) protein. In this context, a ligand is a molecule that specifically binds to a protein and / or a portion of a receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein is usually found on the surface of a cell. Ligand:cognate partner binding can result in modification of the ligand-containing receptor or activate a physiological response, e.g., activation of a signal transduction pathway. In one embodiment, the ligand can be non-native to the genome. In some cases, the ligand has a function that is conserved across at least two species. In one embodiment, the extracellular target binding domain comprises a non-antibody ligand.

[0074] Antibody Reagents In various embodiments, the CARs described herein comprise an antibody reagent or an antigen-binding domain thereof as the extracellular target binding domain.

[0075] As used herein, the term "antibody reagent" refers to a polypeptide that contains at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and that specifically binds to a given antigen. An antibody reagent may comprise an antibody, or a polypeptide that contains the antigen-binding domain of an antibody. In certain embodiments of any of the aspects of the invention, an antibody reagent may comprise a monoclonal antibody, or a polypeptide that contains the antigen-binding domain of a monoclonal antibody. For example, an antibody may contain a heavy (H) chain variable region (referred to herein as V H and a light (L) chain variable region (abbreviated as V LIn another example, an antibody can include 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, CDR, and domain antibody (dAb) fragments (e.g., Wildt et al., Eur J. Immunol. 26(3):629-639, 1996; incorporated herein by reference in its entirety) as well as complete antibodies. Antibodies may have structural characteristics of IgA, IgG, IgE, IgD, or IgM (as well as subtypes and combinations thereof). Antibodies may be from any source, including mouse, rabbit, pig, rat, and primates (human and non-human primates), as well as primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like. Completely human antibody binding domains may be selected, for example, from phage display libraries using methods known to those of skill in the art.

[0076] V H and V L Regions can be further subdivided into regions of hypervariability called "complementarity determining regions (CDRs)" interspersed with more conserved regions called "framework regions (FRs)". The extent of the framework regions and CDRs has been precisely defined (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al., J. Mol. Biol. 196:901-917, 1987, which are incorporated herein by reference in their entireties). Each V H and V L typically contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0077] In some embodiments, the antibody or antibody reagent is not a human antibody or antibody reagent (i.e., the antibody or antibody reagent is murine), but is humanized. A "humanized antibody or antibody reagent" refers to a non-human antibody or antibody reagent that has been modified at the protein sequence level to increase similarity to the antibody or antibody reagent variant that is naturally generated in humans. One approach to humanizing an antibody uses the grafting of murine or other non-human CDRs into a human antibody framework.

[0078] In some embodiments, the extracellular target binding domain of the CAR is linked to the V of an antibody, usually a monoclonal antibody, via a flexible linker peptide. H Domain and V L The CARs comprise or consist essentially of a single chain variable fragment (scFv), created by fusing domains. In various embodiments, the scFv is fused to a transmembrane domain, optionally via a hinge, and to a T cell receptor intracellular signaling domain, such as an engineered intracellular signaling domain as described herein. Antibody binding domains useful for the CARs described herein and methods for selecting and cloning them are well known to those of skill in the art.

[0079] In some embodiments, a CAR useful in the technology described herein comprises at least two antigen-specific targeting regions, an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the CAR comprises a hinge / transmembrane domain as described herein. In such embodiments, the two or more antigen-specific targeting regions target at least two different antigens and can be arranged in tandem and separated by a linker sequence. In another embodiment, the CAR is a bispecific CAR specific for two different antigens.

[0080] For example, a CAR as described herein is a bispecific CAR capable of binding to both CD37 and CD19. The CD37 binding site and the CD19 binding site can each comprise an antibody reagent, such as a single chain variable fragment (scFv).

[0081] Thus, the CD37 binding sequence of the bispecific CAR is, in some embodiments, an antibody reagent. In other embodiments, the antibody reagent is an anti-CD37 scFv. In some embodiments, the VH of the anti-CD37 scFv corresponds to, comprises, 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 more sequence identity thereto, of the sequence of SEQ ID NO: 1. In some embodiments, the VL of the anti-CD37 scFv corresponds to, comprises, 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 more sequence identity thereto, of the sequence of SEQ ID NO: 2. The VH of the anti-CD37 scFv may be located N-terminal to the VL, or alternatively, the VL may be located N-terminal to the VH. The VL and VH domains may optionally be joined via a linker, such as the linker of SEQ ID NO: 3. In some embodiments, the anti-CD37 scFv corresponds to; comprises; or has 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 more sequence identity to the sequence of SEQ ID NO: 4 or 5.

[0082] The CD19 binding sequence of the bispecific CAR is a reagent in some embodiments. In other embodiments, the antibody reagent is an anti-CD19 scFv. In some embodiments, the VH of the anti-CD19 scFv corresponds to, comprises, or has 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 more sequence identity to the sequence of SEQ ID NO: 12. In some embodiments, the VL of the anti-CD19 scFv corresponds to, comprises, or has 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 more sequence identity to the sequence of SEQ ID NO: 13. The VH of the anti-CD19 scFv may be located N-terminal to the VL, or the VL may be located N-terminal to the VH. The VL and VH domains may optionally be joined via a linker, such as the linker of SEQ ID NO: 3. In some embodiments, the anti-CD19 scFv corresponds to or comprises the sequence of SEQ ID NO: 14; 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 more sequence identity to the sequence of SEQ ID NO: 14.

[0083] target / antigen Any cell surface moiety can be targeted by CAR. In most cases, the target is a cell surface polypeptide that is differentially or preferentially expressed on cells that one wishes to target with a T cell response. In this regard, tumor antigens or tumor-associated antigens provide attractive targets, providing a means to target tumor cells while avoiding or at least limiting collateral damage to non-tumor cells or tissues.

[0084] As described above, one target of bispecific CAR is CD37. CD37 is a cell surface protein that contains four hydrophobic transmembrane domains. CD37 is exclusively expressed on immune cells. CD37 is highly expressed on mature B cells and moderately expressed on T cells and myeloid cells. CD37 sequences are known in many species, for example human CD37 (NCBI gene ID:951) polypeptide (e.g., NCBI Ref Seq NP_001035120.1) and mRNA (e.g., NCBI Ref Seq NM_ 001040031.1). CD37 refers to human CD37, including naturally occurring variants, molecules, and alleles thereof. In certain embodiments of any of the aspects of the present invention, for example in livestock applications, CD37 may refer to canine, feline, bovine, equine, porcine, etc. CD37. Homologs and / or orthologs of human CD37 are readily identified for a given species by one of skill in the art using, for example, the NCBI ortholog search function or by searching available sequence data for such species for sequences similar to the reference CD37 sequence.

[0085] The second target of the bispecific CAR is CD19. CD19 is a transmembrane protein expressed in all B lineage cells except plasma cells and follicular dendritic cells. CD19 sequences are known in numerous species, for example human CD19 (NCBI Gene ID: 930) polypeptide (e.g. NCBI GenBank Accession No.: AAB60697.1) and DNA (e.g. NCBI GenBank Accession No.: AH005421.2). CD19 refers to human CD19, including naturally occurring variants, molecules, and alleles thereof. In certain embodiments of any of the aspects of the invention, for example in livestock applications, CD19 may refer to CD19 of feline, canine, bovine, equine, porcine, etc. Homologs and / or orthologs of human CD19 are readily identified for such species by those skilled in the art, for example using the NCBI ortholog search function or searching available sequence data for a given species for sequences similar to the reference CD19 sequence.

[0086] Hinge and transmembrane domains The binding domain of a CAR may be followed by one or more "hinge domains," which serve to position the target binding domain away from the effector cell surface to allow for proper cell / cell contact, target binding, and activation. A CAR optionally comprises one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the bispecific CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8α), CD4, CD28, and CD7, which may be the wild-type hinge region of these molecules or may have been modified. In one embodiment, the hinge domain comprises a CD8α hinge region.

[0087] CD8 is an antigen found preferentially on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and acts as a T cell coreceptor. CD8 consists of alpha (CD8α or CD8a) and beta (CD8β or CD8b) chains. CD8a sequences are known in numerous species, for example human CD8a (NCBI Gene ID:925) polypeptide (e.g., NCBI Ref Seq NP_001139345.1) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12). CD8 refers to human CD8, including naturally occurring variants, molecules, and alleles thereof. In certain embodiments of any of the aspects of the invention, for example in livestock applications, CD8 may refer to canine, feline, bovine, equine, porcine, etc. CD8. Homologs and / or orthologs of human CD8 are readily identified for a given species by one of skill in the art using, for example, the NCBI ortholog search function or by searching available sequence data for such species for sequences similar to the reference CD8 sequence.

[0088] As used herein, a "transmembrane domain" ("TM domain") refers to a portion of a CAR that fuses the extracellular binding portion to the intracellular portion (e.g., the intracellular signaling domain and the costimulatory domain, if present), optionally via a hinge, and anchors the CAR to the plasma membrane of an immune effector cell. A transmembrane domain is a generally hydrophobic region of a CAR that passes through the plasma membrane of a cell. A transmembrane domain can be a transmembrane region or fragment thereof of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Although specific examples are provided herein and used in the Examples, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternative embodiments of the technology. The selected transmembrane region or fragment thereof preferably does not interfere with the intended function of the CAR. When used in connection with a transmembrane domain of a protein or polypeptide, a "fragment thereof" refers to a portion of the transmembrane domain that is sufficient to anchor or attach the protein to a cell surface.

[0089] In some examples, the transmembrane domain of a CAR described herein, or a fragment thereof, is a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT In one embodiment, the transmembrane domain or fragment thereof is derived from or comprises the transmembrane domain of the alpha, beta or zeta chain of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0090] As used herein, "hinge / transmembrane domain" refers to a domain that comprises both the hinge domain and the transmembrane domain. In one embodiment, the hinge / transmembrane domain of the bispecific CAR or a fragment thereof is derived from or comprises the hinge / transmembrane domain of CD8. The CD8 hinge / transmembrane domain may comprise the amino acid sequence of SEQ ID NO: 9 or a variant thereof.

[0091] Costimulatory domain The bispecific CAR described herein may optionally comprise the intracellular domain of a costimulatory molecule, or a costimulatory domain. As used herein, the term "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. For example, the intracellular domain is the intracellular domain of 4-1BB.

[0092] 4-1BB is a membrane receptor protein, also known as CD137, and is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. 4-1BB sequences are known in numerous species, for example, human 4-1BB (also known as TNFRSF9) (NCBI gene ID: 3604) and mRNA (NCBI reference sequence: NM_001561.5). 4-1BB refers to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In certain embodiments of any of the aspects of the present invention, for example in livestock applications, 4-1BB may refer to canine, feline, bovine, equine, porcine, etc. 4-1BB. Homologs and / or orthologs of human 4-1BB are readily identified for such species by those skilled in the art, for example, using the NCBI ortholog search function or searching available sequence data for a given species for sequences similar to the reference 4-1BB sequence.

