Chimeric antigen receptor targeting CD37 and CD19

Abstract

The present invention provides a bispecific chimeric antigen receptor (CAR) targeting CD37 and CD19, and related molecules, immune cells comprising the bispecific chimeric antigen receptor, compositions of the immune cells, and methods of using the same. The present invention also provides methods for treating diseases or disorders such as cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 688,775, filed on June 22, 2018, and U.S. Provisional Application No. 62 / 757,562, filed on November 8, 2018, the contents of which are incorporated herein by reference in their entireties.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on June 19, 2019 is named 51295-018WO3_Sequence_Listing_6.19.19_ST25 and is 56,545 bytes in size. Background Art

[0005] Immunotherapy uses the 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 show the desired specificity, for example, by expressing a chimeric antigen receptor (CAR). CAR provides a method of guiding cytotoxic T cells or NK cells to respond to target cells expressing selected target antigens (most often tumor antigens or tumor-associated antigens), and is an adaptor of the T cell receptor, wherein the antigen binding domain is replaced by the antigen binding domain of an antibody specific to the target antigen. CAR expressed on T cells engages the target antigen on the surface of the target cell to promote the killing of target cells. The use of CAR-expressing T cells for treating diseases is referred to as CAR T cell immunotherapy.

[0006] To date, two CAR T-cell products have been approved for the treatment of relapsed or refractory large cell lymphoma, both targeting CD19: axicabtageneciloleucel with a CD28 co-stimulatory domain and tisagenlecleucel with a 4-1BB co-stimulatory domain. Tisagenlecleucel is also approved for the treatment of relapsed or refractory acute B-cell lymphoblastic leukemia (ALL) in children and young adults. The effective response to anti-CD19 CART cell therapy is in the range of 60%-80%, and approximately 40% of patients achieve long-term complete remission. However, disease relapse due to loss of the CD19 antigen target has been observed in all patient subgroups, including 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 treatments.

[0007] 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 recognition of its complex heterogeneity and discovery of defects in relapse, PTCL remains a clinical conundrum and is poorly treated. While CAR T immunotherapy has demonstrated impressive clinical results in ALL and B-cell NHL, it has not proven successful in treating T-cell malignancies.

[0008] Therefore, new or improved treatments for B and T cell malignancies are needed. Summary of the invention

[0009] The present invention particularly provides bispecific chimeric antigen receptors (CARs) targeting CD37 and CD19 for use in treating a disease or disorder described herein, such as cancer.

[0010] In one aspect, the invention features 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.

[0011] In some embodiments, the CD37 binding domain and / or CD19 binding domain comprises an antibody or an antigen binding fragment thereof, e.g., a single chain variable fragment (scFv). In some embodiments, the CD19 binding domain is located at the N-terminus of the CD37 binding domain, or the CD37 binding domain is located at the N-terminus of the CD19 binding domain.

[0012] In some embodiments, the CAR further comprises (iv) one or more costimulatory domains. In some embodiments, the transmembrane domain includes, for example, a hinge / transmembrane domain of CD8 or 4-1BB. In a specific embodiment, the hinge / transmembrane domain includes a hinge / transmembrane domain of CD8, which optionally includes SEQ ID NO:9 amino acid sequence.

[0013] In further embodiments, the intracellular signaling domain includes an intracellular signaling domain of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 θ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b or CD66d. In specific embodiments, the intracellular signaling domain includes an intracellular signaling domain of CD3 ζ, which optionally includes an amino acid sequence of SEQ ID NO: 11. In some embodiments, the costimulatory domain includes a costimulatory domain of 4-1BB, CD28 or OX-40. In specific embodiments, the costimulatory domain includes a costimulatory domain of 4-1BB, which optionally includes an amino acid sequence of SEQ ID NO: 10.

[0014] In some embodiments, the CAR comprises an amino acid sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to, or contains, the amino acid sequence of SEQ ID NO: 15, 16, 19 or 20.

[0015] In some embodiments, the CD37 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 or containing 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 or containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VH is located at the N-terminus of the VL, or the VL is located at the N-terminus of the VH. In a further embodiment, the CD37 binding domain comprises an amino acid sequence that has 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 contains a sequence thereof.

[0016] In a further embodiment, the CD19 binding domain comprises a heavy chain variable domain (VH), which comprises an amino acid sequence that has 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 a sequence thereof; and a light chain variable domain (VL), which comprises an amino acid sequence that has 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 a sequence thereof. In some embodiments, the CD19 binding domain comprises an amino acid sequence that has 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 contains a sequence thereof.

[0017] In another aspect, the invention features a polynucleotide encoding the CAR of any of the preceding aspects.

[0018] In some embodiments, the polynucleotide further comprises a suicide gene. In some embodiments, the polynucleotide further comprises a sequence encoding a signal sequence.

[0019] In another aspect, the invention features an immune cell comprising the CAR and / or polynucleotide of any of the preceding aspects.

[0020] In some embodiments, the immune cell (eg, human cell) is a T cell or a natural killer (NK) cell.

[0021] In another aspect, the invention features a pharmaceutical composition comprising the immune cell of any of the preceding aspects and a pharmaceutically acceptable carrier.

[0022] In another aspect, the invention features a method of treating cancer in a subject in need thereof, the method comprising administering to the subject the immune cell of any preceding aspect or a pharmaceutical composition thereof.

[0023] In some embodiments, the cancer expresses CD37. In some embodiments, the cancer is a B-cell non-Hodgkin 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)).

[0024] In some embodiments, the subject is unresponsive to anti-CD19 therapy. In further embodiments, the subject is co-administered with anti-CD19 therapy.

[0025] definition

[0026] 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 include the meanings provided below. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed technology, as the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the technology belongs. If there is a clear difference between the usage of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0027] Definitions of commonly used terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, 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, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (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 MKruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (ed.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of each of which are incorporated herein by reference in their entirety.

[0028] The terms "reduce", "reduce", "reduce" or "inhibit" are all used herein to mean a statistically significant amount reduced. In some embodiments, "reduce", "reduce" or "reduce" or "inhibit" generally means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduce" or "inhibit" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. Where appropriate, the reduction may preferably be to an acceptable level within the normal range for individuals without the given disorder.

[0029] The terms "increased", "increase", "enhance" or "activate" are all used herein to mean an increase in a statistically significant amount. In some embodiments, the terms "increased", "increase", "enhance" or "activate" can mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase or any increase between 10%-100%, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase or greater between 2 times and 10 times, compared to a reference level. In the case of a marker or symptom, an "increase" is a statistically significant increase in such a level.

[0030] As used herein, "subject" means a person or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a game animal. Primates include, for example, chimpanzees, crab-eating monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and game animals include, for example, cattle, horses, pigs, deer, bison, buffalo, felines (e.g., house cats), canines (e.g., dogs), foxes, wolves, birds (e.g., chickens), emus, ostriches, and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal (e.g., a primate, such as a human). The terms "individual," "patient," and "subject" are used interchangeably herein.

[0031] Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans may be advantageously used as subjects representing animal models of diseases (e.g., cancer). The subject may be male or female.

[0032] The subject may be a subject who has previously been diagnosed with or identified as having or having a condition (e.g., lymphoma, leukemia, or another type of cancer, etc.) or one or more complications associated with such a condition that requires treatment, and who has optionally undergone treatment for the condition or one or more complications associated with the condition. Alternatively, the subject may also be a subject who has not previously been diagnosed with the condition or related complications. For example, the subject may be a subject who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or a subject who does not exhibit risk factors.

[0033] A "subject in need of treatment" for a particular disorder may be a subject having the disorder, diagnosed as having the disorder, or at risk of developing the disorder.

[0034] A "disease" is a health state in an animal (e.g., a human) in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but the animal's health state is less favorable than it would be in the absence of the disorder. A disorder does not necessarily result in a further decline in the animal's health state if left untreated.

[0035] As used herein, the terms "tumor antigen" and "cancer antigen" are used interchangeably and refer to antigens differentially expressed by cancer cells, and can therefore be utilized to target cancer cells. Cancer antigens are antigens that may potentially stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but not necessarily expressed. These antigens can be characterized as antigens that are usually silent (i.e., not expressed) in normal cells, antigens that are only expressed at certain stages of differentiation, and antigens that are temporarily expressed, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cell genes (such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., mutant p53), and fusion proteins produced by internal deletions or chromosomal translocations). Other cancer antigens can also be encoded by viral genes (such as genes carried on RNA and DNA tumor viruses). Many tumor antigens have been defined for a variety of solid tumors: MAGE 1, 2, and 3 defined by immunity; MART-1 / Melan-A, gp100, carcinoembryonic antigen (CEA), HER2, mucin (i.e., MUC-1), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). In addition, viral proteins, such as those encoded by hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma virus (HPV), have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively.

[0036] As used herein, the term "chimeric" refers to a fusion product of parts of at least two or more different polynucleotide molecules. In one embodiment, the term "chimeric" refers to a gene expression element produced by manipulating known elements or other polynucleotide molecules.

[0037] In some embodiments, "activation" may refer to the state of immune cells (e.g., T or NK cells) that have been sufficiently stimulated to induce detectable cell proliferation. In some embodiments, activation may refer to induced cytokine production. In other embodiments, activation may refer to detectable effector functions. At least, activated T or NK cells as used herein are proliferative T or NK cells.

[0038] As used herein, the terms "specific binding" and "specifically bind" refer to a physical interaction between two molecules, compounds, cells and / or particles, wherein the specificity and affinity of the first entity binding to the second target entity is greater than the specificity and affinity of the first entity binding to the third entity as a non-target. In some embodiments, specific binding can refer to the affinity of the first entity for the second target entity, which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or more of the affinity for the third non-target entity under the same conditions. An agent specific for a given target is an agent that exhibits specific binding to the target under the assay conditions used. Non-limiting examples include antibodies or ligands that recognize and bind to cognate binding partners (e.g., stimulatory and / or co-stimulatory molecules present on T cells) proteins.

[0039] As used herein, "stimulatory ligand" refers to a ligand that can specifically bind to a cognate binding partner (referred to herein as "stimulatory molecule" or "co-stimulatory molecule") on a T cell when present on an antigen presenting cell (APC, such as a macrophage, a dendritic cell, a B cell, an artificial APC, etc.), thereby mediating a primary response of 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 particularly encompass MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0040] 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.

[0041] As used herein, the term "costimulatory ligand" includes molecules on APCs that specifically bind to cognate costimulatory molecules on T cells, thereby providing a signal that mediates T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to the MHC molecule loaded with the peptide. Costimulatory ligands may include, but are not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecules (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 to B7-H3. Co-stimulatory ligands may also include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited 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 ligands that specifically bind to CD83.

[0042] "Costimulatory molecules" refer to cognate binding partners on T cells that specifically bind to costimulatory ligands to mediate costimulatory responses (such as but not limited to proliferation) of T cells. 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.

[0043] In one embodiment, the term "engineered" and its grammatical equivalents as used herein can refer to one or more artificially designed changes of nucleic acids (e.g., nucleic acids in the genome of an organism). In another embodiment, engineering can refer to changes, additions and / or deletions of genes. "Engineered cells" can refer to cells with added, deleted and / or altered genes. The term "cell" or "engineered cells" and its grammatical equivalents as used herein can refer to cells of human or non-human animal origin.

[0044] As used herein, the term "operably linked" refers to a first polynucleotide molecule (such as a promoter) that is linked to a second transcribable polynucleotide molecule (such as a target gene), wherein the polynucleotide molecules are arranged so that the first polynucleotide molecule affects the function of the second polynucleotide molecule. Two polynucleotide molecules may or may not be part of a single continuous polynucleotide molecule, and may or may not be adjacent. For example, if a promoter regulates or mediates transcription of a target gene in a cell, the promoter is operably linked to the target gene.

[0045] In the various embodiments described herein, variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any particular polypeptide are further contemplated. With respect to amino acid sequences, one of ordinary skill will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small percentage of amino acids in the encoding sequence are "conservatively modified variants," wherein the alteration results in the substitution of an amino acid with a chemically similar amino acid and retention of 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.

[0046] A given amino acid may be replaced by a residue having similar physicochemical characteristics, for example, one aliphatic residue for another (such as Ile, Val, Leu or Ala for another), or one polar residue for another (such as between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions, such as substitutions of entire regions having similar hydrophobic characteristics, are well known. Polypeptides containing conservative amino acid substitutions may be tested in any of the assays described herein to confirm that the desired activity, such as ligand-mediated receptor activity and specificity of a native or reference polypeptide, is retained.

[0047] Amino acids can be grouped according to the similarity of the properties of their side chains (in ALLehninger, in Biochemistry, 2nd 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 affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would require exchanging a member of one of these classes for another. Specific conservative substitutions include, for example: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0048] 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 fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to an assay known in the art or described herein below. Functional fragments may comprise conservative substitutions of the sequences disclosed herein.

[0049] In some embodiments, the polypeptides described herein can be variants of polypeptides or molecules as described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained by, for example, mutations of native nucleotide sequences. As referred to herein, "variants" are polypeptides that are substantially homologous to native or reference polypeptides, but due to one or more deletions, insertions or substitutions, their amino acid sequences are different from the amino acid sequences of native or reference polypeptides. The DNA sequence encoding the variant polypeptide encompasses such sequences: when compared to a native or reference DNA sequence, it comprises the addition, deletion or substitution of one or more nucleotides, but encodes a variant protein or fragment thereof that retains the activity of a non-variant polypeptide. A variety of site-specific mutagenesis methods based on PCR are known in the art and can be applied by ordinary technicians.

[0050] Variant amino acid or DNA sequence can be 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 with native or reference sequence. The degree of homology (percent identity) between native sequence and mutant sequence can be determined, for example, by comparing the two sequences using free computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn, using default settings).

[0051] The change of the native amino acid sequence can be completed by any of a variety of techniques known to those skilled in the art. For example, the mutation can be introduced at a specific locus by synthesizing an oligonucleotide containing the mutant sequence, flanking a restriction site that allows connection to a fragment of the native sequence. After connection, the resulting reconstructed sequence encodes an analog with a desired amino acid insertion, substitution or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis program can be used to provide a nucleotide sequence with a change in a specific codon that changes according to a desired substitution, deletion or insertion. Techniques for making such changes 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. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety. It is also common to replace any cysteine ​​residues that are not involved in maintaining the correct conformation of the polypeptide with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds can be added to a polypeptide to improve its stability or promote oligomerization.