[0093] For example, the 4-1BB costimulatory domain of the bispecific CAR described herein may comprise the amino acid sequence of SEQ ID NO: 10 or a variant thereof.

[0094] Intracellular signaling domains Bispecific CARs as described herein include an intracellular signaling domain. "Intracellular signaling domain" refers to a portion of a CAR polypeptide that is involved in introducing the message of an effective CAR binding to a target antigen into the immune effector cell to trigger the effector cell's function, such as activation, proliferation and cytotoxic activity, including cytokine production, release of cytotoxic factors to the CAR-bound target cell, or other cellular response triggered following antigen binding to the extracellular CAR domain. Non-limiting examples of immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling domains that are particularly used in the present technology include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 theta, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0095] CD3 is a T cell coreceptor that facilitates T lymphocyte activation when simultaneously engaged with appropriate costimulation (e.g., binding of costimulatory molecules). The CD3 complex consists of four different chains; mammalian CD3 consists of the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains bind to a molecule known as the T cell receptor (TCR) and CD3ζ to generate an activation signal within the T lymphocyte. 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 comprising an immunoreceptor tyrosine-based activation motif (ITAM) from CD3 zeta (CD3ζ). In some embodiments of any aspect, the ITAM comprises the ITAM triad motif of CD3ζ (ITAM3). In some embodiments of any aspect, the ITAM triad motif of CD3ζ is not mutated and thus comprises a native or wild-type sequence. For example, the CD3ζ sequence of the bispecific CAR described herein comprises the sequence of SEQ ID NO: 11, as described below, or a variant thereof. In various embodiments, such a CAR polypeptide CD3ζ sequence is a native or wild-type sequence.

[0097] More detailed descriptions of CARs and CAR T cells can be found in Maus et al. Blood 2014 123:2624-35; Reardon et al. Neuro-Oncology 2014 16:1441-1458; Hoyos et al. Haematologica 2012 97:1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. Cancer Res 2009 69:4559-4562; and Tamada et al. Clin Cancer Res 2012 18:6436-6445, each of which is incorporated by reference in its entirety.

[0098] In some embodiments, the CAR further comprises a linker domain. As used herein, a "linker domain" or "linker region" refers to an oligo- or polypeptide region of about 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 comprise or consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to one another. For example, the linker of SEQ ID NO: 3. Longer linkers may be used if it is desired to ensure that two adjacent domains do not sterically interfere with one another. The linker may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linker region is T2A from the Thosea asigna virus. Non-limiting examples of linkers that can be used in this technology include P2A and F2A.

[0099] In some embodiments, a CAR as described herein further comprises a reporter molecule, for example to allow for non-invasive imaging (e.g., positron emission tomography PET scan). In bispecific CARs that comprise a reporter molecule, the first extracellular binding domain and the second extracellular binding domain may comprise different reporter molecules or may comprise the same reporter molecule. In bispecific CAR T cells, the first CAR and the second CAR may express different reporter molecules or may express the same reporter molecule. In another embodiment, a CAR as described herein is capable of expressing a reporter molecule alone or in combination with a substrate or chemical (e.g., 9-[4-[ 18 F]fluoro-3-(hydroxymethyl)butyl]guanine ([ 18 In another embodiment, the CAR as described herein further comprises a reporter molecule (e.g., hygromycin phosphotransferase (hph)) that can be imaged in combination with a non-invasive nanoparticle (e.g., a nanoparticle that can be easily imaged using non-invasive techniques).64 Cu 2+ (Gold nanoparticles (GNPs) functionalized with .GAMMA.) The labeling of CAR T cells for non-invasive imaging is reviewed, for example, in Bhatnagar et al., Integr Biol. (Camb). 5(1):231-238, 2013, and Keu et al., Sci Transl. Med. 18; 9(373), 2017, which are incorporated herein by reference in their entireties.

[0100] GFP and mCherry are demonstrated herein as useful fluorescent tags for imaging CARs expressed on T cells or NK cells (e.g., CAR T cells or CAR NK cells). It is envisioned that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. In clinical applications, CARs do not need to contain a fluorescent tag or fluorescent protein.

[0101] In some embodiments, the CAR polypeptide sequence comprises the sequence of SEQ ID NO: 6, 7, 15, 16, 17, 18, 19, or 20. In some embodiments, the CAR polypeptide 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 more sequence identity to the sequence of SEQ ID NO: 6, 7, 15, 16, 17, 18, 19, or 20.

[0102] Nucleic acid encoding CAR Also provided are nucleic acid constructs and vectors encoding the bispecific CAR polypeptides described herein for use in generating bispecific CAR T. In various embodiments, the invention provides constructs comprising separate coding sequences for multiple proteins, each of which is expressed in the bispecific CAR T cells of the invention. These separate coding sequences can be separated from each other by cleavable linker sequences as described herein. For example, a sequence encoding a viral 2A protein (e.g., T2A) can be placed between separate genes and, once transcribed, can direct cleavage of the generated polyprotein. As indicated above, the constructs and vectors of the invention can comprise any of a number of different sequence combinations.

[0103] Furthermore, the polynucleotide of the present invention can include the expression of a suicide gene. This can be done to facilitate external drug-mediated control of the administered cells. For example, the use of a suicide gene allows modified cells to be depleted from the patient, for example in the case of an adverse event. In one example, the FK506 binding domain is fused to the caspase 9 proapoptotic molecule. T cells engineered in this way are made sensitive to the immunosuppressant tacrolimus. Other examples of suicide genes are thymidine kinase (TK), CD20, thymidylate kinase, truncated prostate specific membrane antigen (PSMA), truncated low affinity nerve growth factor receptor (LNGFR), truncated CD19, and modified Fas, which can be induced for conditional excision by administration of specific molecules (e.g., ganciclovir to TK+ cells) or antibodies or antibody-drug conjugates.

[0104] The constructs comprising sequences encoding proteins for expression in the bispecific CAR T cells of the invention can be contained within a vector. In various embodiments, the vector is a retroviral vector. Retroviruses, such as lentiviruses, provide a convenient platform for delivery of nucleic acid sequences encoding genes or chimeric genes of interest. A selected nucleic acid sequence can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, for example, in vitro or ex vivo. Retroviral systems are well known in the art and are described, for example, in U.S. Pat. No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN:978-1-90455-55-4); and Hu and Pathak Pharmacological Reviews 2000 52:493-512, each of which is incorporated herein by reference in its entirety. An efficient lentiviral system for delivery of DNA can be purchased from OriGene; Rockville, MD. In various embodiments, the protein is expressed in T cells or NK cells by transfection or electroporation of an expression vector comprising a nucleic acid encoding the protein using vectors and methods known in the art. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the viral vector is a retroviral vector (e.g., a lentiviral vector), an adenoviral vector, or an adeno-associated viral vector. In alternative embodiments, the CAR polypeptide of any of the bispecific CARs described herein is expressed in mammalian cells via transfection or electroporation of an expression vector comprising a nucleic acid encoding the CAR. Transfection or electroporation methods are known in the art.

[0105] Efficient expression of bispecific CAR polypeptides in immune cells as described herein can be assessed using standard assays that detect mRNA, DNA, or gene products of nucleic acids encoding proteins. For example, RT-PCR, FACS, Northern blotting, Western blotting, ELISA, or immunohistochemistry can be used. The proteins described herein can be constitutively expressed or inducibly expressed. In some examples, the proteins are encoded by recombinant nucleic acid sequences. In one embodiment, the CAR polypeptides described herein are constitutively expressed. In one embodiment, the CAR polypeptides described herein are encoded by recombinant nucleic acid sequences.

[0106] The invention also provides compositions comprising a vector comprising a polynucleotide sequence encoding a bispecific CAR comprising an extracellular domain comprising a sequence that specifically binds to CD37 and CD19, as described herein.

[0107] For example, the invention provides a nucleic acid capable of encoding a CAR polypeptide comprising a CAR polypeptide of SEQ ID NO: 6, 7, 15, 16, 17, 18, 19 or 20, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence of SEQ ID NO: 6, 7, 15, 16, 17, 18, 19 or 20.

[0108] immune cells One aspect of the technology relates to an immune cell comprising any of the bispecific CAR polypeptides described herein (e.g., SEQ ID NO: 15, 16, 19, or 20); or a nucleic acid encoding any of the bispecific CAR polypeptides described herein. In one embodiment, the immune cell comprises an antibody, an antibody reagent, an antigen-binding portion thereof, or any of the bispecific CARs described herein, or a nucleic acid encoding such an antibody, an antibody reagent, an antigen-binding portion thereof, or any of the bispecific CARs described herein. As used herein, "immune cell" refers to a cell that is instrumental in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. Immune cells can be T cells; NK cells; NKT cells; lymphocytes, such as B cells and T cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the immune cell is a T cell. In other embodiments, the immune cell is a NK cell.

[0109] Immune cells (e.g., human immune cells) that can be used in the present invention include autologous cells obtained from the subject to whom the cells are subsequently administered after ex vivo modification and expansion. For example, immune cells can be obtained from an individual who has or has been diagnosed with cancer, an autoimmune disease, or a plasma cell disorder. Immune cells can also be obtained from an allogeneic donor, a non-genetically identical individual of the same species as the intended recipient of the cells. Immune cells useful in the present invention include T cells and natural killer (NK) cells.

[0110] Methods for obtaining T cells and NK cells are known in the art and may be useful for the engineered immune cells described herein. T cells and NK cells are typically obtained from peripheral blood, which is collected from a subject, for example, by venipuncture or withdrawal through an implanted port or catheter. In some cases, blood can be obtained by a process including leukapheresis, in which leukapheresis cells are obtained from the subject's blood and other blood components are returned to the subject. Blood or leukapheresis products (fresh or frozen) are processed to enrich for T cells or NK cells using methods known in the art. Density gradient centrifugation (e.g., using Ficoll) and / or counter-flow centrifugal elutriation can be performed to enrich for mononuclear cells (including T cells or NK cells). In one example, T cells may be further subjected to a T cell stimulation step using CD3 / CD28 antibodies coated on magnetic beads, or artificial antigen presenting cells (aAPCs) expressing, for example, cell surface-bound anti-CD3 and anti-CD28 antibody fragments (see below), to stimulate the T cells and to deplete other cells, e.g., B cells. The T cells of the enriched T cell preparation may then be subjected to genetic modification.

[0111] Instead of peripheral blood, tissues including bone marrow, lymph nodes, spleen, and tumors can be used as sources of T cells and NK cells. The T cells and NK cells can be from humans, primates, hamsters, rabbits, rodents, bovine, porcine, ovine, equine, Yagi, canine, or feline origin, although other mammalian cells may be used. In certain embodiments of any aspect, the T cells and NK cells are human.

[0112] Immune cells, such as T cells or NK cells, can be engineered to include any of the bispecific CAR polypeptides described herein (e.g., SEQ ID NO: 15, 16, 19, or 20); or a nucleic acid encoding any of the CAR polypeptides described herein. In some embodiments, any of the bispecific CAR polypeptides described herein are included in a lentiviral vector. The lentiviral vector is used to express the CAR polypeptide in the cell using infection standard techniques. In some embodiments, the immune cells (e.g., T cells or NK cells) are obtained from an individual who has or has been diagnosed with a cancer that expresses CD37. In some embodiments, the immune cells are obtained from an individual who is non-responsive to anti-CD19 and / or anti-CD20 therapy and / or an individual who is concurrently receiving anti-CD19 and / or anti-CD20 therapy.

[0113] treatment The present invention provides methods and compositions for use in the treatment and prevention of diseases or conditions, including, for example, cancer, an autoimmune disease or disorder, or a plasma cell disease or disorder. These methods include using immune cells (e.g., T cells or NK cells) that contain a bispecific CAR as described herein, and administering the modified immune cells to a subject, for example, to treat cancer. In certain embodiments of any of the aspects of the invention, the modified immune cells (e.g., T cells or NK cells that contain one or more additional modifications as described herein) are stimulated and / or activated prior to administration to a subject.

[0114] As used herein, "condition" includes cancer, infectious disease, autoimmune disease or disorder, plasma cell dyscrasia or disorder, or transplant-related condition. Subjects with a disease or condition are identified by a physician using current methods of diagnosing the disease or condition. Symptoms and / or complications of the disease or condition that characterize and aid in diagnosis of these conditions are well known in the art and include, but are not limited to, fatigue, persistent infection, and persistent bleeding. For example, tests that can aid in the diagnosis of a disease or condition include, but are not limited to, blood screening and bone marrow tests, and are known in the art for a given condition. Family history of a disease or condition, or exposure to risk factors for a disease or condition, can also help determine whether a subject is likely to have a disease or condition, or make a diagnosis of a disease or condition.

[0115] "Cancer" as used herein may refer to its unique characteristic, the hyperproliferation of cells where loss of normal cellular control leads to unregulated growth, lack of differentiation, local tissue invasion, and metastasis, and may be leukemia, lymphoma, multiple myeloma, or solid tumor. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In one embodiment, the leukemia is CLL. Non-limiting examples of lymphomas include B-cell non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt's lymphoma, hairy cell leukemia (HCL), T-cell lymphoma, peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), angioimmunoblastic T-cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL). In one embodiment, the cancer is MCL, DLBCL, FL, Burkitt's lymphoma, PTCL, CTCL, AITL, or ALCL. Non-limiting examples of solid tumors include adrenal cortical tumors, alveolar soft part sarcoma, carcinomas, chondrosarcomas, colorectal carcinomas, desmoid tumors, desmoplastic small round cell tumors, endocrine tumors, yolk sac tumors, epithelioid hemangioendothelioma, Ewing's sarcoma, germ cell tumors (solid tumors), giant cell tumors of bone and soft tissue, glioblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, renal tumors, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcomas (NRSTS), osteosarcoma, paraspinal sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms' tumor. Solid tumors can be found in bone, muscle, or organs and can be sarcomas or carcinomas. It is contemplated that any aspect of the technology described herein can be used to treat any type of cancer, including cancers not enumerated in this application. As used herein, the term "tumor" refers to an abnormal growth of cells or tissues, for example, of malignant or benign type.

[0116] As used herein, an "autoimmune disease" or "autoimmune disorder" is characterized by the inability of one's own immune system to distinguish between foreign and normal cells. As a result, one's own immune system targets one's own normal cells as a program of 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. Other examples of autoimmune related diseases or disorders include, but are not limited to, rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, Graves' disease (overactive thyroid), Hashimoto's thyroiditis (underactive thyroid), celiac disease, Crohn's disease and ulcerative colitis, Guillain-Barre syndrome, primary biliary sclerosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Good Pasteur's disease, Wegener's granulomatosis, rheumatic polyposis, and rheumatoid arthritis. These include myalgia, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), psoriasis, autoimmune Addison's disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anemia, canine polyarthritis, Reiter syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis and fibromyalgia (FM).

[0117] Plasma cells are white blood cells that develop from B lymphocytes and function to produce and release antibodies necessary to fight infection. As used herein, a "plasma cell disorder or disease" is characterized by abnormal proliferation of plasma cells. Abnormal plasma cells have the ability to "crow out" normal plasma cells, thereby resulting in a reduced ability to fight foreign targets, such as viral or bacterial cells. Non-limiting examples of plasma cell disorders include amyloidosis, Waldenstrom's macroglobulinemia, osteosclerosing myeloma (POEMS syndrome), monoclonal gammopathy of undetermined significance (MGUS), and plasma cell myeloma.

[0118] Retroviruses, such as lentiviruses, provide a convenient platform for the delivery of nucleic acid sequences encoding genes or chimeric genes of interest. A selected nucleic acid sequence can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, for example, in vitro or ex vivo. Retroviral systems are well known in the art and are described, for example, in U.S. Pat. No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN:978-1-90455-55-4); and Hu et al., Pharmacological Reviews 52:493-512, 2000, each of which is incorporated herein by reference in its entirety. Efficient lentiviral systems for the delivery of DNA can be purchased from OriGene; Rockville, MD.

[0119] One aspect of the technology described herein relates to a method of treating cancer in a patient in need thereof, the method comprising administering any of the mammalian cells comprising any of the CAR polypeptides described herein.

[0120] Cluster of differentiation (CD) molecules are cell surface markers present on white blood cells. As white blood cells differentiate and mature, their CD profile changes. When white blood cells turn into cancer cells (i.e. lymphoma), their CD profile is important for diagnosing the disease. The treatment and prognosis of certain types of cancer depend on determining the CD profile of the cancer cells. "CDX+", where "X" is a CD marker, indicates that the CD marker is present on the cancer cell, and "CDX-" indicates that the marker is absent. One skilled in the art can assess the presence of CD molecules on cancer cells using standard techniques, for example, immunofluorescence to detect commercially available antibodies bound to CD molecules.

[0121] In some embodiments, the cancer expresses one or more CD37 molecules. The bispecific CARs described herein can be used to treat cancers that express CD37. In some embodiments, the CD37+ cancer is a lymphoma or leukemia. For example, the lymphoma is a B-cell non-Hodgkin's lymphoma (NHL) (e.g., mantle cell lymphoma (MCL), Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, or Burkitt's lymphoma), or a T-cell lymphoma (e.g., peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), angioimmunoblastic T-cell lymphoma (AITL), or anaplastic large cell lymphoma (ALCL)). In another example, the leukemia is chronic lymphocytic leukemia (CLL).

[0122] Furthermore, cancer cells can evolve in response to treatment and change their CD profile to avoid said treatment.For example, a patient with CD19+ leukemia or lymphoma can be treated with anti-CD19 therapy.After treatment, the cancer cells recur or recur after treatment and no longer express CD19 marker, causing CD19 leukemia or lymphoma.As a result, the cancer is no longer targetable by anti-CD19 therapy.

[0123] Also provided are methods of using immune cells comprising a bispecific CAR (e.g., a CAR targeting CD37 and CD19) described herein to treat cancer in subjects who are non-responsive or refractory to anti-CD19 and / or anti-CD20 therapy. In some embodiments, immune cells comprising a bispecific CAR (e.g., a CAR targeting CD37 and CD19) described herein are used to treat cancer in subjects who have a cancer that is CD19- and / or CD20-. In some embodiments, immune cells comprising a bispecific CAR (e.g., a CAR targeting CD37 and CD19) described herein are used to treat cancer in subjects who have a cancer that has relapsed or no longer expresses CD19 or CD20.

[0124] Furthermore, immune cells comprising a bispecific CAR described herein (e.g., a CAR targeting CD37 and CD19) can be used to treat cancer in a subject in need thereof, where the subject is simultaneously administered anti-CD19 and / or anti-CD20 therapy.

[0125] In certain embodiments of any of the aspects of the invention, the immune cells (e.g., T cells or NK cells) comprising the bispecific CAR are stimulated and / or activated prior to administration to a subject.

[0126] Administration In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer, a plasma cell disease or disorder, or an autoimmune disease or disorder with a mammalian cell comprising any of the CAR polypeptides described herein, or a nucleic acid encoding any of the CAR polypeptides described herein. A bispecific CAR T or NK cell as used herein refers to a mammalian T or NK cell that comprises any of the bispecific CAR polypeptides as described, or a nucleic acid encoding any of the bispecific CAR polypeptides.

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

[0128] The term "effective amount" as used herein refers to the amount of bispecific CAR T or NK cells required to alleviate one or more symptoms of a disease or disorder, and relates to a sufficient amount of cell preparation or composition to provide the desired effect. Thus, the term "therapeutically effective amount" refers to an amount of CAR T or NK cells that is sufficient to provide a particular effect against a condition when administered to a typical subject. Effective amount as used herein also includes an amount sufficient to delay the onset of a disease symptom, alter the course of a disease symptom (e.g., delay the progression of a condition), or reverse a condition symptom, in various contexts. Thus, it is usually not practical to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

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

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

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

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

[0133] Dosage As used herein, a "unit dosage form" refers to a dosage amount suitable for one administration. As an example, a unit dosage form may be the amount of a therapeutic agent placed in a delivery device such as a syringe or a drip bag. In one embodiment, a unit dosage form is administered in a single dose. In another embodiment, more than one unit dosage form may be administered simultaneously.

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

[0135] Pharmaceutical compositions comprising the T or NK cells described herein are typically administered at a dose of 10 4 From 10 9 Cell doses were in the range of 10 / kg body weight in some cases. 5 From 10 6 The cells may be administered in doses ranging from 0.1 to 100 mg / kg / day, including all integer values ​​within those ranges. If desired, the T or NK cell compositions may be administered multiple times at these doses. The cells may be administered using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 319:1676, 1988).

[0136] In some embodiments, it may be desirable to administer bispecific CAR T or NK cells to a subject, followed by redrawing blood (or performing apheresis), activating T or NK cells therefrom as described herein, and reinfusing these activated and expanded T or NK cells back into the patient. This process may be performed multiple times every few weeks. In some embodiments, T or NK cells may be activated from a blood draw of 10cc to 400cc. In some embodiments, T or NK cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc.