[0052] As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term "polynucleotide" and "nucleic acid" are used interchangeably herein to indicate the polymer of nucleoside. Generally, polynucleotides are composed of naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) in DNA or RNA connected by phosphodiester bonds. However, the term encompasses molecules (whether or not found in naturally occurring nucleic acids) containing bases, modified skeletons, etc. containing chemical or biological modifications or nucleoside analogs, and for some applications, such molecules may be preferred. When the application relates to polynucleotides, it should be understood that both DNA and RNA are provided, and in each case single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to polynucleotide materials themselves and / or sequence information (i.e., a series of letters used as base abbreviations) that characterize specific nucleic acids biochemically. Unless otherwise indicated, the polynucleotide sequences presented herein are presented in a 5' to 3' direction.

[0053] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. Peptides are relatively short polypeptides, typically about 2 to 60 amino acids in length. The polypeptides used herein typically contain amino acids, such as the 20 most common L-amino acids in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more amino acids in a polypeptide can be modified, for example, by adding chemical entities (such as carbohydrate groups, phosphate groups, fatty acid groups, for conjugation, functionalized linkers, etc.). A polypeptide having a non-polypeptide portion covalently or non-covalently associated therewith is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized by chemical methods (such as conventional solid phase peptide synthesis, etc.). As used herein, the term "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or the sequence information of the biochemical characterization polypeptide (i.e., a series of letters or three letter codes used as abbreviations of the amino acid names). Unless otherwise indicated, the polypeptide sequences presented herein are presented in the direction of N-terminus to C-terminus.

[0054] In some embodiments, the vector comprises a nucleic acid encoding a polypeptide as described herein (e.g., a bispecific CAR polypeptide). In some aspects described herein, the nucleic acid sequence encoding a given polypeptide as described herein or any of its modules is operably connected to the 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 may be viral or non-viral. The term "vector" encompasses any genetic element that can replicate and can transfer a gene sequence to a cell when combined with an appropriate control element. A vector may include, but is not limited to, a cloning vector, an expression vector, a plasmid, a phage, a transposon, a cosmid, an artificial chromosome, a virus, a virion, etc.

[0055] As used herein, the term "expression vector" refers to a vector that guides the expression of RNA or polypeptides from a sequence connected to a transcriptional regulatory sequence on a vector. The expressed sequence will be generally but not necessarily heterologous to the cell. The expression vector may include additional elements, for example, the expression vector may have two replication systems so that it is maintained in two organisms, for example, expressed in human cells, and cloned and amplified in a prokaryotic host. The term "expression" refers to a cell process involving the production of RNA and protein and secretion of protein when appropriate, including but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification, and processing when applicable. "Expression products" include RNA transcribed from a gene and polypeptides obtained by translating mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably connected to a suitable regulatory sequence. The gene may or may not include regions before and after the coding region, such as 5' non-translated (5'UTR) or "leader" sequence and 3'UTR or "tail" sequence, as well as insertion sequences (introns) between each coding segment (exon).

[0056] As used herein, "signal peptide" or "signal sequence" refers to a peptide at the N-terminus of a newly synthesized protein that is used to introduce the nascent protein into the endoplasmic reticulum. In some embodiments, the signal peptide is a CD8 signal peptide.

[0057] 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 viral vector particles. The viral vector may contain nucleic acids encoding polypeptides as described herein to replace non-essential viral genes. The vector and / or particle may be used for the purpose of transferring nucleic acids into cells in vitro or in vivo. Various forms of viral vectors are known in the art.

[0058] "Recombinant vector" means a vector comprising a heterologous nucleic acid sequence or "transgene" that can be expressed in vivo. It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of suitable episomal vectors provides a way to maintain the target nucleotide of the subject in a high copy number of extrachromosomal DNA, thereby eliminating the potential impact of chromosomal integration.

[0059] As used herein, the term "treat, treatment, treating" or "amelioration" refers to therapeutic treatment, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder (e.g., acute lymphoblastic leukemia or other cancers, diseases or disorders). The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. If one or more symptoms or clinical markers are reduced, the treatment is usually "effective". Alternatively, if the progression of the disease is reduced or stopped, the treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or markers, but also includes stopping or at least slowing down the progression or deterioration of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, one or more symptom relief, a reduction in the extent of the disease, a stable disease state (i.e., no deterioration), a delay or slowing of disease progression, an improvement or alleviation of the disease state, relief (whether partial or complete) and / or a reduction in mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief of the symptoms or side effects of the disease (including palliative care).

[0060] As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry). The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for contact with human or animal tissues within the scope of reasonable medical judgment, without producing excessive toxicity, irritation, allergic reactions, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be an artificial or engineered carrier, for example, a carrier in which the naturally occurring active ingredient is not found.

[0061] As used herein, the term "administering" refers to placing a therapeutic or pharmaceutical composition as disclosed herein in a subject by a method or route that results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising agents as disclosed herein can be administered by any appropriate route that results in effective treatment in a subject.

[0062] The term "statistically significant" or "significant" refers to statistical significance, and generally means a difference of two standard deviations (2SD) or greater.

[0063] Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” When used in conjunction with a percentage, the term “about” may mean ±1%.

[0064] As used herein, the term "comprising" means that there may be additional elements in addition to the defined elements presented. The use of "comprising" means inclusion rather than limitation.

[0065] The term "consisting of" refers to the compositions, methods, and corresponding component(s) thereof as described herein, excluding any elements not recited in the description of that embodiment.

[0066] As used herein, the term "consisting essentially of refers to the components required for a given embodiment. The term permits the presence of additional elements that do not materially affect one or more basic and novel or functional characteristics of the described embodiments of the technology.

[0067] Unless the context clearly indicates otherwise, the singular terms "a", "an", and "the" include plural referents. 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 non-limiting examples. Therefore, the abbreviation "eg" is synonymous with the term "for example".

[0068] In some embodiments of any of the aspects, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the germline genetic characteristics of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic characteristics of animals (which may cause animal suffering without any substantial medical benefit to humans or animals), and animals produced by such processes.

[0069] Additional terms are defined within the following description of various aspects and embodiments of the technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1AFACS 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.

[0071] Figure 1B Shown are FACS plots of samples derived from three MCL patients.

[0072] Figure 1C Mean fluorescence intensity (MFI) of CD19 and CD37 on MCL patient cells is shown. Each point represents an individual xenograft sample. (n=3; median is shown).

[0073] Figure 1D Shown are CD19 and CD37 expressions on peripheral blood mononuclear cells (PBMCs) from CLL patients gated on CD3 lymphocytes (n=20; mean±SD is shown; **** indicates p<0.0001 by t-test).

[0074] Figure 1E CD19 and CD37 on CLL samples are shown. Expression levels of CD19 and CD37 in 21 CLL patients were obtained by flow cytometry gated on CD3-CD20+ B cells. Mean values ​​± SD are shown.

[0075] Figure 2A The distribution of CD19 and CD37 antigens on CLL PBMCs is shown. The antibody bound to each cell gated on CD3 cells from each patient sample (ABC) is shown.

[0076] Figure 2B The CD19 and CD37 antigen densities are shown. CD19 range: 24396-70952, mean: 43238; CD37 range: 8992-46550, mean: 23989

[0077] Figure 2C Shown are CD37 immunohistochemistry in primary ALK-negative (left) and ALK-positive (right) ALCL specimens from tissue microarray.

[0078] Figure 3A Schematic representation of two anti-CD37 second-generation CAR constructs with different orientations of humanized murine antibody-derived single-chain variable fragments: light to heavy orientation (CAR-37 LH, top) and heavy to light (CAR-37 HL, bottom).

[0079] Figure 3BShown are representative flow cytometry plots of transduction efficiency of primary human T cells 10 days after activation with CD3 / CD28 beads.

[0080] Figure 3C It is shown that expanded T cells from three healthy donors contained variable CAR-37 expression with average values ​​of 38% (LH) and 75% (HL).

[0081] Figure 3D Ex vivo expansion of CD3 / CD28 bead-activated and target-stimulated T cells from three healthy donors using static culture conditions for 38 days is shown. Each arrow represents antigen stimulation with K562 cells transduced to express CD37 and CD19.

[0082] Figure 3E Activation of Jurkat reporter gene (NFAT-Luc) T cells transduced with different CAR constructs and co-cultured with tumor cells is shown. Luciferase activity was measured after 16 hours. (CD3-CD28 beads: positive control).

[0083] Figure 3F-Figure 3I Shown are whole blood from six normal donors stained with CD45, CD3, CD19, CD16, CD14, CD56, and CD37 antibodies or isotype controls. Figure 3F Shown is a histogram representing CD37 expression gated on CD45+CD19+ B cells. Figure 3G Shown is a histogram representing CD37 expression on the gated CD45+CD16+CD14+ monocyte population. Figure 3H Shown are histograms representing CD37 expression on gated T cells (CD45+CD3+). Fig. 3I Shown is a histogram representing CD37 expression on gated CD45+CD16+CD56+ NK cells.

[0084] Figure 4A Shown are the numbers of CSFE-labeled, unstimulated target T cells measured by flow cytometry after 24 h of coculture with CAR-37 HL, CAR-19, or untransduced T cells at the indicated E:T ratios.

[0085] Figure 4B Shown are the numbers of CSFE-labeled T cells measured by flow cytometry after stimulation with PMA / ionomycin for 6 h followed by coculture with CAR-37 HL, CAR-19, or untransduced T cells at the indicated E:T ratios for 24 h.

[0086] Figure 4CThe number of Jeko-1 CBG-GFP cells measured by flow cytometry after 24 h of co-culture with CAR-37 HL, CAR-19 or untransduced T cells at the indicated E:T ratios is shown. The bars indicate the mean ± SEM counts of triplicates from one normal donor (representative of 3 normal donors).

[0087] Figure 4D Shown is the CD107a and IFNγ production of CAR-37 HL and CAR-19 T cells relative to the culture medium incubated with primary immune cells at an E:T ratio of 1:1 for 6 hours by flow cytometry analysis. The bars show the mean ± SEM percentage of the three normal donors analyzed.

[0088] Figure 5 The cytotoxic capacity of CAR-37 T cells measured after co-culture with the target overnight is shown. 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 resulted in specific killing, while no killing was observed in the control group (UTD). The cytotoxicity assay represents three independent experiments performed with different healthy donors.

[0089] Fig. 6A and Figure 6B Shown with primary CLL ( Fig. 6A ) or MCL PDX( Figure 6B ) tumor samples were incubated with CAR-37 HL, CAR-19 or UTD T cells. 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 mean ± SEM of three donors.

[0090] Figure 6C Cytokine production by CAR-37, CAR-19 or UTD T cells incubated with the indicated tumor cell lines at an E:T ratio of 1:1 for 24 hours is shown; culture supernatants were analyzed by Luminex assay. Significant production of several cytokines was noted in the CAR-37 and CAR-19 groups, but not in UTD. Three normal donors were analyzed, and mean ± SEM is shown.

[0091] Fig.6D Shown is IL-6 production from CAR-37, CAR-19 or UTD T cells incubated with the indicated tumor cell lines at an E:T ratio of 1:1 for 24 hours; culture supernatants were analyzed by Luminex assay. Three normal donors were analyzed, and mean ± SEM are shown.

[0092] Figure 7A-7C A direct comparison of the anti-MCL activity of CAR-37 T cells in an MCL tumor model is shown. Fig. 7A The experimental schematic is shown: NSG mice were injected IV with 1x 10 6 JEKO-1(CBG-GFP) cells were expressed in 4% paraformaldehyde (P < 0.05) cells and tumor burden was monitored by BLI at different time points. On day 0, mice were randomized based on tumor burden (BLI) to receive 1 x 10 6 control T cells (UTD), CAR-37 LH, or CAR-37 HL. Figure 7B Representative bioluminescence images of JEKO-1 growth over time are shown. Figure 7C The mean radiance (p / s / cm2) of the whole mouse at different time points in the three groups is shown. 2 / sr). The graph represents one experiment with five mice per group. Mean ± SD is shown.

[0093] Fig.7D A schematic diagram of the experimental design is shown. NSG mice were injected IV with 1x 10 6 JEKO-1(CBG-GFP) cells were expressed in 4% paraformaldehyde (P < 0.05) cells and tumor burden was monitored by BLI at different time points. On day 0, mice were randomized based on tumor burden (BLI) to receive 2 x 10 6 control T cells (UTD), CAR-37, or CAR-19.

[0094] Fig. 7E Representative bioluminescence images of JEKO-1 growth over time are shown.

[0095] Figure 7F The average flux (photons / second) of all mice in the three groups at different time points is shown. The figure represents two experiments of five mice in each group with CAR T cells obtained from two different healthy donors. Mean ± SD is shown, *** indicates that p < 0.001 was tested by two-way Anova.

[0096] Figure 7G The absolute number of CAR T cells is shown, which was monitored by bleeding and flow cytometry. The absolute CAR T cell counts in peripheral blood on day 14 after CAR T injection are shown. *Indicates p<0.05 by t-test.

[0097] Fig. 8A A schematic diagram of the experimental design is shown. NSG mice were injected IV with 1x 10 6MCL patient-derived cells were used and tumor burden was monitored over time by bioluminescence imaging (BLI). On day 0, mice were randomized based on tumor burden to receive 3x 10 6 control T cells (UTD), CAR-37, or CAR-19.

[0098] Figure 8B Representative BLI of MCL xenografts over time are shown.

[0099] Figure 8C The average flux (photons / second) of all mice in the three groups at different time points is shown. The figure represents two simultaneous experiments of five mice in each group performed with CAR T cells obtained from two different healthy donors and pooled data. Mean values ​​± SD (t test, p < 0.05) are shown.

[0100] Fig.8D The absolute number of CAR T cells in peripheral blood was monitored using flow cytometry. The absolute counts of CAR T cells were plotted on day 14.

[0101] Fig. 9A CD37 expression on PTCL tumor cell lines is shown.

[0102] Fig. 9B Representative FACS plots from patient-derived samples are shown.

[0103] Fig. 10A and Fig. 10B CD69 expression of CART cells after 6 hours of co-culture with the indicated tumor cells at an E:T ratio of 1:1 ( Fig. 10A ) and CD107a degranulation ( Fig. 10B ), as assessed by flow cytometry. Degranulation is relative to a PMA positive control; a representative normal donor is shown.

[0104] Fig. 10C and Fig. 10D The cytotoxicity of CAR-37 T cells is shown in Figure 5, which is in contrast to Hut78 ( Fig. 10C ) and FEPD( Fig. 10D ) Target cells were measured after co-culture overnight at different E:T ratios. Cytotoxicity assays are representative of three independent experiments performed with different healthy donors. Mean ± SEM are shown.

[0105] Fig.11A Schematic diagram of two bispecific second generation CAR constructs with different scFv orders are shown: CAR-19-37 (top) and CAR-37-19 (bottom).

[0106] Fig. 11B Shown are representative flow cytometry plots of transduction efficiency of primary human T cells 10 days after activation with CD3 / CD28 beads.