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

[0138] In certain exemplary aspects, the subject may be subjected to leukapheresis, in which leukocytes are harvested, enriched, or depleted ex vivo to select and / or isolate immune cells of interest, e.g., T cells or NK cells. These immune cell isolates may be expanded, for example, in the case of T cells, by contact with aAPCs as described herein, e.g., aAPCs expressing anti-CD28 and anti-CD3 CDRs, and treated to introduce one or more CAR constructs of the present technology, thereby generating CAR T cells. The subject in need thereof may then be subjected to standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. After or simultaneously with transplantation, the subject may receive an infusion of the expanded CAR T cells or NK cells. In one embodiment, the expanded cells are administered before or after surgery.

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

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

[0141] In some embodiments, a single treatment regimen is required. One or more subsequent doses or treatment regimen administrations may be performed. For example, treatment may be every other week for three months, followed by monthly treatment for six months or more. In some embodiments, no additional treatments are administered after the first treatment.

[0142] The dosage of the compositions as described herein can be determined by a physician and adjusted as necessary to suit the observed therapeutic effect. With regard to the duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when the treatment produces a therapeutic effect and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other changes to 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 age, condition, and sex of the patient, and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of complications.

[0143] Combination therapy The bispecific CAR T or NK cells described herein can be used in combination with other known drugs and therapies. For example, the subject can be further administered anti-CD19 therapy and / or anti-CD20 therapy. In one embodiment, the subject is resistant to anti-CD19 therapy and / or anti-CD20 therapy. In another embodiment, the subject is administered anti-CD19 therapy and / or anti-CD20 therapy simultaneously.

[0144] As used herein, administered "in combination" means that two (or more) different therapies are delivered to a subject during the course of the subject's suffering from a disorder, for example, two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated or after the treatment is stopped for other reasons. In some embodiments, when the delivery of the second therapeutic agent begins, the delivery of one therapeutic agent is still occurring, so there is an overlap in terms of administration. This may be referred to herein as "simultaneous" or "concurrent" delivery. In other embodiments, the delivery of one therapeutic agent ends before the delivery of the other begins. In either case of the embodiment, the combined administration makes the therapeutic agents more effective. For example, the second therapeutic agent is more effective. For example, the same effect is seen with less of the second therapeutic agent than would be seen if the second therapeutic agent were administered without the first therapeutic agent being present, or the second therapeutic agent significantly reduces symptoms, or a similar situation is seen with the first therapeutic agent. In some embodiments, the delivery is such that the reduction in symptoms, or other parameters related to the disorder, is greater than would be observed with one therapeutic agent delivered in the absence of the other. The effect of the two therapeutic agents may be partially additive, fully additive, or more than additive. The delivery may be such that the effect of the first therapeutic agent delivered is still detectable when the second therapeutic agent is delivered. The bispecific CAR T or NK cells described herein and at least one additional therapeutic agent may be administered simultaneously, in the same or different compositions, or sequentially. For sequential administration, the CAR-expressing immune cells described herein may be administered first and the additional agent may be administered second, or the order of administration may be reversed. The bispecific CAR T or NK therapy and / or other therapeutic agents, procedures, or modalities may be administered during periods of active disorder or during periods of remission or less active disease. The CAR T or NK therapy may be administered before another treatment, simultaneously with another treatment, after another treatment, or during remission of the disorder.

[0145] When administered in combination, the bispecific CAR T or NK cells and additional agent (e.g., second or third agent), or all of them, can be administered in amounts or dosages that are greater than, less than, or the same as the amount or dosage of each agent used individually, e.g., as a monotherapy. In some embodiments, the amount or dosage of the bispecific CAR T or NK cells, additional agent (e.g., second or third agent), or all of them administered is (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) less than the amount or dosage of each agent used individually. In other embodiments, the amount or dosage of the bispecific CAR T or NK cells, additional agent (e.g., second or third agent), or all of them that results in a desired effect (e.g., treatment of cancer) is (e.g., at least 20%, at least 30%, at least 40%, or at least 50% less) than the amount or dosage of each agent required individually to achieve the same therapeutic effect. In further embodiments, the bispecific CAR T or NK cells described herein can be used in a treatment regimen in combination with surgery, chemotherapy, radiation therapy, mTOR pathway inhibitors, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, or peptide vaccines, such as those described in Izumoto et al., J. Neurosurg. 108:963- 971, 2008.

[0146] For example, the bispecific CAR T or NK cells described herein can be used in combination with anti-CD19 therapy. Examples of anti-CD19 therapy include, but are not limited to, blinatumomab, cortuximab ravtansine, MOR208, MEDI-551, denintuzumab mafodotin, taplitumomaba paptox, XmAb 5871, MDX-1342, AFM11, DI-B4, axicabtagene ciloleucel, and tisagenlecleucel. Furthermore, the bispecific CAR T or NK cells described herein can be used in combination with anti-CD20 therapy. Examples of anti-CD20 therapy include, but are not limited to, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tiuxetan, tositumomab, ublituximab, ocaratuzumab, IMMU-106, and GA-101.

[0147] In further embodiments, the bispecific CAR T or NK cells described herein can 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-CTLA-4 inhibitors (ipilimumab and tremelimumab), anti-PD-L1 inhibitors (atezolizumab, avelomab, durvalumab, MSB0010718C, MEDI4736 and MPDL3280A), and anti-TIM3 inhibitors.

[0148] In further embodiments, the bispecific CAR T or NK cells described herein can 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, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), antimetabolites (e.g., cefotaxime ... These include immunomodulatory agents such as, for example, folate antagonists, pyrimidine analogues, purine analogues and adenosine deaminase inhibitors (e.g., fludarabine), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin or bortezomib), thalidomide or thalidomide derivatives (e.g., lenalidomide). Common chemotherapy agents that may be 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®), and carboplatin (Calplatin®). cisplatin (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (ActinomycinD, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), ibrutinib, (IMBRUVICA®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (EL SPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Mylotarg, paclitaxel (Taxol®), Phoenix (Yttrium 90 / MX-DTPA), pentostatin, polipheprosan 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 mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, and triazenes: uracil mustard (AminouracilMustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune TM), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (also known as actinomycin-D; Cosmegen®); melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard; Alkeran®); altretamine (also known as hexamethylmelamine (HMM); Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Bus ulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU; CeeNU®); cisplatin (also known as CDDP; Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (also known as DTIC, DIC, and imidazole carboxamide; DTIC-Dome®); altretamine (also known as hexamethylmelamine (HMM).These include: Hexalen®; ifosfamide (Ifex®); prednumustine (Prednumustine); procarbazine (Matulane®); mechlorethamine (nitrogen mustard, also known as mustine and mechlorethamine hydrochloride, Mustargen®); streptozocin (Zanosar®); thiotepa (thiophosphamide, also known as TESPA and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine HC1 (Treanda®).Exemplary mTOR inhibitors include, for example, temsirolimus; ridafolimus (formerly 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-ll,36-dioxa-4-azatricyclo[30.3.1.04'9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication WO 03 / 064383; everolimus (Afinitor® or RADOOl); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3; emsirolimus, (5-{2,4-bis[(35,)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-JJpyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-l-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-La-aspartyl-L-serine-, inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplary immunomodulatory agents include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (a mixture of human cytokines including interleukin-1, interleukin-2, and interferon-gamma, CAS 951209-71-5, available from IRX Therapeutics). Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomes (daunorubicin citrate liposomes, DaunoXome®); mitoxantrone (DHAD, Novantron®); TM); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; rabidomycin; and desacetylrabidomycin. Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). Exemplary proteosome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, (5)-4-methyl-N-((5)-l-(((5)-4-methyl-l-((R)-2-methyloxiran-2-yl)-l-oxopentan-2-yl)amino)-l-oxo-3-phenylpropan-2-yl)-2-((5,)-2-(2-morpholinoacetamido)-4-phenylbutanal). ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(11S')-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0149] Those skilled in the art can readily identify the chemotherapeutic agent to be used (see, 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).

[0150] In one embodiment, the bispecific CAR T or NK cells described herein are administered to a subject in combination with a molecule that reduces the level and / or activity of a molecule that targets GITR and / or modulates GITR function, a molecule that reduces the Treg cell population, an mTOR inhibitor, a GITR agonist, a kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a CDK4 inhibitor, and / or a BTK inhibitor.

[0151] Effectiveness For example, the efficacy of bispecific CAR T or NK cells in treating a condition described herein or for inducing a response described herein (e.g., reduction in cancer cells) can be determined by a skilled clinician. However, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, if other clinically recognized symptoms are improved, or if a desired response is induced, for example, by at least 10% after treatment with the methods described herein, the treatment is considered to be an "effective treatment" as that term is used herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or occurrence of a condition treated by the methods described herein, or any other suitable measurable parameter. Treatment with the methods described herein can reduce the level of a marker or symptom of a condition by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0152] Efficacy can also be measured by the absence of deterioration of an individual as assessed by hospitalization or the need for medical intervention (i.e., disease progression is halted). Methods for measuring these indicators are known to those of skill in the art and are described herein.

[0153] Treatment includes the treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) alleviating the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means an amount sufficient to provide effective treatment for the disease, as that term is defined herein, when administered to a subject in need thereof. The efficacy of an agent can be determined by assessing physical indicators of the condition or desired response. It is within the ability of one of ordinary skill in the art to monitor the efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. The efficacy of a given approach can be evaluated in animal models of the conditions described herein, e.g., the treatment of cancer. When using experimental animal models, the efficacy of the treatment is demonstrated when a statistically significant change in the marker is observed.

[0154] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methods described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior art or for any other reason. All statements as to the date or contents of these documents are based on the information available to the applicant and do not constitute an admission as to the accuracy of the dates or contents of these documents.

[0155] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as will be recognized by those skilled in the relevant art. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, using the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure. Furthermore, some changes can be made to protein structures without affecting the type or amount of biological or chemical action, due to considerations of biological functional equivalence. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0156] Certain elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages in order to fall within the scope of the present disclosure.

[0157] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any way. EXAMPLES

[0158] The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments can be implemented given the description provided herein. Examples 1-11 provide the materials and methods used in the experiments described herein, Examples 12-19 demonstrate the results, and Example 20 provides a sequence listing.

[0159] Example 1 Primary Human T Cell Culture For expansion of primary T lymphocytes, bulk human T cells were activated using anti-CD3 / CD28 Dynabeads (LifeTechnologies) (day 0) and subsequently transduced with a lentiviral vector encoding a CAR 24 h later. Starting on day 0 of culture, T cells were cultured in medium supplemented with 20 IU / ml rhIL-2 and measured every 2-3 days to reach a concentration of 0.5 × 10 6 For functional assays, CAR T cells were cryopreserved on days 8-10 of culture, and upon thawing, they were either immediately stimulated with antigen or injected into mice.

[0160] Example 2 Cell lines and culture conditions JEKO-1, RAJI and wild-type K562 parental cells were purchased from American Type Culture Collection (ATCC). K562 cells were engineered to express CD37 and CD19 (K562-CD37-CD19). For some assays, cell lines were engineered to constitutively express click beetle green (CBG) luciferase / enhanced GFP (eGFP) and then sorted on a FACSAria (BD Biosciences®) to obtain ≧99% pure populations (CBG-GFP+). Cell lines were cultured in RPMI medium containing 10% fetal bovine serum (FBS), penicillin and streptomycin.

[0161] Example 3 Flow Cytometry The following antibodies were used: CD37-APC (clone MB-1, eBioscience®), CD37-BV711 (clone MB-371, BD Biosciences®), CD19-Pacific Blue (clone HIB19, Biolegend®), CD19-FITC (clone 4G7, BD), CD5-BUV737 (clone UCHT2, BD), CD20-APC Cy7 (clone 2H7, Biolegend®), CD79b-PE (clone CB3-1, eBioscience®), CD3-BV786 (clone SK7, BD), CD3-BV605 (clone OKT3, Biolegend®), CD45-PeCy7 (clone HI30, Biolegend®), CD16-PE (clone B73.1, Biolegend®), CD14-Pacific Blue (clone HCD14, Biologend®), CD56-APC (clone HCD56, Biolegend®), CD33-BV510 (clone P67.6, Biolegend®), CD107a-AF700 (clone H4A3, BD Biosciences®), CD69-APC (clone FN50, Biolegend®), and IFNγ-FITC (clone GZ-4, eBioscience®). Cells were stained for 30 min at 4°C in the dark and washed twice in PBS with 2% FBS. DAPI was added prior to acquisition to gate on live cells. Antigen density was measured using antibody binding per cell (ABC) and analyzed using Quantum TM Calculations were performed using Simply Cellular (Bangs Laboratories).

[0162] Example 4 Fratricide Assay Human T cells purified from anonymous human healthy donor leukopacks were activated with Cell Stimulation Cocktail (eBioscience®, Catalog No. 00-4970-03) for 6 hours. Activated and non-activated T cells were labeled with CFSE (ThermoFisher®, Catalog No. C34554) according to the manufacturer's instructions and co-cultured with CAR-37, CAR-19 or non-transduced T cells generated from the same normal donor. After 24 hours, the number of CFSE-positive cells in each condition was counted using flow cytometry.

[0163] Example 5 Immune Cell Isolation, Differentiation, and Co-Culture Assays PBMCs from three normal donors were isolated using Ficoll-Paque PLUS (GE Healthcare, C987R36) and monocytes were purified using the StemCell® kit (cat. no. 19359). M1 / M2 macrophages were generated in vivo as previously described (Zhang et al., PLoS One. 11(4):e0153550, 2016). Monocytes, macrophages, NK cells and T cells were cultured with CAR-37 CAR-19 or non-transduced T cells at a 1:1 E:T ratio for 6 hours and CD107a and IFNγ production was measured by flow cytometry. PMA / ionomycin was used as a positive control. Values ​​were normalized to culture medium. Graphs represent fold change.

[0164] Example 6 Cytotoxicity and Cytokine Assays For cytotoxicity assay, CAR T cell effector cells were co-cultured with CBG luciferase expressing tumor target at the indicated ratio for 16 hours. Luciferase activity was measured using Synergy Neo2 Luminescence Microplate Reader (Biotek®). For soluble cytokine analysis, effector cells were co-cultured with tumor target at 1:1 ratio for 24 hours.

[0165] CAR-37 and CAR-19 T cells were normalized for CAR expression by adding non-transduced but cultured and activated T cells from the same donor to obtain the same percentage of CAR+ T cells in each sample. For cytotoxicity assays, percent specific lysis was calculated by the following equation: % specific lysis = (total RLU / target cell only RLU) x 100. For cytokine assays, cell-free supernatants were analyzed for cytokine expression using Luminex arrays (Luminex Corp, FLEXMAP 3D®) according to the manufacturer's instructions. All samples were measured in technical duplicates. Duplicates were averaged before graphing using GraphPad Prism® 7 (version 7.0). In addition, all assays were performed in biological duplicates or triplicates, as indicated by N in each experiment, based on the number of unique healthy donor T cells tested.

[0166] Example 7 Jurkat reporter activation assay Jurkat (NFAT-Luc) reporter cells (Signosis, SL-0032) were transfected with different CAR constructs. They were co-cultured with B-cell lymphoma tumor cells or Nalm6 leukemia cells at an E:T ratio of 1:1 for 24 hours. Anti-CD3 / CD28 beads were used as positive control and medium as negative control. Luciferase activity was measured after 16 hours using a Synergy Neo2 Luminescence Microplate Reader (Biotek®). Relative activation was calculated with PMA.

[0167] Example 8 Immunohistochemistry Paraffin sections were deparaffinized in xylene, followed by a series of ethanol washes followed by H 2 The slides were rehydrated with 200 mL of 0.01 M sodium citrate buffer, pH 6.0, for 15 min. Antigen retrieval was performed by microwave heating the slides for 15 min. After washing with phosphate-buffered saline containing 0.1% Tween-20 (PBS-T), endogenous peroxidase activity was detected by 3% 0.01 M sodium citrate buffer, pH 6.0. 2O 2 The slides were then washed again with PBS-T and blocked with Novolink Protein Block for 30 min at 25° C. After an additional wash with PBS-T, the slides were incubated in PBS-T containing 5% goat serum and mouse anti-CD37 (Invitrogen®, Cat. No. MA5-15492) diluted 1:150 for 1 h at 25° C. After washing with PBS-T, the slides were incubated with Cell Signaling Technology Signal Stain Boost IHC Mouse Detection Reagent for 30 min at 25° C., washed again with PBS-T, and incubated in DAB Diluent (Vector Labs) containing DAB chromogen. After staining development, the slides were washed again with PBS-T and counterstained with hematoxylin.

[0168] Example 9 TMA Structure Formalin-fixed, paraffin-embedded tissues involving peripheral T-cell lymphoma (PTCL) were retrieved from the archives of the Department of Pathology at Brigham and Women's Hospital. Cores (0.6 mm diameter) from donor blocks were transferred to recipient blocks for tissue microarray construction at the Tissue Microarray Core of the Dana Farber / Harvard Cancer Center and used to prepare 4-micron sections for immunohistochemical staining studies.

[0169] Example 10 In vivo studies NOD-SCID-γ-chain- / - (NSG) (The Jackson Laboratory®) were engrafted with JEKO-1 cell line or patient-derived tumor cells via the described route of administration. After tumor engraftment was confirmed by luminescence, cryopreserved CAR-37, CAR-19, or non-transduced T cells were injected intravenously. Tumor burden was monitored periodically using an Ami Spectral Imaging device and analyzed with IDL software v. 4.3.1 after intraperitoneal injection of D-luciferin substrate solution (30 mg / mL). Animals were euthanized according to the experimental protocol or when they reached pre-specified endpoints defined by the IACUC.

[0170] Example 11 Statistical Analysis Unless otherwise stated, two-tailed Student's t-test or two-way analysis of variance was used for normal data with equal variance. Significance was considered when P<0.05. Analyses were performed using GraphPad Prism® 7 (version 7.0).

[0171] Example 12 CAR construction and T cell culture transduction Two anti-CD37 CAR constructs were synthesized and cloned into a third generation lentiviral plasmid backbone under the restriction of the human EF-1α promoter. All of the CARs have a CD8 hinge, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. The vectors also contained a second transgene encoding the fluorescent reporter mCherry to facilitate enumeration of transduction efficiency. Human T cells were purified from anonymous human healthy donor leukopacks purchased from the MGH blood bank under an IND-exempt protocol (StemCell Technologies®, Catalog No. 15061). Details of the T cell culture are provided in Example 1.

[0172] Example 13 CD37 is highly expressed in human lymphomas. Using flow cytometry, we investigated the expression of CD37 and CD19 in leukocytes and non-Hodgkin's lymphoma cell lines Nalm6, Jeko-1, and Raji (Figure 1A), and in patient-derived MCL cell lines (Figures 1B and 1C), and in primary patient CLL cells (Figure 1D). We also produced K562 cells transfected with both CD37 and CD19 to use as artificial antigen-presenting cells for in vitro stimulation and as a positive control for cytotoxicity assays (Figure 1A). Pre-B cell-derived leukemia cells (Nalm6) expressed CD19 but not CD37 (Figure 1A), and all lymphoma cells expressed both CD19 and CD37 (Figures 1A-1C). In patient-derived MCL samples, we recorded a high and uniform expression of CD37, even higher than CD19, based on the mean fluorescence intensity of gated positive cells, although it is recognized that this difference may reflect differences in antibody binding or fluorophore brightness (Figure 1C).

[0173] We next assessed CD37 and CD19 expression in PBMCs from 21 patients with chronic lymphocytic leukemia by flow cytometry (Figure 1D), again documenting a higher and more uniform expression of CD37 among CD3-negative lymphocytes compared to CD19. When gated on CD3-CD20+ B cells, CD37 expression remains high and uniform (Figure 1E). To determine the level of antigen density for both antigens, CD19 and CD37 expression in these 21 samples was quantified using beads (Figure 2A). We observed that the antigen density per cell was higher for CD19 than for CD37 (for CD19, mean = 31,829 ± 3,212 antibody bound (ABC) per cell, compared to 29,680 ± 3232 ABC for CD37 (Figure 2B, Table 1).

[0174] TIFF0007611705000001.tif116170

[0175] Based on 29 samples of bone marrow aspirates, lymph node biopsies, and peripheral blood from patients with hematologic malignancies, we confirmed expression of CD37 on B-cell lymphomas and normal B cells but not on hematopoietic stem cells from normal donors or on hematogones (Table 2).