[0107] Fig. 11C Shown are expanded T cells from two healthy donors showing variable CAR expression with average values ​​of 19% (CAR-19-37) and 48% (CAR-37-19).

[0108] Fig. 12A Activation of Jurkat reporter gene (NFAT-Luc) T cells transduced with different CAR constructs and co-cultured with tumor cells is shown. Luciferase activity was measured after 16 hours. (CD3-CD28 beads: positive control).

[0109] Fig. 12B Shown is the ex vivo expansion of CD3 / CD28 bead-activated and target-stimulated T cells over 30 days in two healthy donors.

[0110] Fig. 12C Shown are the cytotoxic capacity 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. The cytotoxicity assays are representative of two independent experiments performed with different healthy donors.

[0111] Fig.12D Figure 2 shows tumor burden over time in NSG mice. NSG mice were injected IV with 1x10 6 JEKO-1(CBG-GFP) cells were added and tumor burden was monitored by BLI. On day 0, mice were randomized based on tumor burden (BLI) to receive 2x 10 6 Control T cells (UTD), CAR-37, CAR-19, CAR-19-37 or CAR-37-19. All CAR T cell groups are normalized to have the same % CAR+ cells and untransduced cells. The average flux (photons / second) of all mice at different time points is shown. The figure shows an experiment of six mice per group.

[0112] Fig.12E Shown are the absolute counts of CAR T cells, which were enumerated in peripheral blood by flow cytometry at the indicated time points. The absolute counts of CAR T cells are shown as mean ± SEM. DETAILED DESCRIPTION

[0113] The present invention provides bispecific chimeric antigen receptor (CAR) polypeptides targeting CD37 and CD19. Also described are nucleic acid molecules encoding bispecific CARs, vectors comprising the nucleic acid molecules, and methods for preparing and using the same. In addition, methods for treating diseases or disorders (e.g., cancer) described herein with bispecific CARs and related molecules described herein are provided.

[0114] Chimeric Antigen Receptor

[0115] The technology described herein provides bispecific CARs targeting CD37 and CD19 for use, for example, in immunotherapy for treating cancer.

[0116] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered T cell receptor that specifically transplants a ligand or antigen to 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. CAR is also referred to as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor. In addition, the term "CAR" includes bispecific CARs as described herein.

[0117] CAR places a chimeric extracellular target binding domain (which specifically binds to a target expressed on the surface of a cell to be targeted to obtain a T cell response, such as a polypeptide) on a construct comprising a transmembrane domain of a T cell receptor molecule and one or more intracellular domains. 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 to be targeted to obtain a T cell response. The characteristics of the intracellular signaling domain of CAR may vary as known in the art and disclosed herein, but when the chimeric target / antigen binding domain binds to a target / antigen on the surface of a target cell, the chimeric target / antigen binding domain sensitizes the receptor to signal activation.

[0118] Regarding the intracellular signaling domain, the so-called "first generation" CAR includes a CAR that only provides a CD3zeta (CD3ζ) signal after antigen binding. The so-called "second generation" CAR includes a CAR that provides both co-stimulation (e.g., CD28 or CD137) and activation (CD3ζ) domains, while the so-called "third generation" CAR includes a CAR that provides multiple co-stimulation (e.g., CD28 and CD137) domains and activation domains (e.g., CD3ζ). In various embodiments, CAR is selected to have high affinity or avidity for the target / antigen-for example, compared to naturally occurring T cell receptors, antibody-derived targets or antigen binding domains will generally have higher affinity and / or avidity for the target antigen. This characteristic, combined with the high specificity of antibodies that can be selected, provides high-specificity T cell targeting performed by CAR T cells.

[0119] As used herein, "CAR T cells" or "CAR-T" refers to T cells expressing CAR. Similarly, "CAR NK cells" refers to NK cells expressing CAR. When expressed in T or NK cells, CAR has the ability to redirect the specificity and reactivity of T or NK cells against the selected target in a non-MHC restricted manner using the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T or NK cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape.

[0120] As used herein, the term "extracellular target binding domain" refers to a polypeptide found outside the cell that is sufficient to promote binding to a target. The extracellular target binding domain will specifically bind to its binding partner, i.e., the target. As a non-limiting example, the extracellular target binding domain may include an antigen binding domain of an antibody or ligand that recognizes or binds to a cognate binding partner (e.g., CD37 or CD19) protein. In this article, a ligand is a molecule that specifically binds to a portion of a protein and / or a receptor. The cognate binding partner of the ligand that can be used in the methods and compositions described herein can generally be found on the cell surface. Ligand: cognate partner binding can result in changes in the receptor with the ligand, or activate a physiological response, such as activating a signal transduction pathway. In one embodiment, the ligand may be non-natural to the genome. Optionally, the ligand has a conserved function on at least two species. In one embodiment, the extracellular target binding domain comprises a non-antibody ligand.

[0121] Antibody Reagents

[0122] In various embodiments, the CAR described herein comprises an antibody agent or an antigen binding domain thereof as an extracellular target binding domain.

[0123] As used herein, the term "antibody reagent" refers to a polypeptide that comprises at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. The antibody reagent may comprise an antibody or a polypeptide comprising the antigen binding domain of an antibody. In some embodiments of any aspect, the antibody reagent may comprise a monoclonal antibody or a polypeptide comprising the antigen binding domain of a monoclonal antibody. For example, an antibody may comprise a heavy (H) chain variable region (abbreviated herein as V H ) and light (L) chain variable region (abbreviated herein as V L). In another example, the antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDR and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur J. Immunol. 26(3): 629-639, 1996; which is incorporated herein by reference in its entirety) as well as complete antibodies. The antibody may have structural features of IgA, IgG, IgE, IgD or IgM (and subtypes and combinations thereof). The antibody may be from any source, including mice, rabbits, pigs, rats and primates (human and non-human primates) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, etc. Methods known to those of ordinary skill in the art may be used, for example, to select complete human antibody binding domains from phage display libraries.

[0124] V H and V L The V region can be further subdivided into regions of high variability, termed "complementarity determining regions" ("CDRs"), interspersed with more conserved regions, termed "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; each of which is incorporated by reference in its entirety). Each V region is a member of a family of proteins, termed "complementarity determining regions" ("CDRs"), termed "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; each of which is incorporated by reference in its entirety). H and V L It is usually composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0125] In some embodiments, the antibody or antibody reagent is not a human antibody or antibody reagent (i.e., the antibody or antibody reagent is a mouse), but has been humanized. "Humanized antibody or antibody reagent" refers to a non-human antibody or antibody reagent that has been modified at the protein sequence level to increase its similarity to an antibody or antibody reagent variant naturally produced in the human body. One method for humanizing antibodies is to transplant mouse or other non-human CDRs onto a human antibody framework.

[0126] In some embodiments, the extracellular target binding domain of the CAR comprises or consists essentially of a single-chain variable fragment (scFv) that is a V fragment of an antibody (usually a monoclonal antibody) fused via a flexible linker peptide. H and V L In various embodiments, the scFv is optionally fused to the transmembrane domain via a hinge and fused to the T cell receptor intracellular signaling domain (e.g., an engineered intracellular signaling domain as described herein). Antibody binding domains that can be used for CARs described herein and the ways in which they are selected and cloned are well known to those of ordinary skill in the art.

[0127] In some embodiments, the CAR that can be used for the technology described herein comprises at least two antigen-specific targeting regions, an extracellular domain, a transmembrane domain, and an intracellular signaling domain. Optionally, 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 series and separated by a linker sequence. In another embodiment, CAR is a bispecific CAR that is specific for two different antigens.

[0128] For example, a CAR as described herein is a bispecific CAR that can bind both CD37 and CD 19. The CD37 binding site and the CD19 binding site can each comprise an antibody reagent, such as a single chain variable fragment (scFv).

[0129] Therefore, in some embodiments, the CD37 binding sequence of the bispecific CAR is 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 a sequence of SEQ ID NO: 1, 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 greater sequence identity with the sequence of SEQ ID NO: 1. In some embodiments, the VL of the anti-CD37 scFv corresponds to, comprises a sequence of SEQ ID NO: 2, 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 greater sequence identity with the sequence of SEQ ID NO: 2. The VH of the anti-CD37 scFv can be located at the N-terminus of the VL, or the VL can be located at the N-terminus of the VH. The VL and VH domains can be optionally connected via a linker, such as the linker of SEQ ID NO: 3. In some embodiments, the anti-CD37 scFv corresponds to the sequence of SEQ ID NO: 4 or 5; comprises the sequence of SEQ ID NO: 4 or 5; 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 greater sequence identity with the sequence of SEQ ID NO: 4 or 5.

[0130] In some embodiments, the CD19 binding sequence of the bispecific CAR is an antibody reagent. In other embodiments, the antibody reagent is an anti-CD19 scFv. In some embodiments, the VH of the anti-CD19 scFv corresponds to, comprises a sequence of SEQ ID NO: 12, or comprises a sequence with SEQ ID NO: 12 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 greater sequence identity. In some embodiments, the VL of the anti-CD19 scFv corresponds to, comprises a sequence of SEQ ID NO: 13, or comprises a sequence with SEQ ID NO: 13 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 greater sequence identity. The VH of the anti-CD19 scFv may be located at the N-terminus of the VL, or the VL may be located at the N-terminus of the VH. The VL and VH domains may optionally be connected via a linker, such as the linker of SEQ ID NO: 3. In some embodiments, the anti-CD19 scFv corresponds to the sequence of SEQ ID NO: 14; 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 greater sequence identity with the sequence of SEQ ID NO: 14.

[0131] Target / Antigen

[0132] Any cell surface part can be targeted by CAR. Most commonly, the target will be a cell surface polypeptide that is differentially expressed or preferentially expressed on cells that are desired to be targeted to obtain a T cell response. In this regard, tumor antigens or tumor-associated antigens provide attractive targets, providing a means of targeting tumor cells while avoiding or at least limiting collateral damage to non-tumor cells or tissues.

[0133] As noted above, one target of bispecific CAR is CD37. CD37 is a cell surface protein containing four hydrophobic transmembrane domains. CD37 is expressed only on immune cells. It is highly expressed on mature B cells and moderately expressed on T cells and myeloid cells. CD37 sequences of many species are known, 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 may refer to human CD37, including naturally occurring variants, molecules and alleles thereof. In some embodiments of any aspect, for example, in veterinary applications, CD37 may refer to CD37 of, for example, dogs, cats, cattle, horses, pigs, etc. Homologs and / or orthologs of human CD37 are readily identified for a given species by one skilled in the art, for example, using the NCBI ortholog search function or searching available sequence data for sequences similar to a reference CD37 sequence for such species.

[0134] The second target of bispecific CAR is CD19. CD19 is a transmembrane protein expressed in all B lineage cells (except plasma cells) and follicular dendritic cells. CD19 sequences of many species are known, for example, human CD19 (NCBI gene ID: 930) polypeptide (e.g., NCBI GenBank accession number: AAB60697.1) and DNA (e.g., NCBI GenBank accession number: AH005421.2). CD19 may refer to human CD19, including its naturally occurring variants, molecules and alleles. In some embodiments of any aspect, for example, in veterinary applications, CD19 may refer to CD19 of, for example, cats, dogs, cattle, horses, pigs, etc. By those skilled in the art, for example, using the NCBI ortholog search function or searching for available sequence data for sequences similar to the reference CD19 sequence for a given species, it is easy to identify homologs and / or orthologs of human CD19 for such species.

[0135] Hinge and transmembrane domains

[0136] The binding domain of CAR is optionally followed by one or more "hinge domains", which play a role in positioning the target binding domain away from the effector cell surface to achieve appropriate cell / cell contact, target binding and activation. CAR optionally includes one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. The exemplary hinge domains suitable for bispecific CAR described herein include hinge regions derived from the extracellular region of type 1 membrane proteins (such as CD8 (e.g., CD8α), CD4, CD28 and CD7), which may be wild-type hinge regions from these molecules or may be altered. In one embodiment, the hinge domain includes a CD8α hinge region.

[0137] CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-to-cell interactions within the immune system and acts as a T cell co-receptor. CD8 consists of α (CD8α or CD8a) and β (CD8β or CD8b) chains. CD8a sequences of many species are known, 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 may refer to human CD8, including naturally occurring variants, molecules and alleles thereof. In some embodiments of any aspect, for example, in veterinary applications, CD8 may refer to CD8 of, for example, dogs, cats, cattle, horses, pigs, etc. By those skilled in the art, for example, using the NCBI ortholog search function or searching for available sequence data for sequences similar to the reference CD8 sequence for a given species, it is easy to identify homologs and / or orthologs of human CD8 for such species.

[0138] As used herein, "transmembrane domain" ("TM domain") refers to a portion of CAR that optionally fuses the extracellular binding portion with the intracellular portion (e.g., intracellular signaling domain and co-stimulatory domain (if present)) via a hinge, and anchors CAR to the plasma membrane of immune effector cells. The transmembrane domain is typically a hydrophobic region of CAR that spans the plasma membrane of the cell. The transmembrane domain can be a transmembrane region or fragment thereof of a transmembrane protein (e.g., type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Although specific examples are provided herein and used in the embodiments, other transmembrane domains will be clear to those skilled in the art and can be used in combination with alternative embodiments of the present technology. The selected transmembrane region or fragment thereof will preferably not interfere with the intended function of CAR. As used with respect to the transmembrane domain of a protein or polypeptide, "its fragment" refers to a portion of a transmembrane domain that is sufficient to anchor or attach a protein to a cell surface.

[0139] In some examples, the transmembrane domain of the CAR described herein or a fragment thereof comprises a transmembrane domain selected from the following transmembrane domains: α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, 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 AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C. In one embodiment, the transmembrane domain or fragment thereof is derived from or comprises the transmembrane domain of CD8.

[0140] As used herein, "hinge / transmembrane domain" refers to a domain comprising both a hinge domain and a transmembrane domain. In one embodiment, the hinge / transmembrane domain of the bispecific CAR or its fragment is derived from or comprises the hinge / transmembrane domain of CD8. The CD8 hinge / transmembrane domain may include the amino acid sequence of SEQ ID NO: 9 or a variant thereof.

[0141] Costimulatory domain

[0142] The bispecific CAR described herein optionally includes 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. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors, which provide a second signal required for the effective activation and function of T lymphocytes when binding antigens. Illustrative examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70. For example, the intracellular domain is the intracellular domain of 4-1BB.

[0143] 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. The 4-1BB sequences of many species are known, for example, human 4-1BB, also known as TNFRSF9 (NCBI gene ID: 3604) and mRNA (NCBI reference sequence: NM_001561.5). 4-1BB may refer to human 4-1BB, including its naturally occurring variants, molecules and alleles. In some embodiments of any aspect, for example, in veterinary applications, 4-1BB may refer to 4-1BB such as dogs, cats, cattle, horses, pigs, etc. By those skilled in the art, for example, using the NCBI ortholog search function or searching for available sequence data for a given species for a sequence similar to a reference 4-1BB sequence, it is easy to identify homologs and / or orthologs of human 4-1BB for such species.

[0144] For example, the 4-1BB co-stimulatory domain of the bispecific CAR described herein may include the amino acid sequence of SEQ ID NO: 10 or a variant thereof.

[0145] Intracellular signaling domain

[0146] Bispecific CAR as described herein includes intracellular signaling domain. "Intracellular signaling domain" refers to a part of CAR polypeptide, which participates in the information transduction of effective CAR combined with target antigen to the inside of immune effector cells to induce effector cell function (e.g., activation, cytokine production, proliferation and cytotoxic activity), including releasing cytotoxic factors to the target cells combined with CAR, or other cellular responses triggered after antigen binding to the extracellular CAR domain. Non-limiting examples of intracellular signaling domains containing activation motifs (ITAMs) based on immunoreceptor tyrosine used in the technology include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 θ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b and CD66d.

[0147] CD3 is a T cell co-receptor that promotes T lymphocyte activation when engaged simultaneously with appropriate co-stimulation (e.g., binding of co-stimulatory molecules). The CD3 complex consists of 4 different chains; mammalian CD3 consists of one CD3γ chain, one CD3δ chain, and two CD3ε chains. These chains associate with molecules called T cell receptors (TCRs) and CD3ζ, thereby generating activation signals in T lymphocytes. The complete TCR complex contains the TCR, CD3ζ, and the complete CD3 complex.

[0148] 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, ITAM comprises three motifs of the ITAM (ITAM3) of CD3ζ. In some embodiments of any aspect, the three motifs of the ITAM of CD3ζ are not mutated, and thus include a natural or wild-type sequence. For example, the CD3ζ sequence of the bispecific CAR described herein comprises SEQ ID NO:11 sequence or a variant thereof, as shown below. In various embodiments, the CD3ζ sequence of such CAR polypeptides is a natural or wild-type sequence.

[0149] A more detailed description of CAR and CAR T cells can be found in the following documents: 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 herein by reference in its entirety.

[0150] In some embodiments, CAR also includes a joint domain. "Joint domain" or "joint region" as used herein refers to an oligo or polypeptide region of about 2 to 100 amino acids in length, which connects any domain / region of CAR as described herein together. In some embodiments, the joint may include or be composed of flexible residues (such as glycine and serine) so that adjacent protein domains are free to move relative to each other, for example, SEQ ID NO: 3 joints. When it is desired to ensure that two adjacent domains do not interfere with each other spatially, a longer joint may be used. The joint may be cleavable or non-cleavable. Examples of cleavable joints include 2A joints (e.g., T2A), 2A-like joints or their functional equivalents and combinations thereof. In some embodiments, the joint region is T2A derived from Thosea asigna virus. Non-limiting examples of joints that can be used in this technology include P2A and F2A.

[0151] In some embodiments, CAR as described herein further comprises a reporter molecule, for example to allow non-invasive imaging (e.g., positron emission tomography PET scanning). In a bispecific CAR comprising a reporter molecule, the first extracellular binding domain and the second extracellular binding domain may include different or identical reporter molecules. In a bispecific CAR T cell, the first CAR and the second CAR may express different or identical reporter molecules. In another embodiment, CAR as described herein further comprises a reporter molecule that can be expressed alone or with a substrate or chemical substance (e.g., 9-[4-[ 18 F]fluoro-3-(hydroxymethyl)butyl]guanine ([ 18 In another embodiment, the CAR as described herein further comprises nanoparticles that can be easily imaged using non-invasive techniques (e.g., using 64 Cu 2+Functionalized gold nanoparticles (GNPs). Labeling of CAR T cells for non-invasive imaging is reviewed in, for example, 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 entirety.

[0152] GFP and mCherry are shown herein to be fluorescent tags for imaging CAR expressed on T cells or NK cells (e.g., CAR T cells or CAR NK cells). It is expected that substantially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, CAR does not have to include fluorescent tags or fluorescent proteins.

[0153] 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 greater sequence identity to the sequence of SEQ ID NO: 6, 7, 15, 16, 17, 18, 19 or 20.

[0154] Nucleic acid encoding CAR

[0155] Also provided are nucleic acid constructs and vectors encoding bispecific CAR polypeptides described herein for producing bispecific CAR T cells. In various examples, the present invention provides constructs, each including independent coding sequences for a variety of proteins to be expressed in the bispecific CAR T cells of the present invention. These independent 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 the cleavage of the polyprotein produced can be guided during transcription. As noted above, the constructs and vectors of the present invention may include any of a variety of different sequence combinations.

[0156] In addition, the polynucleotides of the present invention may include the expression of suicide genes. This may facilitate external drug-mediated control of the administered cells. For example, by using suicide genes, in the case of, for example, adverse events, the modified cells may be depleted from the patient. In one example, the FK506 binding domain is fused to a caspase 9 pro-apoptotic molecule. T cells engineered in this manner are made sensitive to the immunosuppressive drug 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 may be triggered by administering specific molecules (e.g., ganciclovir to TK+ cells) or antibodies or antibody-drug conjugates to conditional ablation.

[0157] A construct including a sequence encoding a protein expressed in the bispecific CAR T cells of the present invention may be included in a vector. In various examples, the vector is a retroviral vector. Retroviruses (such as lentiviruses) provide a convenient platform for delivering nucleic acid sequences encoding target genes or chimeric genes. The selected nucleic acid sequence can be inserted into a vector and packaged in 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 in, for example, U.S. Patent No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu and Pathak Pharmacological Reviews 200052: 493-512; Each of the documents described is incorporated herein by reference in its entirety. The lentiviral system for effective DNA delivery can be purchased from OriGene in Rockville, Maryland. In various embodiments, the protein is expressed in T or NK cells by transfecting or electroporating an expression vector containing a nucleic acid encoding a 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 polypeptides of any bispecific CAR described herein are expressed in mammalian cells via transfection or electroporation of an expression vector containing a nucleic acid encoding a CAR. Transfection or electroporation methods are known in the art.

[0158] Standard assays for detecting mRNA, DNA or gene products of nucleic acids encoding proteins can be used to assess the effective expression of bispecific CAR polypeptides as described herein in immune cells. For example, RT-PCR, FACS, northern blot, western blot, ELISA or immunohistochemistry can be used. The proteins described herein can be constitutively expressed or inducibly expressed. In some examples, the protein is encoded by a recombinant nucleic acid sequence. In one embodiment, the CAR polypeptides described herein are constitutively expressed. In one embodiment, the CAR polypeptides described herein are encoded by a recombinant nucleic acid sequence.

[0159] As described herein, the present invention also provides a composition comprising a vector comprising a polynucleotide sequence encoding a bispecific CAR, wherein the bispecific CAR comprises an extracellular domain comprising a sequence that specifically binds to CD37 and CD19.

[0160] For example, the present invention provides a nucleic acid that can encode a CAR polypeptide of SEQ ID NO: 6, 7, 15, 16, 17, 18, 19 or 20, or a CAR polypeptide comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to the sequence of SEQ ID NO: 6, 7, 15, 16, 17, 18, 19 or 20.

[0161] Immune cells

[0162] One aspect of the technology relates to an immune cell comprising any bispecific CAR polypeptide described herein (e.g., SEQ ID NO: 15, 16, 19 or 20); or a nucleic acid encoding any bispecific CAR polypeptide described herein. In one embodiment, the immune cell comprises an antibody, an antibody reagent, an antigen binding portion thereof, or any bispecific CAR described herein, or a nucleic acid encoding such an antibody, an antibody reagent, an antigen binding portion thereof, or any bispecific CAR described herein. As used herein, "immune cell" refers to a cell that plays a role in an 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. The immune cell may be a T cell; 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.

[0163] Immune cells (e.g., human immune cells) that can be used in the present invention include autologous cells, which are obtained from a subject to which the cells will be subsequently administered after ex vivo modification and amplification. For example, immune cells can be obtained from individuals suffering from or diagnosed with cancer, autoimmune diseases, or plasma cell disorders. Immune cells can also be obtained from allogeneic donors, which are non-genetically identical individuals of the same species as the intended recipient of the cells. Immune cells that can be used in the present invention include T cells and natural killer (NK) cells.

[0164] Methods for obtaining T cells and NK are known in the art and can be used for engineered immune cells as described herein.T cells and NK cells are generally obtained from peripheral blood, which is collected from a subject by, for example, venipuncture or withdrawal by implantation of a port or catheter. Optionally, blood can be obtained by a method including leukocyte apheresis, wherein leukocytes are obtained from the blood of the subject, and other blood components are returned to the subject. Blood or leukocyte apheresis products (fresh or cryopreserved) are processed using methods known in the art to enrich T cells or NK cells. For example, density gradient centrifugation (using, for example, Ficoll) and / or countercurrent centrifugal elutriation can be performed to enrich monocytes (including T cells or NK cells). In one example, for T cells, a T cell stimulation step can be further performed to stimulate T cells and exhaust other cells (for example, B cells), and the T cell stimulation step uses, for example, CD3 / CD28 antibodies coated on magnetic beads or expression, for example, artificial antigen presenting cells (aAPC) of anti-CD3 and anti-CD28 antibody fragments bound to cell surfaces (see below). The T cells of the enriched T cell preparation can then be subjected to genetic modification.

[0165] As an alternative to peripheral blood, tissues including bone marrow, lymph nodes, spleen and tumors can be used as sources of T cells and NK cells. T cells and NK cells can be from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs or cats, but any other mammalian cells can be used. In certain embodiments of any aspect, T cells and NK cells are human.

[0166] Immune cells (e.g., T cells or NK cells) can be engineered to include any bispecific CAR polypeptides described herein (e.g., SEQ ID NO: 15, 16, 19 or 20); or nucleic acids encoding any CAR polypeptides described herein. In some embodiments, any bispecific CAR polypeptides described herein are contained in a lentiviral vector. Using standard infection techniques, lentiviral vectors are used to express CAR polypeptides in cells. In some embodiments, the immune cells (e.g., T cells or NK cells) are obtained from individuals suffering from or diagnosed with cancer expressing CD37. In some embodiments, immune cells are obtained from individuals who are unresponsive to anti-CD19 and / or anti-CD20 therapy and / or receive anti-CD19 and / or anti-CD20 therapy simultaneously.

[0167] Treatment

[0168] The present invention provides methods and compositions for treating and preventing diseases and disorders, including, for example, cancer, autoimmune diseases or disorders or plasma cell diseases or disorders. These methods include using immune cells (e.g., T cells or NK cells) comprising bispecific CAR as described herein, and applying modified immune cells to subjects to treat, for example, cancer. In some embodiments of any of the aspects, modified immune cells (e.g., including one or more additionally modified T cells or NK cells as described herein) are stimulated and / or activated before being applied to a subject.

[0169] As used herein, "disorder" includes cancer, infectious diseases, autoimmune diseases or disorders, plasma cell diseases or disorders, or disorders related to transplantation. Subjects suffering from a disease or condition can be identified by a physician using current methods for diagnosing the disease or condition. Symptoms and / or complications of the disease or condition that characterize these conditions and help diagnose are well known in the art, and include, but are not limited to, fatigue, persistent infection, and persistent bleeding. Tests that can help diagnose, for example, a disease or condition include, but are not limited to, blood screening and bone marrow testing, and are known in the art for a given condition. A family history of a disease or condition, or risk factors for exposure to a disease or condition, can also help determine whether a subject is likely to suffer from the disease or condition, or make a diagnosis of the disease or condition.

[0170] As used herein, "cancer" may refer to an over-proliferation of cells, a unique trait, loss of normal cell control, resulting in unregulated growth, lack of differentiation, local tissue infiltration and metastasis, and may be a 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 lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt 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 lymphoma, PTCL, CTCL, AITL, or ALCL. Non-limiting examples of solid tumors include adrenocortical tumors, alveolar soft tissue sarcomas, carcinomas, chondrosarcomas, colorectal cancer, ligamentous tumors, desmoplastic small round cell tumors, endocrine tumors, endodermal sinus tumors, epithelioid hemangioendothelioma, Ewing's sarcoma, germ cell tumors (solid tumors), giant cell tumors of bone and soft tissue, glioblastomas, hepatoblastomas, hepatocellular carcinomas, melanomas, nephroblastomas, neuroblastomas, non-rhabdomyosarcoma soft tissue sarcomas (NRSTS), osteosarcomas, paravertebral sarcomas, renal cell carcinomas, retinoblastomas, rhabdomyosarcomas, synovial sarcomas, and Wilms' tumors. Solid tumors can be found in bones, muscles, or organs, and can be sarcomas or cancers. It is contemplated that any aspect of the technology described herein can be used to treat all types of cancers, including cancers not listed in the present application. As used herein, the term "tumor" refers to, for example, an abnormal growth of cells or tissues of a malignant or benign type.

[0171] As used herein, "autoimmune disease" or "autoimmune disorder" is characterized by the inability of a person's immune system to distinguish between foreign cells and healthy cells. This causes the immune system to target healthy cells in the human body for programmed cell death. Non-limiting examples of autoimmune diseases or disorders include inflammatory arthritis, type 1 diabetes, multiple sclerosis, psoriasis, inflammatory bowel disease, SLE and vasculitis, allergic inflammation, such as allergic asthma, atopic dermatitis and contact hypersensitivity. Other examples of autoimmune-related diseases or disorders (but not to be construed as being limited to) include rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, Graves' disease (hyperthyroidism), Hashimoto's thyroiditis (underactive thyroid), celiac disease, Crohn's disease and ulcerative colitis, Guillain-Barre syndrome, primary biliary cirrhosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjögren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporomandibular dysplasia, leukemia ... Arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), psoriasis, autoimmune Addison's disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anemia, canine polyarthritis, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, and fibromyalgia (FM).

[0172] Plasma cells are white blood cells produced by B lymphocytes, whose function is to produce and release antibodies needed to resist infection. As used herein, the characteristic of "plasma cell disorder or disease" is the abnormal proliferation of plasma cells. Abnormal plasma cells can "crowd out" healthy plasma cells, which leads to reduced ability to resist foreign bodies (such as viruses or bacterial cells). Non-limiting examples of plasma cell disorders include amyloidosis, Waldenstrom's macroglobulinemia, osteosclerotic myeloma (POEMS syndrome), monoclonal gammopathy of unknown significance (MGUS) and plasma cell myeloma.