[0176] TIFF0007611705000002.tif237170TIFF0007611705000003.tif239170TIFF0007611705000004.tif237170TIFF0007611705000005.tif234170

[0177] In this dataset, there were no samples from patients with T-cell malignancies. Therefore, CD37 expression was evaluated in primary peripheral T-cell lymphomas by performing immunohistochemical staining on tissue microarrays containing triplicate cores of 67 PTCL samples from nine different subtypes. Overall, positive staining was seen in at least a subset of cells in patient samples from each subtype, including 15 of 16 AILT, 1 of 1 ALK+ anaplastic large cell lymphoma (ALCL), 6 of 13 ALK-negative ALCL, 2 of 6 adult T-cell leukemia, 18 of 23 PTCL, 1 of 1 enteropathy-associated T-cell lymphoma, 4 of 4 extranodal NK / T-cell lymphoma, nasal type, 1 of 1 T-cell prolymphocytic leukemia, and 1 of 2 hepatosplenic gamma / delta T-cell lymphoma, unless otherwise specified. Typical strong staining of CD37 in PTCL (ALCL) samples is shown in FIG. 2C.

[0178] Example 14 Generation of anti-CD37 CAR T cells Two anti-CD37 chimeric antigen receptors were designed, consisting of anti-CD37 scFv and CD8 transmembrane domain in tandem with 4-1BB intracellular signaling domain and CD3ζ (Figure 3A). The scFvs were synthesized in both variable heavy and light chain orientations to generate CAR-37 LH and CAR-37 HL. To facilitate evaluation of transduction efficiency, the mCherry fluorescent reporter gene was incorporated after a 2A ribosomal skip sequence at the C-terminus of the CAR sequence. Highly efficient gene transfer into primary activated human T cells was obtained with both constructs using third generation self-inactivating lentiviral vectors (Figures 3B and 3C). CAR-37 T cells showed proliferation after initial priming with anti-CD3 / CD28 beads for the first 10 days, comparable to anti-CD19 CAR T cells (CAR-19). As a comparison, CAR-19 T cells were generated based on the same scaffold with CD8 transmembrane domain and 4-1BB and CD3ζ intracellular signaling domains. We found that CAR-37 T cells could be proliferated long-term through repeated antigen stimulation with irradiated K562 cells transduced to express CD37 and CD19 (Figure 3D). We next tested the activation of CAR using Jurkat reporter (NFAT-luciferase) T cells. After transducing different CAR constructs into Jurkat reporter cells, we co-cultured the cells with various stimuli including anti-CD3 / CD28 beads, B cell lymphoma tumor cells, Nalm6 leukemia cells, or medium as a negative control. By measuring fluorescence, we demonstrated specific T cell activation and NFAT-mediated fluorescence in response to antigen stimulation (Figure 3E). In this assay, anti-CD37 CAR in LH orientation appeared to initiate activation more robustly than anti-CD37 CAR in HL orientation and at similar levels as anti-CD19 CAR in response to CD19-expressing tumors. However, there is no known threshold or optimal amount of NFAT translocation that corresponds to an optimal CAR construct. These data indicate that anti-CD37 CARs mediate T cell activation signals in response to specific antigen stimulation and are capable of long-term growth in response to antigen stimulation.

[0179] Since CD37 has been reported to be expressed on T cells and other hematopoietic mononuclear cells, we collated the expression of CD37 on whole blood immune cells of healthy donors (Figure 3F-3I). High expression of CD37 was observed on B cells, with minimal expression on monocytes, but not on NK cells or T cells. However, since many T cell markers change with activation, we tested the possibility of fratricide by co-culturing CAR-37 T cells with activated or non-activated CFSE-labeled T cells of the same donor. After 24 h, the number of target T cells was analyzed. Despite the expected cytotoxicity against Jeko-1 target cells in the same experiment, no significant differences were detected in the number of labeled resting T cells (Figure 4A) or activated T cells (Figure 4B) co-cultured with CAR-37 T cells compared to those cultured with CAR-19 T cells (Figure 4C). We then tested the degranulation and IFNγ production of CAR-37 T cells against monocytes, NK cells, and in vitro differentiated M1 or M2 macrophages (Figure 4D). No significant differences in degranulation or IFNγ production were observed between CAR-37 and CAR-19 T cells, indicating no evidence of immune cytotoxicity induced by CAR-37 T cells.

[0180] Example 15 CAR-37 T cells exhibit robust effector function in response to CD37-positive tumor cells in vitro To define the antitumor activity of CAR-37 T cells, cytotoxicity assays were performed against a panel of lymphoma cell lines. CAR-37 T cells were co-cultured with Jeko-1, OSU-CLL, Raji or K562-CD37-CD19 cells at various effector-to-target ratios for 16 h (Figure 5). All CAR-37 T cell effectors were able to lyse target cells, but in contrast to what was observed in the Jurkat activation assay, the heavy-light chain configuration was preferred over the light-heavy configuration of anti-CD37 CAR T cells. This difference was clear and consistent with all tumor lines tested. Notably, CAR-37 HL or CAR-19 transduced T cells demonstrated comparable cytolytic activity against these target tumor cells, all of which express both antigens.

[0181] Next, we analyzed cytokine production in response to antigen stimulation. We compared the distinct patterns of cytokines produced by different CAR constructs after stimulation with target cells. CAR-37 T cells demonstrated antigen-specific production of Th1-type cytokines TNF-α, IFN-γ, IL-2, and GM-CSF after in vitro stimulation with tumor cell lines (Figures 6C and 6D), primary CLL (Figure 6A), and MCL patient-derived xenograft (PDX) samples (Figure 6B). Consistent with the cytotoxicity assays, these experiments demonstrated improved antigen-specific effector function of CAR-37 HL compared to CAR-37 LH.

[0182] Example 16 CAR-37 T cells eradicate MCL tumors in vivo As in vitro assays indicate that CAR-37 HL may be superior to CAR-37 LH but are not conclusive, these two formats were compared in a xenogeneic model of MCL. NSG mice were injected intravenously with luciferase-expressing Jeko-1 (CBG-GFP+) cells. Seven days later, disease burden in all mice was assessed by bioluminescence imaging (BLI) and CAR-37 or untransduced (UTD) T cells were administered by tail vein injection. By day 14, there was partial disease control in CAR-37 LH-treated animals, whereas CAR-37 HL-treated mice had complete eradication of disease (Figures 7A-7C), thus confirming the superior antigen-induced effector function of CAR-37 HL over CAR-37 LH, and CAR-37 HL was subsequently selected for further experiments and for direct comparison with CAR-19 T cells in vivo.

[0183] As shown, CAR T cells were injected into NSG mice 7 days after intravenous injection of the MCL cell line Jeko-1 (CBG-GFP+) (Figure 7D). Serial imaging of luminescence to assess tumor burden showed rapid and complete elimination of tumors by day 14 for both CAR-37 and CAR-19 T cells. By day 7, tumor volume was dramatically reduced (Figures 7E and 7F). Flow cytometry confirmed the persistence of CAR T-cells in the peripheral blood (Figure 7G). More CAR-37 T cells persisted at day 7 (p<0.05).

[0184] Although tumor cell lines are quite useful for evaluating the efficacy of CAR T cells, they often do not fully represent the heterogeneity and biology of primary patient tumors. In contrast, PDX models, in which tumor cells are derived directly from the patient and cultured for only 2-3 passages, are considered to more closely resemble the clinical context. We evaluated the efficacy of CAR-37 (HL configuration) in a PDX model of MCL. Ten NSG mice were injected with luciferase-expressing MCL-PDX cells. After confirming disease engraftment and tumor burden similar to the Jeko-1 model by BLI, CAR-37, CAR-19 or non-transduced T cells were injected (Figure 8A). We observed that CAR-37 T cells were able to eliminate tumors significantly more quickly than CAR-19, in as little as 12 days (p<0.05 at day 12) (Figures 8B and 8C). Flow cytometric evaluation of peripheral blood collected on day 14 confirmed the persistence of CAR T cells in the blood (Figure 8D). Collectively, these results demonstrate that CAR-37 T cells mediate significant antitumor effects against B-cell NHL in vivo, both in tumor lines and PDX models of MCL.

[0185] Example 17 Targeting CD37 in T-cell lymphoma Surface expression of CD37 in PTCL lines and PTCL PDX samples was analyzed by flow cytometry. Three cell lines (Hut78, Fedp, and Seax) and five PDX samples were identified that expressed CD37 on the cell surface at various levels (Figures 9A and 9B, Table 3).

[0186] TIFF0007611705000006.tif51170

[0187] We tested the activation of CAR-37 T cells after co-culture with these PTCL samples. The CD69 activation marker was analyzed by flow cytometry of gated CAR+ T cells. After 6 hours of co-culture with PTCL target cells, we observed a robust upregulation of CD69, which indicated CAR-37 T cell activation (Figure 10A). Interestingly, the degree of CD69 upregulation was independent of the proliferation level of CD37 antigen on the target cells. We then tested the degranulation of CAR-37 T cells by measuring the expression of CD107a in CAR-37 cells after incubation with PTCL target cells (Figure 10B). Consistent with the CD69 assay, we observed that CAR-37 T cells degranulated in response to PTCL cells. This indicated the activation of CAR-37 T cells in response to PTCL. Finally, we observed that CAR-37 T cells effectively lyse the CD37-positive PTCL cell lines Hut78 and Fepd, as determined by cytotoxicity assays performed at various E:T ratios (Figures 10C and 10D). Collectively, these experiments demonstrated that CAR-37 T cells can be activated against and lyse T cell lymphoma cells.

[0188] Example 18 Bispecific CAR T cells against CD19 and CD37 A possible means to avoid antigen escape is to generate T cells capable of recognizing multiple antigens (Ruella et al., J. Clin. Invest. 126:3814-26, 2016; Zah et al., Cancer Immunology Research. 4:498-508, 2016). Dual targeting of CD19 and CD37 was previously explored with a biligand immunoliposome approach that yielded effective results in B-CLL cells (Yu et al., Biomaterials. 34:6185-6193, 2013). We designed a bispecific CAR that could efficiently trigger T cell activation when CD19 or CD37, alone or in combination, are present on target cells. Two constructs were generated and tested with anti-CD19 and anti-CD37 scFvs linked in tandem in different orders in the second generation 4-1BB-CD3ζ vector (Figure 11A). Even when the same MOI was used, CAR19-37 T cells were observed to have a lower transduction efficiency compared to CAR37-19 (Figures 11B and 11C).