[0173] Retroviruses (such as lentiviruses) provide a convenient platform for delivering nucleic acid sequences encoding target genes or chimeric genes. The selected nucleic acid sequence can be inserted into a vector and packaged in 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 in, for example, U.S. Patent No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu et al., Pharmacological Reviews 52: 493-512, 2000; all of which are incorporated herein by reference in their entirety. Slow virus systems for effective DNA delivery can be purchased from OriGene in Rockville, Maryland.

[0174] One aspect of the technology described herein relates to a method of treating cancer in a subject in need thereof, the method comprising administering a cell of any mammalian cell comprising any of the CAR polypeptides described herein.

[0175] Cluster of differentiation (CD) molecules are cell surface markers present on leukocytes. As leukocytes differentiate and mature, their CD spectrum changes. In the case where leukocytes become cancer cells (i.e., lymphoma), their CD spectrum is important for diagnosing the disease. The treatment and prognosis of certain types of cancer depend on determining the CD spectrum of cancer cells. "CDX+", where "X" is a CD marker, indicates that the CD marker is present in cancer cells, while "CDX-" indicates that the marker is not present. Those skilled in the art will be able to use standard techniques, such as using immunofluorescence to detect commercially available antibodies that bind to CD molecules, to assess the CD molecules present on cancer cells.

[0176] In some embodiments, the cancer expresses one or more CD molecules. The bispecific CAR described herein can be used to treat cancers expressing CD37. In some embodiments, the CD37+ cancer is lymphoma or leukemia. For example, the lymphoma is B cell non-Hodgkin lymphoma (NHL) (e.g., mantle cell lymphoma (MCL), Burkitt's lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma or Burkitt's lymphoma) or 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).

[0177] In addition, cancer cells can evolve in response to treatment to change their CD profiles, thereby escaping the treatment. For example, patients with CD19+ leukemia or lymphoma can be treated with anti-CD19 therapy. After treatment, cancer cells may relapse or relapse after treatment and no longer express the CD19 marker, resulting in a CD19 leukemia or lymphoma. Therefore, the cancer will no longer be targeted by anti-CD19 therapy.

[0178] Provided is a method for treating cancer in a subject who is unresponsive or refractory to anti-CD19 and / or anti-CD20 therapy using an immune cell comprising a bispecific CAR as described herein (e.g., a CAR targeting CD37 and CD19). In some embodiments, immune cells comprising a bispecific CAR as described herein (e.g., a CAR targeting CD37 and CD19) are used to treat cancer in a subject with CD19- and / or CD20- cancer. In some embodiments, immune cells comprising a bispecific CAR as described herein (e.g., a CAR targeting CD37 and CD19) are used to treat cancer in a subject with relapsed and no longer expressing CD19 or CD20 cancer.

[0179] In addition, immune cells comprising a bispecific CAR as described herein (e.g., a CAR targeting CD37 and CD19) can be used to treat cancer in a subject in need thereof, wherein the subject is concurrently administered anti-CD19 and / or anti-CD20 therapy.

[0180] In some embodiments of any of the aspects, 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.

[0181] Application

[0182] In some embodiments, the methods described herein relate to treating a subject suffering from or diagnosed with cancer, plasma cell disease or disorder, or autoimmune disease or disorder with a mammalian cell comprising any CAR polypeptide described herein or a nucleic acid encoding any CAR polypeptide described herein. As used herein, bispecific CAR T or NK cells refer to mammalian T or NK cells comprising any bispecific CAR polypeptide as described or a nucleic acid encoding any bispecific CAR polypeptide.

[0183] The compositions described herein can be administered to subjects suffering from or diagnosed with a disease. In some embodiments, the methods described herein include administering to a subject an effective amount of bispecific CAR T or NK cells described herein to alleviate the symptoms of the disease. As used herein, "reducing the symptoms of a disease" is to improve any disease or symptoms associated with the disease. Compared to an equivalent untreated control, this reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique. A variety of methods for administering the compositions described herein to a subject are known to those skilled in the art. In one embodiment, the compositions described herein are administered systemically or topically. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the tumor site.

[0184] As used herein, the term "effective amount" refers to the amount of bispecific CAR T or NK cells required to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of cell preparations or compositions to provide the desired effect. Therefore, the term "therapeutically effective amount" refers to the amount of bispecific CAR T or NK cells sufficient to provide a specific anti-disease effect when administered to a typical subject. In various cases, the effective amount as used herein will also include an amount sufficient to delay the development of disease symptoms, change the course of symptomatic disease (such as but not limited to slowing the progression of the disease) or reverse the symptoms of the disease. Therefore, it is generally not feasible to specify an exact "effective amount". However, for any given case, a person of ordinary skill in the art can determine the appropriate "effective amount" using only routine experiments.

[0185] Effective amount, toxicity and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell culture or experimental animals. The dosage may vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods showing a large therapeutic index are preferred. Initially, the therapeutically effective dose can be estimated from a cell culture assay. In addition, a dosage can be formulated in an animal model to achieve a circulating plasma concentration range including an IC50 (i.e., a concentration at which bispecific CAR T or NK cells achieve half-maximal inhibition of symptoms) as determined in cell culture or in an appropriate animal model. The level in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dose can be monitored by a suitable bioassay, such as an assay for bone marrow testing, etc. The dosage can be determined by a physician and adjusted as needed to suit the observed therapeutic effect.

[0186] In one aspect of the technology, the technology described herein relates to a pharmaceutical composition comprising a bispecific CAR T or NK cell as described herein, and an optional pharmaceutically acceptable carrier. The active ingredient of the pharmaceutical composition comprises at least a bispecific CAR T or NK cell as described herein. In some embodiments, the active ingredient of the pharmaceutical composition is substantially composed of a bispecific CAR T or NK cell as described herein. In some embodiments, the active ingredient of the pharmaceutical composition is composed of a bispecific CAR T or NK cell as described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulations include saline and aqueous buffer solutions, Ringer's solution, and serum components, such as serum albumin, HDL, and LDL. Terms (such as "excipient", "carrier", "pharmaceutically acceptable carrier", etc.) are used interchangeably herein.

[0187] In some embodiments, the pharmaceutical composition comprising bispecific CAR T or NK cells as described herein can be a parenteral dosage form. Since the administration of parenteral dosage forms usually bypasses the patient's natural defenses against contaminants, the components other than the bispecific CAR T or NK cells themselves are preferably sterile or can be sterilized before administration to the patient. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry products to be dissolved or suspended in a pharmaceutically acceptable injection vehicle, suspensions for injection, and emulsions. Any of these can be added to the bispecific CAR T or NK cell preparation before administration.

[0188] Suitable vehicles that can be used to provide parenteral dosage forms of bispecific CAR T or NK cells as disclosed are well known to those skilled in the art. Examples include, but are not limited to, saline solutions, dextrose 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, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles, such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0189] dose

[0190] The term "unit dosage form" as used herein refers to a dosage for a suitable single administration. For example, a unit dosage form can be an amount of therapeutic agent placed in a delivery device (e.g., a syringe or an intravenous drip bag). In one embodiment, the unit dosage form is administered in a single administration. In another embodiment, more than one unit dosage form can be administered simultaneously.

[0191] In some embodiments, the bispecific CAR T or NK cell T cells described herein are administered as a monotherapy, i.e., another treatment for the disorder is not concurrently administered to the subject.

[0192] Typically, the pharmaceutical composition comprising the T or NK cells described herein can be taken as 10 4 Up to 10 9 cells / kg body weight, in some cases 10 5 Up to 10 6 The T or NK cell compositions may be administered at a dose of 1 cell / kg body weight, including all integer values ​​within those ranges. If desired, the T or NK cell compositions may also be administered multiple times at these doses. The cells may be administered using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 319: 1676, 1988).

[0193] In some aspects, it may be desirable to administer bispecific CAR T or NK cells to a subject, and then perform a subsequent blood draw (or blood sampling), activate T or NK cells therefrom as described herein, and re-inject these activated and amplified T or NK cells into the patient. This process can be performed multiple times every few weeks. In some aspects, T or NK cells can be activated from 10cc to 400cc of drawn blood. In some aspects, T or NK cells are activated from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc of drawn blood.

[0194] Mode of administration may include, for example, intravenous (iv) injection or infusion. Compositions described herein may be administered to patients via arteries, intratumoral, intranodular or intramedullary. In some embodiments, compositions of T or NK cells may be injected directly into tumors, lymph nodes or infection sites. In one embodiment, compositions described herein are administered to body cavities or body fluids (e.g., ascites, pleural fluid, peritoneal fluid or cerebrospinal fluid).

[0195] In a specific exemplary aspect, the subject can undergo leukapheresis, wherein ex vivo collection, enrichment or depletion of leukocytes to select and / or separate target immune cells, such as T cells or NK cells. These immune cell isolates can be, for example, in the case of T cells, by contacting with aAPC as described herein (such as aAPC expressing anti-CD28 and anti-CD3 CDR) and amplified, and processed so that one or more CAR constructs of the technology can be introduced, thereby producing CAR T cells. Subjects in need can then undergo standard treatment using high-dose chemotherapy, followed by peripheral blood stem cell transplantation. After transplantation or simultaneously therewith, the subject can receive an infusion of amplified CAR T cells or NK cells. In one embodiment, the amplified cells are administered before or after surgery.

[0196] In some embodiments, prior to administering one or more CAR T or NK cells as described herein, lymphodepletion is performed on the subject. In such embodiments, lymphodepletion may include administering one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0197] The dosage of the above treatments administered to a patient will vary with the exact nature of the condition being treated and the recipient of the treatment. Dosage scales for human administration can be made according to practices accepted in the art.

[0198] In some embodiments, a single treatment regimen is desired. In other cases, one or more subsequent doses or treatment regimens may be administered. For example, after biweekly treatment for three months, treatment may be repeated monthly for six months or a year or longer. In some embodiments, no additional treatment is administered after the initial treatment.

[0199] The dosage of the composition as described herein can be determined by a physician and adjusted as needed to suit the observed therapeutic effect. Regarding the duration and frequency of treatment, the subject is usually monitored by a skilled clinician to determine when the treatment provides a therapeutic benefit, and to determine whether to administer other cells, stop treatment, resume treatment, or make other changes to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. Typically, the dosage will vary with the patient's age, illness, and sex, and can be determined by those skilled in the art. In the case of any complications, the dosage can also be adjusted by an individual physician.

[0200] Combination therapy

[0201] The bispecific CAR T or NK cells described herein can be used in combination with other known agents and therapies. For example, anti-CD19 therapy and / or anti-CD20 therapy can be further administered to the subject. In one embodiment, the subject is resistant to anti-CD19 and / or anti-CD20 therapy. In another embodiment, the subject is administered anti-CD19 and / or anti-CD20 therapy simultaneously.

[0202] As used herein, "combination" administration means delivering two (or more) different treatments to a subject during the course of the subject suffering from a disorder, for example, the two or more treatments are delivered after the subject is diagnosed with the disorder and before the disorder is cured or eliminated or the treatment is stopped for other reasons. In some embodiments, when the delivery of the second treatment is started, the delivery of one treatment is still in progress, so that there is overlap in terms of administration. This is sometimes referred to as "simultaneous" or "concurrent" delivery in this article. In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of either case, the treatment is more effective due to combined administration. For example, compared with the administration of the second treatment in the absence of the first treatment, the second treatment is more effective, for example, an equivalent effect is observed with less second treatment, or the second treatment alleviates symptoms to a greater extent, or a similar situation is observed with the first treatment. In some embodiments, delivery causes a reduction in symptoms or other parameters associated with the disorder to be greater than the reduction observed by delivering one treatment in the absence of another treatment. The effects of the two treatments can be partially additive, completely additive, or greater than additive. The delivery can be such that when the second treatment is delivered, the effect of the first treatment delivered is still detectable. The bispecific CAR T or NK cells described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions or in sequence. For sequential administration, the immune cells expressing CAR described herein can be administered first, and then additional agents can be administered, or the order of administration can be reversed. Bispecific CAR T or NK therapy and / or other therapeutic agents, procedures or methods can be administered during an active disorder, or during a disease with less activity during remission. CAR T or NK therapy can be administered before another treatment, simultaneously with the treatment, after treatment, or during disorder remission.

[0203] When administered in combination, bispecific CAR T or NK cells and additional agents (e.g., second or third agents) or all, can be administered in an amount or dosage higher than, lower than or equal to the amount or dosage of each agent used alone (e.g., as a monotherapy). In certain embodiments, the amount or dosage of bispecific CAR T or NK cells, additional agents (e.g., second or third agents) or all is lower than (e.g., at least 20%, at least 30%, at least 40% or at least 50%) the amount or dosage of each agent used alone. In other embodiments, the amount or dosage of bispecific CAR T or NK cells, additional agents (e.g., second or third agents) or all resulting in a desired effect (e.g., cancer treatment) is lower than (e.g., at least 20%, at least 30%, at least 40% or at least 50% lower) the amount or dosage of each individual agent required to achieve the same therapeutic effect. In further embodiments, the bispecific CAR T or NK cells described herein can be used in combination with treatment regimens such as surgery, chemotherapy, radiation, mTOR pathway inhibitors, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, 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.

[0204] For example, bispecific CAR T or NK cells described herein can be used in combination with anti-CD19 therapy. The example of anti-CD19 therapy includes but is not limited to blinatumomab, levofloxacin-cotuximab, MOR208, MEDI-551, Martin-dinintozumab, Patto-taritumomab, XmAb 5871, MDX-1342, AFM11, DI-B4, sirolece-Acatajin and tesalen. In addition, bispecific CAR T or NK cells described herein can be used in combination with anti-CD20 therapy. The example of anti-CD20 therapy includes but is not limited to rituximab, ocrelizumab, otuzumab, ofatumumab, ibritumomab tiuxetan, tiuxetan, tositumomab, urituximab, otuzumab, IMMU-106 and GA-101.

[0205] In a further embodiment, 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 (atelizumab, aveiromonab, durvalumab, MSB0010718C, MEDI4736 and MPDL3280A) and anti-TIM3 inhibitors.