[0189] To determine whether bispecific CAR T cells could be activated in response to either antigen, CAR-19, CAR-37, bispecific CAR19-37, and bispecific CAR37-19, we generated Jurkat NFAT-Luc reporter cell lines and analyzed activation after overnight co-culture with target cells. Bispecific CAR T cells showed high NFAT activation when either a single antigen or both antigens were present on the surface of target cells, and there was no discernible difference in the activation signal of bispecific CAR T cells when they were activated by CD19 alone or CD37 alone (Figure 12A). Bispecific CAR T cells showed comparable proliferation after initial priming with anti-CD3 / CD28 beads for the first 10 days (Figure 12B). Similarly, we found that bispecific CAR T cells could be expanded long-term through repeated antigen stimulation with K562 expressing CD19 and CD37 (Figure 12B). In cytotoxicity assays with primary human CAR T cells, we observed that the bispecific CAR T cells responded to a single target or both targets, with no discernible differences in cytotoxicity against CD19 or CD37 (Figure 12C). Finally, the bispecific constructs were tested in vivo against Jeko-1 tumor cells in comparison to conventional CAR-37 and CAR-19 T cells. By day 14 post-treatment, there was complete disease eradication in all groups except for CAR-19-37, which showed partial disease control (Figure 12D). This discrepancy may be related to the structural differences between the two antigens and the order of the scFvs in the CAR constructs. The persistence of CAR T cells in the peripheral blood was confirmed by flow cytometry by day 35 of treatment (Figure 12E). At all time points tested, there were no discernible differences between CAR-37, CAR-19, and the optimal tumor-clearing bispecific CAR37-19. Interestingly, the suboptimal CAR 19-37 remained more abundant at day 35, which may reflect persistent antigen stimulation by the tumor.Collectively, these data demonstrate that bispecific CAR T cells caused specific target lysis of cells expressing one or both antigens on their surface, and that bispecific CAR T cells were as effective as the monospecific CAR alone against MCL tumors in vivo.

[0190] Example 19 CAR immunotherapy with NK cells CD37 expression has been reported in PTCL (Pereira et al., Mol. Cancer. Ther. 14(7):1650-60, 2015) and many B-NHLs, and is therefore a promising target for CAR T cell immunotherapy. However, while activated T cells can express CD37, natural killer (NK) cells do not. Thus, NK cells are attractive cytotoxic cells for CAR T cell therapy, including CD19 / CD37-CAR or CD37-CAR. NK cells mediate antitumor effects without the risk of GvHD and are short-lived compared to T cells. This makes NK cells, with or without CAR-37, a promising source of allogeneic cell therapy products. Previous preclinical studies have redirected CAR-modified primary human NK cells against a variety of antigens, including CD19, CD20, and HER2, and donor-derived and haploidentical NK cells modified with anti-CD19 CARs have entered clinical trials for B-cell ALL (NCT00995137, NCT01974479). Self-targeting of T cells could potentially be mitigated by using NK cells instead and by using NK as an allogeneic source.

[0191] Example 20 Sequence scFv sequence Anti-CD37 scFv VH-VL (SEQ ID NO:4) comprising VH (amino acids 1-116 (SEQ ID NO:1)), a linker region (amino acids 117-136 (SEQ ID NO:3)), and VL (amino acids 137-244 (SEQ ID NO:2)).

[0192] AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR (SEQ ID NO: 4)

[0193] VH (SEQ ID NO:1 (amino acids 1-116 of SEQ ID NO:4)) AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS (SEQ ID NO: 1)

[0194] Linker region (SEQ ID NO:3 (amino acids 117-136 of SEQ ID NO:4)) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3)

[0195] VL (SEQ ID NO:2 (amino acids 137-244 of SEQ ID NO:4)) DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR (SEQ ID NO: 2)

[0196] Anti-CD37 scFv VL-VH (SEQ ID NO:5) comprising a VL (amino acids 1-108 (SEQ ID NO:2)), a linker region (amino acids 109-128 (SEQ ID NO:3)), and a VH (amino acids 129-244 (SEQ ID NO:1)).

[0197] DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS (SEQ ID NO: 5)

[0198] VL (SEQ ID NO:2 (amino acids 1-108 of SEQ ID NO:5)) DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR (SEQ ID NO: 2)

[0199] Linker region (SEQ ID NO:3 (amino acids 109-128 of SEQ ID NO:5)) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3)

[0200] VH (SEQ ID NO:1 (amino acids 129-244 of SEQ ID NO:5)) AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS (SEQ ID NO: 1)

[0201] An anti-CD19 scFv (SEQ ID NO:14) comprising a VL (amino acids 1-107 (SEQ ID NO:13)), a linker region (amino acids 108-128 (SEQ ID NO:3)), and a VH (amino acids 129-247 (SEQ ID NO:12)).

[0202] EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 14)

[0203] VL (SEQ ID NO:13 (amino acids 1-107 of SEQ ID NO:14)) EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK (SEQ ID NO: 13)

[0204] Linker region (SEQ ID NO:3 (amino acids 108-128 of SEQ ID NO:14) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3)

[0205] VH (SEQ ID NO: 12 (amino acids 129-247 of SEQ ID NO: 14)) QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 12)

[0206] DNA sequencing pMGH8(CAR-37 LH)-CD8 signal / anti-CD37 LH / CD8 hinge+TM / 4-1BB / CD3 zeta (SEQ ID NO:7), comprising the CD8 signal sequence (amino acids 1-21 (SEQ ID NO:8)); anti-CD37 LH (amino acids 22-265 (SEQ ID NO:5)); CD8 hinge and TM domain (amino acids 266-334 (SEQ ID NO:9)); 4-1BB (amino acids 335-376 (SEQ ID NO:10)); and CD3 zeta (amino acids 377-488 (SEQ ID NO:11)).

[0207] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGT KVEIKRGGGGSGGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCAR SVGPMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 7)

[0208] CD8 signal sequence (SEQ ID NO:8 (amino acids 1-21 of SEQ ID NO:7)) MALPVTALLLPLALLLHAARP (SEQ ID NO: 8)

[0209] Anti-CD37 LH (SEQ ID NO:5 (amino acids 22-265 of SEQ ID NO:7)) DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS (SEQ ID NO: 5)

[0210] CD8 hinge and TM domain SEQ ID NO:9 (amino acids 266-334 of SEQ ID NO:7) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 9)

[0211] 4-1BB (SEQ ID NO:10 (amino acids 335-376 of SEQ ID NO:7)) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 10)

[0212] CD3ζ (SEQ ID NO:11 (amino acids 377-488 of SEQ ID NO:7)) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11)

[0213] pMGH8(CAR-37 HL)-CD8 signal / anti-CD37 HL / CD8 hinge+TM / 4-1BB / CD3ζ (SEQ ID NO:6), comprising the CD8 signal sequence (amino acids 1-21 (SEQ ID NO:8)); anti-CD37 HL (amino acids 22-265 (SEQ ID NO:4)); CD8 hinge and TM domain (amino acids 266-334 (SEQ ID NO:9)); 4-1BB (amino acids 335-376 (SEQ ID NO:10)); and CD3ζ (amino acids 377-488 (SEQ ID NO:11)).

[0214] MALPVTALLLPLALLLHAARPAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVG PMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYF CQHHSDNPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence number 6)

[0215] CD8 signal sequence (SEQ ID NO:8 (amino acids 1-21 of SEQ ID NO:6)) MALPVTALLLPLALLLHAARP (SEQ ID NO: 8)

[0216] Anti-CD37 HL (SEQ ID NO:4 (amino acids 22-265 of SEQ ID NO:6)) AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR (SEQ ID NO: 4)

[0217] CD8 hinge and TM domain (SEQ ID NO:9 (amino acids 266-334 of SEQ ID NO:6)) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 9)

[0218] 4-1BB (SEQ ID NO:10 (amino acids 335-376 of SEQ ID NO:6)) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 10)

[0219] CD3ζ (SEQ ID NO:11 (amino acids 377-488 of SEQ ID NO:6)) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11)

[0220] pMGH95(CAR-19-37)-CD8 signal / anti-CD19 / anti-CD37 HL / CD8 hinge+TM / 4-1BB / CD3 zeta (SEQ ID NO:15) comprising: CD8 signal sequence (amino acids 1-21 (SEQ ID NO:8)); anti-CD19 (amino acids 22-268 (SEQ ID NO:14)); linker (amino acids 269-288 (SEQ ID NO:3)); anti-CD37 HL (amino acids 289-532 (SEQ ID NO:4)); CD8 hinge+TM (amino acids 533-601 (SEQ ID NO:9)); 4-1BB (amino acids 602-643 (SEQ ID NO:10)), CD3 zeta (amino acids 644-755 (SEQ ID NO:11)).

[0221] (SEQ ID NO:15)

[0222] CD8 signal sequence (SEQ ID NO:8 (amino acids 1-21 of SEQ ID NO:15)) MALPVTALLLPLALLLHAARP (SEQ ID NO: 8)

[0223] Anti-CD19 (SEQ ID NO:14 (amino acids 22-268 of SEQ ID NO:15) EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 14)

[0224] Linker region (SEQ ID NO:3 (amino acids 269-288 of SEQ ID NO:15) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3)

[0225] Anti-CD37 HL (SEQ ID NO:4 (amino acids 289-532 of SEQ ID NO:15)) AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR (SEQ ID NO: 4)

[0226] CD8 hinge and TM domain (SEQ ID NO:9 (amino acids 533-601 of SEQ ID NO:15)); TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 9)

[0227] 4-1BB (SEQ ID NO: 10 (amino acids 602-643 of SEQ ID NO: 15)), KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 10)

[0228] CD3ζ (SEQ ID NO:11 (amino acids 644-755 of SEQ ID NO:15)) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11)

[0229] pMGH96(CAR-37-19)-CD8 signal / anti-CD37 HL / anti-CD19 / CD8 hinge+TM / 4-1BB / CD3 zeta (SEQ ID NO:16) comprising CD8 signal sequence (amino acids 1-21 (SEQ ID NO:8)); anti-CD37 HL (amino acids 22-265 (SEQ ID NO:4)); linker (amino acids 266-285 (SEQ ID NO:3)); anti-CD19 (amino acids 286-532 (SEQ ID NO:14)); CD8 hinge+TM (amino acids 533-601 (SEQ ID NO:9)); 4-1BB (amino acids 602-643 (SEQ ID NO:10)), CD3 zeta (amino acids 644-755 (SEQ ID NO:11)).

[0230] (SEQ ID NO:16)

[0231] CD8 signal sequence (SEQ ID NO:8 (amino acids 1-21 of SEQ ID NO:16)); MALPVTALLLPLALLLHAARP (SEQ ID NO: 88)

[0232] anti-CD37 HL (SEQ ID NO:4 (amino acids 22-265 of SEQ ID NO:16)); AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR (SEQ ID NO: 4)

[0233] Linker region (SEQ ID NO:3 (amino acids 266-285 of SEQ ID NO:16)); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3)

[0234] anti-CD19 (SEQ ID NO:14 (amino acids 286-532 of SEQ ID NO:16)); EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 14)

[0235] CD8 hinge and TM domain (SEQ ID NO:9 (amino acids 533-601 of SEQ ID NO:16)); TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 9)

[0236] 4-1BB (SEQ ID NO: 10 (amino acids 602-643 of SEQ ID NO: 16)), KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 10)

[0237] CD3ζ (SEQ ID NO:11 (amino acids 644-755 of SEQ ID NO:16)) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11)

[0238] The sequences of the present application and the corresponding SEQ ID NOs are shown in Table 4.