[0206] 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., alemtuzumab, gemtuzumab, rituximab, tositumomab); antimetabolites (including, for example, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)); mTOR inhibitors; TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists; proteasome inhibitors (e.g., aclarubicin A, gliotoxin, or bortezomib); immunomodulators, such as thalidomide or thalidomide derivatives (e.g., lenalidomide). General chemotherapeutic agents contemplated for combination therapy include anastrozole Bicalutamide Bleomycin sulfate Busulfan Busulfan Injection Capecitabine N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin Carmustine Chlorambucil Cisplatin Cladribine Cyclophosphamide ( or ), cytosine arabinoside, cytosine arabinoside Cytarabine liposome injection Dacarbazine Dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride Daunorubicin Citrate Liposome Injection Dexamethasone, docetaxel Doxorubicin Hydrochloride Etoposide Fludarabine phosphate 5-Fluorouracil Flutamide Tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea Ibrutinib, Idabi Ifosfamide Irinotecan L-Asparaginase Calcium folinate, melphalan 6-Mercaptopurine Methotrexate Mitoxantrone Gemtuzumab (mylotarg), paclitaxel Phoenix (Yttrium90 / MX-DTPA), Pentostatin, Polyphenylpropanone 20 with Carmustine implant Tamoxifen citrate Teniposide 6-thioguanine, thiotepa, tirapazamine Topotecan Hydrochloride for Injection Vinblastine Vincristine Vinorelbine Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: Aminouracil mustard Uracil nitrogen ), Nitrogen mustard hydrochloride Cyclophosphamide ( Revimmune TM ), ifosfamide Melphalan Chlorambucil Propirizine Triethylenemelamine triethylenethiophosphoramine, temozolomide Thiotepa Busulfan Carmustine Lomustine Streptozotocin and dacarbazine Additional exemplary alkylating agents include, but are not limited to, oxaliplatin Temozolomide ( and ); Dactinomycin (also known as actinomycin-D, ); Melphalan (also known as L-PAM, L-myristolysin, and phenylalanine mustard, ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), ); Carmustine Bendamustine Busulfan and ); Carboplatin Lomustine (also known as CCNU, ); cisplatin (also known as CDDP, and -AQ); Chlorambucil Cyclophosphamide ( and ); Dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), ); ifosfamide Prednumustine; Procarbazine Dichloromethyldiethylamine (also known as nitrogen mustard, mustine and nitrogen mustard hydrochloride, ); Streptozotocin Thiotepa (also known as thiophosphoramide, TESPA and TSPA, ); Cyclophosphamide and bendamustine HC1 Exemplary mTOR inhibitors include, for example, temsirolimus; lirolimus (formally known as deferolimus, (1R, 2R, 45)-4-[(2R)-2[(1R, 95, 125, 15R, 16E, 18R, 19R, 21R, 235, 24E, 26E, 28Z, 305, 325, 35R)-1, 18-dihydroxy-19, 30-dimethoxy- 15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04'9]triacontria-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383); everolimus ( or RADOOl); rapamycin (AY22989, ); simapimod (CAS 164301-51-3); temsirolimus (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-(i]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholine]-4-yl]methoxy]butyl]-L-arginylglycyl-La-aspartyl-L-serine-, inner salt (SF1126, CAS 936487-67-1) and XL765. Exemplary immunomodulators include, for example, afutuzumab (available from pegfilgrastim Lenalidomide (CC-5013, ); thalidomide 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 ( and ); Bleomycin Daunorubicin (daunorubicin hydrochloride, daunomycin and daunorubicin hydrochloride, ); daunorubicin liposomes (daunorubicin citrate liposomes, ); Mitoxantrone (DHAD, ); Ellence TM ); Idabi ( Idamycin ); Mitomycin C geldanamycin; heramicin; ravidomycin; and deacetyl ravidomycin. Exemplary vinca alkaloids include, for example, vinorelbine tartrate Vincristine Vindesine ); Vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, and ); and vinorelbine Exemplary proteosome inhibitors include bortezomib Carfilzomib (PX-171-007, (5)-4-methyl-N-((5)-1-(((5)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((5,)-2-(2-morpholinoacetamido)-4-phenylbutyramido)-pentanamide); Marizomib (NP T0052); ixazomib citrate (MLN-9708); dilanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(llS')-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0207] The chemotherapeutic agents used can be readily identified by one skilled in the art (see, e.g., Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Chapter 85 in Principles of Cancer Therapy, Harrison's Principles of Internal Medicine, 18th ed.; 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).

[0208] 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.

[0209] effect

[0210] The efficacy of bispecific CAR T or NK cells in, for example, treating a condition described herein or inducing a reaction as described herein (e.g., cancer cell reduction) can be determined by a skilled clinician. However, as used herein, treatment is considered to be "effective treatment", if one or more signs or symptoms of the condition described herein are changed in a beneficial manner, then after treatment according to the methods described herein, other clinically acceptable symptoms are improved, or even improved, or the desired reaction is induced, for example, at least 10%. Efficacy can be assessed, for example, by measuring the markers, indicators, symptoms and / or morbidity of the condition treated according to the methods described herein, or any other appropriate measurable parameter. Treatment according to the methods described herein can reduce the level of markers or symptoms of the condition, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.

[0211] Efficacy can also be measured by failure of an individual to deteriorate as assessed by hospitalization or need for medical intervention (ie, halting disease progression). Methods of measuring these indicators are known to those of skill in the art and / or described herein.

[0212] Treatment includes any 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 worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing regression of symptoms. An effective amount for treating a disease means an amount sufficient to result in effective treatment of the disease when administered to a subject in need thereof, as that term is defined herein. The efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the capabilities of those skilled in the art to monitor the efficacy of dosing and / or treatment by measuring any one or any combination of such parameters. The efficacy of a given method can be evaluated in an animal model of a condition described herein (e.g., treatment of cancer). When an experimental animal model is used, the efficacy of the treatment is demonstrated when a statistically significant change in a marker is observed.

[0213] All patents and other publications cited in this application (including references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methods described in such publications that can be used in conjunction with the technology described herein. These publications are provided solely because their disclosure precedes 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 about dates or representations about the contents of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0214] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the disclosed precise form. Although the specific embodiments and embodiments of the present disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. For example, although the method steps or functions are presented in a given order, alternative embodiments can perform functions in different orders, or functions can be performed substantially simultaneously. The teachings of the present disclosure provided herein can be appropriately applied to other programs or methods. Various embodiments described herein can be combined to provide additional embodiments. If necessary, the various aspects of the present disclosure can be modified to provide another embodiment of the present disclosure using the composition, function and concept of the above references and applications. In addition, due to the consideration of the equivalence of biological functions, some changes can be made to the protein structure without affecting the type or amount of biological or chemical effects. According to the detailed description, these and other changes can be made to the disclosure. All such modifications are intended to be included within the scope of the appended claims.

[0215] The specific elements of any of the foregoing embodiments may be combined or substituted for the elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need to exhibit such advantages to fall within the scope of the present disclosure.

[0216] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting.

[0217] Example

[0218] The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments can be implemented according to the description provided herein. It should be noted that Examples 1-11 provide materials and methods for the experiments described herein, while Examples 12-19 show the results and Example 20 provides the sequences.

[0219] Example 1. Primary human T cell culture

[0220] For primary T lymphocyte expansion, bulk human T cells were activated (day 0) using anti-CD3 / CD28 Dynabeads (Life Technologies) and subsequently transduced with lentiviral vectors encoding CAR 24 hours later. Starting from day 0 of culture, T cells were cultured in medium supplemented with 20 IU / ml rhIL-2 and counted every 2-3 days at 0.5x10 6 T cells were maintained at a constant cell concentration of 100 μg / mL. For functional assays, CAR T cells were cryopreserved on day 8-10 of culture and stimulated with antigen or injected into mice immediately after thawing.

[0221] Example 2. Cell lines and culture conditions

[0222] JEKO-1, RAJI and wild-type parental K562 cells were purchased from the 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 stained on a FACSAria (BD Biosciences). ) to obtain a population with a purity of ≥99% (CBG-GFP+). The cell line was cultured in RPMI medium containing 10% fetal bovine serum (FBS), penicillin and streptomycin.

[0223] Example 3. Flow cytometry

[0224] The following antibodies were used: CD37-APC (clone MB-1 ), CD37-BV711 (clone MB-371, BD ), CD19-Pacific Blue (clone HIB19, ), CD19-FITC (clone 4G7, BD), CD5-BUV737 (clone UCHT2, BD), CD20-APC Cy7 (clone 2H7, ), CD79b-PE (clone CB3-1, ), CD3-BV786 (clone SK7, BD), CD3-BV605 (clone OKT3, ), CD45-PeCy7 (clone HI30, ), CD16-PE (clone B73.1, ), CD14-PacificBlue (clone HCD14, ), CD56-APC (clone HCD56, ), CD33-BV510 (clone P67.6, ), CD107a-AF700 (clone H4A3, BD ), CD69-APC (clone FN50, ) and IFNγ-FITC (clone GZ-4, ). The cells were stained for 30 min at 4°C in the dark and washed twice in PBS containing 2% FBS. DAPI was added to gate live cells before acquisition. The antibody bound to each cell (ABC) was measured and analyzed using Quantum TM Antigen density was calculated by Simply Cellular (Bangs Laboratories).

[0225] Example 4. Mutual killing assay

[0226] Cell stimulation mixture ( Human T cells purified from leukopaks of anonymous healthy human donors (catalog number 00-4970-03) were activated for 6 hours. Following the manufacturer's instructions, CFSE ( C34554) were used to label activated and non-activated T cells and co-cultured with CAR-37, CAR-19 or non-transduced T cells generated from the same normal donor. After 24 hours, flow cytometry was used to count the number of CFSE-positive cells in each condition.

[0227] Example 5. Immune cell isolation, differentiation and co-culture assay

[0228] PBMCs from three normal donors were isolated using Ficoll-Paque PLUS (GE Healthcare, C987R36) and Kit (Cat. No. 19359) purifies monocytes. M1 / M2 macrophages are generated in vitro as previously described (Zhang et al., PLoS One. 11 (4): e0153550, 2016). Monocytes, macrophages, NK and T cells are cultured with CAR-37CAR-19 or untransduced T cells at an E:T ratio of 1:1 for 6 hours, and CD107a and IFNγ are produced by flow cytometry. PMA / ionomycin is used as a positive control. Values ​​are normalized with respect to culture medium, and the figure represents the fold change.

[0229] Example 6. Cytotoxicity and cytokine assays

[0230] For cytotoxicity assays, CAR T cell effector cells were co-cultured with tumor targets expressing CBG luciferase at the indicated ratios for 16 h. Luciferase activity was measured. For analysis of soluble cytokines, effector cells were co-cultured with tumor targets at a 1:1 ratio for 24 h.

[0231] By adding untransduced but cultured and activated T cells from the same donor, CAR-37 and CAR-19 T cells were normalized for CAR expression to achieve the same proportion of CAR+ T cells in each sample. For cytotoxicity assays, the percentage of specific lysis was calculated by the following equation: % specific lysis = (total RLU / target cell RLU only) x 100. For cytokine assays, Luminex arrays (Luminex Corp, FLEXMAP ) were used to analyze the cytokine expression of cell-free supernatants. All samples were measured in duplicate. 7 (version 7.0) were averaged before plotting. In addition, all assays were performed with biological duplicates or triplicates or more, as indicated by N in each experiment, based on the number of unique healthy donor T cells tested.

[0232] Example 7. Jurkat reporter gene activation assay

[0233] Jurkat (NFAT-Luc) reporter cells (Signosis, SL-0032) were transduced with different CAR constructs. They were co-cultured with B cell lymphoma tumor cells or Nalm6 leukemia cells at a 1:1 E:T ratio for 24 h; anti-CD3 / CD28 beads were used as positive control, and culture medium was used as negative control. After 16 h, the expression of 50 μM chromatin was detected by a Synergy Neo2 luminescence microplate reader. Luciferase activity was measured. Relative activation was calculated with respect to PMA.

[0234] Example 8. Immunohistochemistry

[0235] Paraffin sections were dewaxed with xylene and then washed with a series of ethanol followed by HO. 2 O for rehydration. Antigen retrieval was performed by irradiating the slides with microwaves for 15 min in 0.01 M sodium citrate buffer (pH 6.0). After washing with phosphate-buffered saline containing 0.1% Tween 20 (PBS-T), the slides were washed with 3% H 2 O 2 Endogenous peroxidase activity was quenched for 10 min. The slides were then washed again with PBS-T and blocked with Novolink Protein Block for 30 min at 25°C. After additional washes with PBS-T, the slides were incubated with 5% goat serum and mouse anti-CD37 ( MA5-15492) in PBS-T at 25°C for 1 hour. After washing with PBS-T, the slides were incubated with Cell Signaling Technology Signal Stain Enhanced IHC Mouse Detection Reagent at 25°C for 30 min, washed again with PBS-T, and incubated with DAB Diluent (Vector Laboratories) containing DAB chromogen. After staining development, the slides were washed again with PBS-T and counterstained with hematoxylin.

[0236] Example 9. TMA Construction

[0237] Formalin-fixed paraffin-embedded tissues involved in peripheral T-cell lymphoma (PTCL) were retrieved from the archives of the Department of Pathology at the Brigham and Women's Hospital. Cores (0.6 mm in diameter) from donor blocks were transferred to recipient blocks to create tissue microarrays at the Tissue Microarray Core at the Dana Farber / Harvard Cancer Center and used to prepare 4-micron sections for immunohistochemical staining studies.

[0238] Example 10. In vivo studies

[0239] JEKO-1 cell line or patient-derived tumor cells were transplanted into NOD-SCID-γ chain- / - (NSG) (The Jackson ). After tumor engraftment was confirmed by luminescence, cryopreserved CAR-37, CAR-19 or non-transduced T cells were injected intravenously. After intraperitoneal injection of D-luciferin substrate solution (30 mg / mL), tumor burden was regularly monitored using an Ami Spectrum Imager and analyzed using IDL software v.4.3.1. Animals were euthanized according to the experimental protocol or when the animals reached a pre-specified endpoint defined by IACUC.

[0240] Example 11. Statistical analysis

[0241] Unless otherwise stated, for normal data, 2-tailed Student's t-test or two-way Anova test was used under equal variance. P < 0.05 was considered significant. 7 (version 7.0) for analysis.

[0242] Example 12. Construction of CAR and T cell culture transduction

[0243] Two anti-CD37 CAR constructs were synthesized and cloned into a third-generation lentiviral plasmid backbone under the regulation of the human EF-1α promoter. All CARs carry a CD8 hinge, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. The vectors also contain a second transgene encoding the fluorescent reporter mCherry to facilitate calculation of transduction efficiency. Human T cells (StemCell) were purified from anonymous human healthy donor leukocytes purchased from the MGH Blood Bank under an IND exemption agreement. Catalog No. 15061). Example 1 provides details of T cell culture.

[0244] Example 13. CD37 is highly expressed in human lymphoma

[0245] Flow cytometry was used to examine the expression of leukemia and non-Hodgkin's lymphoma cell lines Nalm6, Jeko-1, and Raji ( Figure 1A ) and patient-derived MCL lines ( Figure 1B and Figure 1C ) and primary patient CLL cells ( Figure 1D ) in CD37 and CD19 expression. K562 cells transduced with both CD37 and CD19 were also generated to serve as positive controls for artificial antigen presenting cells stimulated in vitro and for cytotoxicity assays ( Figure 1A Although pre-B cell-derived leukemia cells (Nalm6) express CD19, they do not express CD37 ( Figure 1A ), but all lymphoma cells express both CD19 and CD37 ( Figure 1A-Figure 1C In patient-derived MCL samples, based on the mean fluorescence intensity of gated positive cells, it was noted that CD37 expression was high and uniform, even higher than that of CD19 ( Figure 1C ), but recognize that this difference may reflect differences in antibody binding or brightness of the fluorophore.

[0246] Next, CD37 and CD19 expression was evaluated by flow cytometry on PBMCs derived from 21 chronic lymphocytic leukemia patients ( Figure 1D ), and again noted that CD37 expression was higher and more uniform in CD3-negative lymphocytes compared to CD19. When gating on CD3-CD20+ B cells, CD37 expression remained high and uniform ( Figure 1E To determine the antigen density levels of both antigens, the expression of CD19 and CD37 on these 21 samples was quantified using beads ( Figure 2A A higher antigen density on a per cell basis was observed for CD19 compared to CD37 (mean ABC for CD19 = 31,829 ± 3,212, compared to 29,680 ± 3232 for CD37) ( Figure 2B , Table 1).

[0247] Table 1.

[0248]

[0249]

[0250] Based on 29 samples of bone marrow aspirates, lymph node biopsies, and peripheral blood from patients with hematological malignancies, CD37 expression was confirmed on B-cell lymphomas and normal B cells, but not on hematopoietic stem cells or primitive blood cells from normal donors (Table 2).

[0251]

[0252]

[0253]

[0254] In this dataset, there were no samples from patients with T-cell malignancies. Therefore, CD37 expression in primary peripheral T-cell lymphomas was assessed by immunohistochemical staining on tissue microarrays containing triplicate cores of 67 PTCL samples from 9 different subtypes. Overall, positive staining was observed in at least one subset of cells in patient samples from each subtype, including 15 of 16 AILTs, 1 of 1 ALK+ anaplastic large cell lymphoma (ALCL), 6 of 13 ALK-negative ALCL, 2 of 6 adult T-cell leukemias, 18 of 23 PTCLs (not otherwise specified), 1 of 1 enteropathy-associated T-cell lymphoma, 4 of 4 extranodal NK / T-cell lymphomas (nasal type), 1 of 1 T-cell lymphocytic leukemia, and 1 of 2 hepatosplenic gamma / delta T-cell lymphomas. Representative strong staining of CD37 in PTCL (ALCL) samples was Figure 2C Shown in.

[0255] Example 14. Generation of anti-CD37 CAR T cells

[0256] Two anti-CD37 chimeric antigen receptors were designed, which consisted of an anti-CD37 scFv and a CD8 transmembrane domain in tandem with a 4-1BB intracellular signaling domain and CD3ζ ( Figure 3A ). scFvs were synthesized in both directions of the variable heavy chain and light chain to generate CAR-37 LH and CAR-37 HL. To facilitate the evaluation of transduction efficiency, the mCherry fluorescent reporter gene was incorporated after the 2A ribosomal skipping sequence at the C-terminus of the CAR sequence. Using a third-generation self-inactivating lentiviral vector, efficient gene transfer to primary activated human T cells was obtained with both constructs ( Figure 3B and Figure 3C ). CAR-37 T cells showed comparable expansion to anti-CD19 CAR T cells (CAR-19) within the first 10 days after initial priming with anti-CD3 / CD28 beads. As a comparator, CAR-19 T cells were generated based on the same backbone, which has a CD8 transmembrane domain and 4-1BB and CD3ζ intracellular signaling domains. It was found that CAR-37 T cells can undergo long-term expansion by repeated antigen stimulation with irradiated K562 cells transduced to express CD37 and CD19 ( Figure 3D). Next, activation of CAR using Jurkat reporter (NFAT-luciferase) T cells was tested. After transduction of Jurkat reporter cells with different CAR constructs, cells were co-cultured with various stimuli including anti-CD3 / CD28 beads, B cell lymphoma tumor cells, Nalm6 leukemia cells, or culture medium as a negative control. Measurement of luminescence demonstrated specific T cell activation in response to antigen stimulation and NFAT-mediated luminescence ( Figure 3E ). In this assay, the LH-oriented anti-CD37 CAR appeared to initiate activation more robustly than the HL-oriented anti-CD37 CAR and had a similar level of response to CD19-expressing tumors as the anti-CD19 CAR. However, there is no known threshold or optimal amount of NFAT translocation corresponding to the optimal CAR construct. These data suggest that the anti-CD37 CAR mediates T cell activation signals in response to specific antigenic stimulation and can undergo long-term growth in response to antigenic stimulation.

[0257] Since CD37 has been reported to be expressed on T cells and other hematopoietic mononuclear cells, the expression of CD37 on whole blood immune cells from healthy donors was investigated ( Figure 3F-Figure 3I ). High expression of CD37 was observed on B cells, with minimal expression on monocytes, but not on NK cells or T cells. However, many T cell markers change with activation, and the possibility of mutual killing was therefore tested by co-culturing CAR-37 T cells with activated or non-activated CFSE-labeled T cells from the same donor. After 24 hours, target T cell counts were analyzed. Despite the expected cytotoxicity against Jeko-1 target cells in the same experiment, labeled resting T cells ( Figure 4A ) or labeled activated T cells ( Figure 4B No significant differences were detected in the counts of Figure 4C Next, we 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, suggesting that there was no sign of CAR-37 T cell-induced immune cytotoxicity.

[0258] Example 15. CAR-37 T cells exhibit potent effector functions in response to CD37-positive tumor cells in vitro

[0259] To clarify 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 different 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 more favorable than the light-reconstructed configuration for anti-CD37CAR T cells. This difference was significant and consistent with all tumor lines tested. It is noteworthy that T cells transduced with CAR-37 HL or CAR-19 exhibited equivalent cytolytic activity against these target tumor cells, all of which expressed both antigens.

[0260] Next, we analyzed cytokine production in response to antigen stimulation. We compared the different patterns of cytokine production by different CAR constructs after stimulation with target cells. Figure 6C and Fig.6D ), primary CLL ( Fig. 6A ) and MCL patient-derived xenograft (PDX) samples ( Figure 6B )After in vitro stimulation, CAR-37 T cells displayed antigen-specific production of Th1-type cytokines TNF-α, IFN-γ, IL-2, and GM-CSF. Consistent with the cytotoxicity assay, these experiments demonstrated improved antigen-specific effector function of CAR-37 HL compared to CAR-37 LH.

[0261] Example 16. CAR-37 T cells eradicate MCL tumors in vivo

[0262] Since in vitro assays suggested that CAR-37 HL might, but was not definitely, superior to CAR-37 LH, the two modalities were compared in a xenogeneic model of MCL. NSG mice were injected intravenously with Jeko-1 (CBG-GFP+) cells expressing luciferase. Seven days later, disease burden was assessed in all mice by bioluminescence imaging (BLI) and CAR-37 or untransduced (UTD) T cells were administered by tail vein injection. By day 14, disease was partially controlled in CAR-37 LH-treated animals and completely eradicated in CAR-37 HL-treated mice ( Figure 7A-7C ), thus confirming that the antigen-induced effector function of CAR-37 HL is superior to that of CAR-37 LH, and the CAR-37 HL was then selected for further experiments and direct comparison with CAR-19 T cells in vivo.

[0263] As shown in the figure, CAR T cells were injected into NSG mice seven days after intravenous injection of the MCL cell line Jeko-1 (CBG-GFP+) ( Fig.7D Continuous imaging of luminescence to assess tumor burden demonstrated rapid and complete tumor elimination by day 14 for both CAR-37 and CAR-19 T cells, with a significant reduction in tumor volume by day 7 ( Fig. 7E and Figure 7F CAR T cell persistence was confirmed in peripheral blood by flow cytometry ( Figure 7G ), among which CAR-37 T cells had higher persistence at day 7 (p<0.05).

[0264] Although tumor cell lines are quite useful in evaluating the efficacy of CAR T cells, they are often not fully representative of the heterogeneity and biological characteristics of primary patient tumors. In contrast, PDX models, in which tumor cells are believed to be directly derived from patients and cultured for only 2-3 generations, are more similar to the clinical setting. The efficacy of CAR-37 (HL configuration) in a PDX model of MCL was evaluated. Ten NSG mice were injected with MCL-PDX cells expressing luciferase. After confirming disease engraftment and tumor burden similar to the Jeko-1 model by BLI, CAR-37, CAR-19, or untransduced T cells ( Fig. 8A ). It was observed that CAR-37 T cells were able to clear tumors in only 12 days, significantly faster than CAR-19 (p<0.05 at the 12-day time point) ( Figure 8B and Figure 8C Flow cytometric evaluation of peripheral blood collected on day 14 confirmed the persistence of CAR T cells in the blood ( Fig.8D ). Collectively, these results demonstrate that CAR-37 T cells mediate significant antitumor effects against B-cell NHL in vivo in both tumor cell lines and PDX models of MCL.

[0265] Example 17. Targeting CD37 on T-cell lymphoma

[0266] The surface expression of CD37 in PTCL lines and PDX samples of PTCL was analyzed by flow cytometry. Three cell lines (Hut78, Fedp, and Seax) and five PDX samples ( Fig. 9A and Fig. 9B , Table 3).

[0267] Table 3.

[0268]

[0269]

[0270] CAR-37 T cells were tested for activation after co-culture with these PTCL samples. CD69 activation marker was analyzed on gated CAR+ T cells by flow cytometry; robust upregulation of CD69 was observed after 6 hours of co-culture with PTCL target cells, indicating CAR-37 T cell activation ( Fig. 10A Interestingly, the extent of CD69 upregulation was independent of the expression level of CD37 antigen on target cells. Next, degranulation of CAR-37 T cells was tested by measuring the expression of CD107a in CAR-37 cells after incubation with PTCL target cells ( Fig. 10B ). Consistent with the CD69 assay, CAR-37 T cells were observed to degranulate in response to PTCL cells, indicating that CAR-37 T cells were activated in response to PTCL. Finally, CAR-37 T cells were observed to effectively lyse the CD37-positive PTCL cell lines Hut78 and Fepd, as determined by cytotoxicity assays performed at different E:T ratios ( Fig. 10C and Fig. 10D ). Taken together, these experiments demonstrate that CAR-37 T cells target and activate T-cell lymphoma cells and cause them to lyse.

[0271] Example 18. Bispecific CAR T cells targeting CD19 and CD37

[0272] A possible strategy to avoid antigen escape is to generate T cells that can recognize multiple antigens (Ruella et al., J.Clin.Invest.126:3814-26,2016; Zah et al., Cancer Immunology Research.4:498-508,2016). The dual targeting of CD19 and CD37 has been previously studied with a dual ligand immunoliposome method that is effective for B-CLL cells (Yu et al., Biomaterials.34:6185-6193,2013). A bispecific CAR is designed that will effectively trigger T cell activation when CD19 or CD37 is present on target cells alone or in combination. Two constructs carrying anti-CD19 and anti-CD37 scFvs connected in series in different orders were generated and tested in the second generation 4-1BB-CD3ζ vector ( Fig.11A ). It was observed that even with the same MOI, the transduction efficiency of CAR19-37 T cells was lower than that of CAR37-19 ( Fig. 11B and Fig. 11C ).

[0273] 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 Jurkat NFAT-Luc reporter cell lines were generated and analyzed for activation after overnight co-culture with target cells. Bispecific CAR T cells showed high NFAT activation when either or both antigens were present on the target cell surface, and there was no apparent difference in their activation signature when bispecific CAR T cells were activated by CD19 alone or CD37 alone ( Fig. 12A Bispecific CAR T cells showed comparable expansion within the first 10 days after initial priming with anti-CD3 / CD28 beads ( Fig. 12B ). Similarly, it was found that bispecific CAR T cells can undergo long-term expansion through repeated antigen stimulation with K562 expressing CD19 and CD37 ( Fig. 12B In cytotoxicity assays using primary human CAR T cells, it was observed that bispecific CAR T cells responded to either a single target or both targets, and there was no significant difference in cytotoxicity against CD19 or CD37 ( Fig. 12C Finally, the bispecific constructs were tested in vivo against Jeko-1 tumor cells, where they were compared to conventional CAR-37 and CAR-19 T cells. At day 14 after treatment, all groups had completely eradicated the disease, except for CAR-19-37, which showed partial control of the disease ( Fig.12D This difference may be related to the structural differences between the two antigens and the order of the scFv in the CAR construct. On day 35 of treatment, CAR T cell persistence in peripheral blood was confirmed by flow cytometry ( Fig.12E ). There were no significant differences between CAR-37, CAR-19, and the best tumor-clearing bispecific CAR37-19 at all time points examined. Interestingly, the suboptimal CAR 19-37 had greater persistence at day 35, which may reflect continued antigenic stimulation of the tumor. Taken together, these data suggest that bispecific CAR T cells cause specific target cell lysis of cells expressing one or both antigens on their surface and that they are as effective as monospecific CARs alone against MCL tumors in vivo.

[0274] Example 19. CAR immunotherapy using NK cells

[0275] CD37 expression has been reported in PTCL (Pereira et al., Mol. Cancer. Ther. 14 (7): 1650-60, 2015) and many B-NHLs, and therefore CD37 expression is a promising target for CAR T cell immunotherapy. However, activated T cells can express CD37, while natural killer (NK) cells cannot. Therefore, NK cells are attractive cytotoxic cells for CAR T cell therapy (including CD19 / CD37-CAR or CD37-CAR). NK cells mediate anti-tumor effects without the risk of GvHD, and are short-lived relative to T cells. This also 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 different antigens, including CD19, CD20, and HER2, and anti-CD19 CAR-modified, donor-derived, and haploid NK cells have entered clinical trials for B-cell ALL (NCT00995137, NCT01974479). T cell self-targeting can potentially be mitigated by using NK cells instead and using NK as an allogeneic source.

[0276] Example 20. Sequence

[0277] scFv sequences

[0278] Anti-CD37 scFv VH-VL (SEQ ID NO:4), which comprises 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)).

[0279] AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGG GSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR(SEQ ID NO:4)

[0280] VH (SEQ ID NO: 1 (amino acids 1-116 of SEQ ID NO: 4))

[0281] AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS(SEQ ID NO:1)

[0282] Linker region (SEQ ID NO: 3 (amino acids 117-136 of SEQ ID NO: 4))

[0283] GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:3)

[0284] VL (SEQ ID NO: 2 (amino acids 137-244 of SEQ ID NO: 4))

[0285] DIQMTQSPSSSLSASVGDRVTITTCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR(SEQ ID NO:2)

[0286] Anti-CD37 scFv VL-VH (SEQ ID NO:5), which comprises VL (amino acids 1-108 (SEQ ID NO:2)), a linker region (amino acids 109-128 (SEQ ID NO:3)), and VH (amino acids 129-244 (SEQ ID NO:1)).