[0239] TIFF0007611705000007.tif252170TIFF0007611705000008.tif255170TIFF0007611705 000009.tif255170TIFF0007611705000010.tif255170TIFF0007611705000011.tif33170

[0240] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs. 1. A chimeric antigen receptor (CAR) comprising: (i) an extracellular domain comprising a CD37-binding domain and a CD19-binding domain, (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

[0241] 2. The CAR of paragraph 1, wherein the CD37 binding domain and / or the CD19 binding domain comprises an antibody, or an antigen-binding fragment thereof.

[0242] 3. The CAR of paragraph 1 or 2, wherein the CD37 binding domain and / or the CD19 binding domain comprises a single chain variable fragment (scFv).

[0243] 4. A CAR described in any one of paragraphs 1 to 3, wherein the CD19 binding domain is located N-terminal to the CD37 binding domain.

[0244] 5. A CAR described in any one of paragraphs 1 to 3, wherein the CD37 binding domain is located N-terminal to the CD19 binding domain.

[0245] 6.(iv) A CAR described in any one of paragraphs 1 to 5, further comprising one or more costimulatory domains.

[0246] 7. The CAR of any one of paragraphs 1 to 6, wherein the transmembrane domain comprises a hinge / transmembrane domain.

[0247] 8. The CAR of paragraph 7, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of CD8 or 4-1BB.

[0248] 9. The CAR of paragraph 8, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of CD8, which may comprise the amino acid sequence of SEQ ID NO:9.

[0249] 10. The CAR of any one of paragraphs 1 to 9, wherein the intracellular signaling domain comprises an intracellular signaling domain of TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3theta, CD3epsilon, CD3zeta, CD22, CD79a, CD79b, or CD66d.

[0250] 11. The CAR of paragraph 10, wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, which may comprise the amino acid sequence of SEQ ID NO:11.

[0251] 12. The CAR of any one of paragraphs 6 to 11, wherein the costimulatory domain comprises a 4-1BB, CD28, or OX-40 costimulatory domain.

[0252] 13. The CAR of paragraph 12, wherein the costimulatory domain comprises a costimulatory domain of 4-1BB, which may comprise the amino acid sequence of SEQ ID NO: 10.

[0253] 14. A CAR described in any one of paragraphs 1 to 13, comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15, 16, 19, or 20.

[0254] 15. The CAR of paragraph 14, comprising the amino acid sequence of SEQ ID NO: 15, 16, 19, or 20.

[0255] 16. The CAR of any one of paragraphs 1 to 15, wherein the CD37 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1, and a light chain variable domain (VL) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0256] 17. The CAR of paragraph 16, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO: 2.

[0257] 18. The CAR of paragraph 16 or 17, wherein the VH is positioned N-terminal to the VL.

[0258] 19. The CAR of paragraph 16 or 17, wherein the VL is positioned N-terminal to the VH.

[0259] 20. A CAR described in any one of paragraphs 1 to 19, wherein the CD37 binding domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 or 5.

[0260] 21. The CAR described in paragraph 20, wherein the CD37 binding domain comprises the amino acid sequence of SEQ ID NO: 4 or 5.

[0261] 22. The CAR of any one of paragraphs 1 to 21, wherein the CD19 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12, and a light chain variable domain (VL) comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0262] 23. The CAR of paragraph 22, wherein the VH comprises the amino acid sequence of SEQ ID NO: 12 and the VL comprises the amino acid sequence of SEQ ID NO: 13.

[0263] 24. A CAR described in any one of paragraphs 1 to 23, wherein the CD19 binding domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14.

[0264] 25. The CAR of paragraph 24, wherein the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 14.

[0265] 26. A polynucleotide encoding a CAR according to any one of paragraphs 1 to 25.

[0266] 27. The polynucleotide of paragraph 26, further comprising a suicide gene.

[0267] 28. The polynucleotide of paragraph 26 or 27, further comprising a sequence encoding a signal sequence.

[0268] 29. An immune cell comprising a CAR according to any one of paragraphs 1 to 25 and / or a polynucleotide according to any one of paragraphs 26 to 28.

[0269] 30. The immune cell described in paragraph 29, which is a T cell or a natural killer (NK) cell.

[0270] 31. The immune cell of paragraph 29 or 30, which is a human cell.

[0271] 32. A pharmaceutical composition comprising an immune cell according to any one of paragraphs 29 to 31 and a pharma- ceutically acceptable carrier.

[0272] 33. A method for treating cancer in a subject in need thereof, comprising administering to the subject an immune cell described in any one of paragraphs 29 to 31 or a pharmaceutical composition described in paragraph 32.

[0273] 34. The method of paragraph 33, wherein the cancer comprises cells that express CD37.

[0274] 35. The method of paragraph 34, wherein the cancer is B-cell non-Hodgkin's lymphoma, T-cell lymphoma, or leukemia.

[0275] 36. The method of paragraph 35, wherein the B-cell non-Hodgkin's lymphoma is mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), or Burkitt's lymphoma.

[0276] 37. The method of paragraph 35, wherein the T-cell lymphoma is peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), angioimmunoblastic T-cell lymphoma (AITL), or anaplastic large cell lymphoma (ALCL).

[0277] 38. The method of paragraph 35, wherein the leukemia is chronic lymphocytic leukemia (CLL).

[0278] 39. The method of any one of paragraphs 33 to 38, wherein the subject is non-responsive to anti-CD19 therapy.

[0279] 40. The method of any one of paragraphs 33 to 39, wherein the subject is co-administered an anti-CD19 therapy.

[0280] Other embodiments The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but such illustration and description are not intended to limit the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entireties by reference.

Claims

1. A chimeric antigen receptor (CAR) comprising: (i) an extracellular domain comprising a CD37-binding domain and a CD19-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

2. The CAR of claim 1, wherein the CD37 binding domain and / or the CD19 binding domain comprises an antibody, or an antigen-binding fragment thereof.

3. The CAR of claim 2, wherein the CD37 binding domain and / or the CD19 binding domain comprises a single chain variable fragment (scFv).

4. The CAR of claim 1, wherein the CD19 binding domain is located N-terminal to the CD37 binding domain.

5. The CAR of claim 1, wherein the CD37 binding domain is located N-terminal to the CD19 binding domain.

6. (iv) the CAR of claim 1, further comprising one or more costimulatory domains.

7. The CAR of claim 1, wherein the transmembrane domain comprises a hinge / transmembrane domain.

8. The CAR of claim 7, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of CD8 or 4-1BB.

9. The CAR of claim 8, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of CD8.

10. The CAR of claim 1, wherein the intracellular signaling domain comprises an intracellular signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d.

11. The CAR of claim 10, wherein the intracellular signaling domain comprises an intracellular signaling domain of CD3ζ.

12. The CAR of claim 6, wherein the costimulatory domain comprises a costimulatory domain of 4-1BB, CD28, or OX-40.

13. The CAR of claim 12, wherein the costimulatory domain comprises a 4-1BB costimulatory domain.

14. The CAR of claim 1, comprising the amino acid sequence of SEQ ID NO:

20.

15. The CD37 binding domain is a heavy chain variable domain (VH) comprising VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NO: 1; and a light chain variable domain (VL) comprising VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NO:

2.

16. The CAR of claim 15, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO:

2.

17. The CAR of claim 15, wherein the VH is positioned N-terminal to the VL.

18. The CAR of claim 15, wherein the VL is positioned N-terminal to the VH.

19. The CAR of claim 1, wherein the CD37 binding domain comprises the amino acid sequence of SEQ ID NO: 4 or 5.

20. The CD19 binding domain is a heavy chain variable domain (VH) comprising VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NO: 12; and a light chain variable domain (VL) comprising VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NO:

13.

21. The CAR of claim 20, wherein the VH comprises the amino acid sequence of SEQ ID NO: 12 and the VL comprises the amino acid sequence of SEQ ID NO:

13.

22. The CAR of claim 1, wherein the CD19 binding domain comprises the amino acid sequence of SEQ ID NO:

14.

23. A polynucleotide encoding the CAR of claim 1.

24. 24. The polynucleotide of claim 23, further comprising a suicide gene.

25. 24. The polynucleotide of claim 23, further comprising a sequence encoding a signal sequence.

26. An immune cell comprising the CAR of claim 1 and / or a polynucleotide encoding the CAR of claim 1.

27. The immune cell of claim 26, which is a T cell or a natural killer (NK) cell.

28. The immune cell of claim 26, which is a human cell.

29. 27. A pharmaceutical composition comprising the immune cells of claim 26 and a pharma- ceutically acceptable carrier.

30. 27. A medicament for treating cancer in a subject in need thereof comprising an immune cell according to claim 26 or a pharmaceutical composition comprising the immune cell according to claim 26.

31. The pharmaceutical agent of claim 30, wherein the cancer comprises cells that express CD37.

32. The pharmaceutical composition of claim 31 , wherein the cancer is B-cell non-Hodgkin's lymphoma, T-cell lymphoma, or leukemia.

33. The pharmaceutical composition of claim 32, wherein the B-cell non-Hodgkin's lymphoma is mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), or Burkitt's lymphoma.

34. The pharmaceutical composition of claim 32, wherein the T-cell lymphoma is peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), angioimmunoblastic T-cell lymphoma (AITL), or anaplastic large cell lymphoma (ALCL).

35. The pharmaceutical composition of claim 32, wherein the leukemia is chronic lymphocytic leukemia (CLL).

36. The pharmaceutical composition of claim 30, wherein the subject is non-responsive to anti-CD19 therapy.

37. The pharmaceutical composition of claim 30, wherein the subject is co-administered an anti-CD19 therapy.

38. 27. Use of an immune cell according to claim 26 or a pharmaceutical composition comprising an immune cell according to claim 26 for the manufacture of a medicament for treating cancer in a subject in need thereof.

39. A CAR as described in claim 1, wherein the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 14 and the CD37 binding domain comprises the amino acid sequence of SEQ ID NO: 4 or 5.

40. A CAR described in claim 1, comprising the amino acid sequence of sequence number 15.

41. A CAR described in claim 1, comprising the amino acid sequence of SEQ ID NO:

16.

42. A CAR described in claim 1, comprising the amino acid sequence of SEQ ID NO: 19.

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