[0287] DIQMTQSPSSSLSASVGDRVTITTCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSG GGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS(SEQ ID NO:5)

[0288] VL (SEQ ID NO: 2 (amino acids 1-108 of SEQ ID NO: 5))

[0289] DIQMTQSPSSSLSASVGDRVTITTCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR(SEQ ID NO:2)

[0290] Linker region (SEQ ID NO: 3 (amino acids 109-128 of SEQ ID NO: 5))

[0291] GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:3)

[0292] VH (SEQ ID NO: 1 (amino acids 129-244 of SEQ ID NO: 5))

[0293] AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS(SEQ ID NO:1)

[0294] Anti-CD19 scFv (SEQ ID NO: 14) comprising VL (amino acids 1-107 (SEQ ID NO: 13)), a linker region (amino acids 108-128 (SEQ ID NO: 3)), and VH (amino acids 129-247 (SEQ ID NO: 12)).

[0295] EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGG GSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID NO:14)

[0296] VL (SEQ ID NO: 13 (amino acids 1-107 of SEQ ID NO: 14))

[0297] EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK(SEQ ID NO:13)

[0298] Linker region (SEQ ID NO: 3 (amino acids 108-128 of SEQ ID NO: 14)

[0299] GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:3)

[0300] VH (SEQ ID NO: 12 (amino acids 129-247 of SEQ ID NO: 14))

[0301] QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID NO:12)

[0302] CAR sequence

[0303] pMGH8(CAR-37 LH)-CD8 signal / anti-CD37 LH / Cd8 hinge+TM / 4-1BB / CD3ζ (SEQ ID NO:7), which comprises 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 domains (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)).

[0304] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:7)

[0305] CD8 signal sequence (SEQ ID NO:8 (amino acids 1 - 21 of SEQ ID NO:7))

[0306] MALPVTALLLPLALLLHAARP (SEQ ID NO:8)

[0307] Anti - CD37 L - H (SEQ ID NO:5 (amino acids 22 - 265 of SEQ ID NO:7))

[0308] DIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSS (SEQ ID NO:5)

[0309] CD8 hinge and TM domain SEQ ID NO:9 (amino acids 266-334 of SEQ ID NO:7)

[0310] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:9)

[0311] 4-1BB (SEQ ID NO: 10 (amino acids 335-376 of SEQ ID NO: 7))

[0312] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:10)

[0313] CD3ζ (SEQ ID NO: 11 (amino acids 377-488 of SEQ ID NO: 7))

[0314] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:11)

[0315] pMGH8(CAR-37 HL)-CD8 signal / anti-CD37 HL / CD8 hinge+TM / 4-1BB / CD3ζ (SEQ ID NO:6), which comprises 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 domains (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)).

[0316] MALPVTALLLPLALLLHAARPAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:6)

[0317] CD8 signal sequence (SEQ ID NO:8 (amino acids 1 - 21 of SEQ ID NO:6))

[0318] MALPVTALLLPLALLLHAARP (SEQ ID NO:8)

[0319] Anti - CD37 H - L (SEQ ID NO:4 (amino acids 22 - 265 of SEQ ID NO:6))

[0320] AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR (SEQ ID NO:4)

[0321] CD8 hinge and TM domain (SEQ ID NO:9 (amino acids 266-334 of SEQ ID NO:6))

[0322] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:9)

[0323] 4-1BB (SEQ ID NO: 10 (amino acids 335-376 of SEQ ID NO: 6))

[0324] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:10)

[0325] CD3ζ (SEQ ID NO: 11 (amino acids 377-488 of SEQ ID NO: 6))

[0326] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:11)

[0327] pMGH95 (CAR-19-37) - CD8 signal / anti-CD19 / anti-CD37 HL / Cd8 hinge + TM / 4-1BB / CD3ζ (SEQ ID NO: 15), which contains 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)), CD3ζ (amino acids 644-755 (SEQ ID NO: 11)).

[0328] MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:15)

[0329] CD8 signal sequence (SEQ ID NO:8 (amino acids 1 - 21 of SEQ ID NO:15))

[0330] MALPVTALLLPLALLLHAARP (SEQ ID NO:8)

[0331] Anti - CD19 (SEQ ID NO:14 (amino acids 22 - 268 of SEQ ID NO:15))

[0332] EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID NO:14)

[0333] Linker region (SEQ ID NO:3 (amino acids 269 - 288 of SEQ ID NO:15)

[0334] GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:3)

[0335] Anti - CD37 H - L (SEQ ID NO:4 (amino acids 289 - 532 of SEQ ID NO:15))

[0336] AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR(SEQ ID NO:4)

[0337] CD8 hinge and TM domain (SEQ ID NO:9 (amino acids 533 - 601 of SEQ ID NO:15));

[0338] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:9)

[0339] 4 - 1BB (SEQ ID NO:10 (amino acids 602 - 643 of SEQ ID NO:15)),

[0340] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:10)

[0341] CD3ζ (SEQ ID NO: 11 (amino acids 644-755 of SEQ ID NO: 15))

[0342] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:11)

[0343] pMGH96 (CAR-37-19) - CD8 signal / anti-CD37 HL / anti-CD19 / Cd8 hinge + TM / 4-1BB / CD3ζ (SEQ ID NO: 16), which contains 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)), CD3ζ (amino acids 644-755 (SEQ ID NO: 11)).

[0344] MALPVTALLLPLALLLHAARPAVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:16)

[0345] CD8 signal sequence (SEQ ID NO:8 (amino acids 1 - 21 of SEQ ID NO:16));

[0346] MALPVTALLLPLALLLHAARP (SEQ ID NO:88)

[0347] Anti - CD37 H - L (SEQ ID NO:4 (amino acids 22 - 265 of SEQ ID NO:16));

[0348] AVQLVQSGAEVKKPGSSVKVSCKASGYSFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTTYNRKFKGRVTLTVDKSSSTAYMELSSLRSEDTAVYYCARSVGPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSYLAWYQQKPGKAPKLLVSSAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQHHSDNPWTFGQGTKVEIKR(SEQ ID NO:4)

[0349] Linker region (SEQ ID NO:3 (amino acids 266 - 285 of SEQ ID NO:16));

[0350] GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:3)

[0351] Anti - CD19 (SEQ ID NO:14 (amino acids 286 - 532 of SEQ ID NO:16));

[0352] EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID NO:14)

[0353] CD8 hinge and TM domain (SEQ ID NO:9 (amino acids 533 - 601 of SEQ ID NO:16));

[0354] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:9)

[0355] 4 - 1BB (SEQ ID NO:10 (amino acids 602 - 643 of SEQ ID NO:16)),

[0356] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:10)

[0357] CD3ζ (SEQ ID NO: 11 (amino acids 644-755 of SEQ ID NO: 16)).

[0358] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:11)

[0359] Table 4 provides a table of the sequences of the present application and the corresponding SEQ ID NO:.

[0360] Table 4. Sequences of this application

[0361]

[0362]

[0363]

[0364]

[0365] Some embodiments of the technology described herein may be defined according to any of the following numbered paragraphs:

[0366] 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.

[0367] 2. The CAR according to paragraph 1, wherein the CD37 binding domain and / or the CD19 binding domain comprises an antibody or an antigen-binding fragment thereof.

[0368] 3. The CAR according to paragraph 1 or 2, wherein the CD37 binding domain and / or the CD19 binding domain comprises a single-chain variable fragment (scFv).

[0369] 4. The CAR according to any one of paragraphs 1-3, wherein the CD19 binding domain is located at the N-terminus of the CD37 binding domain.

[0370] 5. The CAR according to any one of paragraphs 1-3, wherein the CD37 binding domain is located at the N-terminus of the CD19 binding domain.

[0371] 6. The CAR of any one of paragraphs 1-5, wherein the CAR further comprises (iv) one or more co-stimulatory domains.

[0372] 7. The CAR of any one of paragraphs 1-6, wherein the transmembrane domain comprises a hinge / transmembrane domain.

[0373] 8. The CAR of paragraph 7, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of CD8 or 4-1BB.

[0374] 9. The CAR according to paragraph 8, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of CD8, which optionally comprises the amino acid sequence of SEQ ID NO:9.

[0375] 10. The CAR of any one of paragraphs 1-9, wherein the intracellular signaling domain comprises the intracellular signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3ε, CD3ζ, CD22, CD79a, CD79b or CD66d.

[0376] 11. The CAR according to paragraph 10, wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, which optionally comprises the amino acid sequence of SEQ ID NO:11.

[0377] 12. A CAR according to any one of paragraphs 6-11, wherein the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB, CD28 or OX-40.

[0378] 13. The CAR according to paragraph 12, wherein the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB, which optionally comprises the amino acid sequence of SEQ ID NO: 10.

[0379] 14. The CAR according to any one of paragraphs 1-13, wherein the CAR comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 15, 16, 19 or 20.

[0380] 15. The CAR according to paragraph 14, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 15, 16, 19 or 20.

[0381] 16. The CAR of any one of paragraphs 1-15, wherein the CD37 binding domain comprises a heavy chain variable domain (VH), the heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1; and a light chain variable domain (VL), the light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0382] 17. The CAR according to 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.

[0383] 18. The CAR according to paragraph 16 or 17, wherein the VH is located at the N-terminus of the VL.

[0384] 19. The CAR according to paragraph 16 or 17, wherein the VL is located at the N-terminus of the VH.

[0385] 20. The CAR of any one of paragraphs 1-19, wherein the CD37 binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4 or 5.

[0386] 21. The CAR of paragraph 20, wherein the CD37 binding domain comprises the amino acid sequence of SEQ ID NO: 4 or 5.

[0387] 22. The CAR of any one of paragraphs 1-21, wherein the CD19 binding domain comprises a heavy chain variable domain (VH), the heavy chain variable domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12; and a light chain variable domain (VL), the light chain variable domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 13.

[0388] 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.

[0389] 24. The CAR of any one of paragraphs 1-23, wherein the CD19 binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14.

[0390] 25. The CAR of paragraph 24, wherein the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 14.

[0391] 26. A polynucleotide encoding the CAR according to any one of paragraphs 1-25.

[0392] 27. The polynucleotide according to paragraph 26, further comprising a suicide gene.

[0393] 28. The polynucleotide of paragraph 26 or 27, further comprising a sequence encoding a signal sequence.

[0394] 29. An immune cell comprising a CAR according to any one of paragraphs 1-25 and / or a polynucleotide according to any one of paragraphs 26-28.

[0395] 30. The immune cell of paragraph 29, wherein the immune cell is a T cell or a natural killer (NK) cell.

[0396] 31. The immune cell of paragraph 29 or 30, wherein the immune cell is a human cell.

[0397] 32. A pharmaceutical composition comprising the immune cell according to any one of paragraphs 29-31 and a pharmaceutically acceptable carrier.

[0398] 33. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the immune cell of any one of paragraphs 29-31 or the pharmaceutical composition of paragraph 32.

[0399] 34. The method of paragraph 33, wherein the cancer comprises cells expressing CD37.

[0400] 35. The method of paragraph 34, wherein the cancer is B-cell non-Hodgkin lymphoma, T-cell lymphoma, or leukemia.

[0401] 36. The method of paragraph 35, wherein the B-cell non-Hodgkin lymphoma is mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), or Burkitt's lymphoma.

[0402] 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).

[0403] 38. The method of paragraph 35, wherein the leukemia is chronic lymphocytic leukemia (CLL).

[0404] 39. The method according to any one of paragraphs 33-38, wherein the subject is unresponsive to anti-CD19 therapy.

[0405] 40. The method of any one of paragraphs 33-39, wherein the subject is co-administered with anti-CD19 therapy.

[0406] Other Implementations

[0407] Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety. Sequence Listing <110> The General Hospital Corporation <120> Chimeric antigen receptor targeting CD37 and CD19 <130> 51295-018WO3 <150> US 62 / 757,562 <151> 2018-11-08 <150> US 62 / 688,775 <151> 2018-06-22 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 1 Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 2 <211> 108 <212> PRT <213> artificial sequence <220> <223> Synthesis structure <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45 Ser Ser Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 3 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 4 <211> 244 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 4 Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 145 150 155 160 Thr Ser Glu Asn Val Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Val Ser Ser Ala Lys Thr Leu Ala Glu 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys 210 215 220 Gln His His Ser Asp Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg <210> 5 <211> 244 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45 Ser Ser Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 130 135 140 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 145 150 155 160 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 165 170 175 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 180 185 190 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 195 200 205 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser <210> 6 <211> 488 <212> PRT <213> artificial sequence <220> <223> Synthesis structure <400> 6 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Ser Phe Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln 50 55 60 Gly Leu Glu Trp Met Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr 65 70 75 80 Tyr Asn Arg Lys Phe Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 145 150 155 160 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 165 170 175 Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr Leu Ala Trp 180 185 190 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val Ser Ser Ala 195 200 205 Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 210 215 220 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 225 230 235 240 Ala Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp Thr Phe Gly 245 250 255 Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 325 330 335 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 340 345 350 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 355 360 365 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 7 <211> 488 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 7 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu 35 40 45 Asn Val Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu Leu Val Ser Ser Ala Lys Thr Leu Ala Glu Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln His His 100 105 110 Ser Asp Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val 145 150 155 160 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Ser Phe Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln 180 185 190 Gly Leu Glu Trp Met Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr 195 200 205 Tyr Asn Arg Lys Phe Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser 210 215 220 Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 325 330 335 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 340 345 350 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 355 360 365 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 8 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 8 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 9 <211> 69 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 9 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 35 40 45 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 50 55 60 Ile Thr Leu Tyr Cys 65 <210> 10 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 10 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 11 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 11 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 12 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 13 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 14 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr 130 135 140 Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 145 150 155 160 Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 165 170 175 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser 180 185 190 Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys 195 200 205 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys 210 215 220 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 225 230 235 240 Thr Leo Val Thr Val Ser Ser 245 <210> 15 <211> 755 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 145 150 155 160 Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser 165 170 175 Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 180 185 190 Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln 195 200 205 Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn 210 215 220 Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 225 230 235 240 Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp 245 250 255 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 260 265 270 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 275 280 285 Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 290 295 300 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 305 310 315 320 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 325 330 335 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 340 345 350 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 355 360 365 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 370 375 380 Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 385 390 395 400 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 405 410 415 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 420 425 430 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 435 440 445 Thr Ser Glu Asn Val Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 450 455 460 Gly Lys Ala Pro Lys Leu Leu Val Ser Ser Ala Lys Thr Leu Ala Glu 465 470 475 480 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 485 490 495 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys 500 505 510 Gln His His Ser Asp Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 515 520 525 Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 530 535 540 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 545 550 555 560 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 565 570 575 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 580 585 590 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 595 600 605 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 610 615 620 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 625 630 635 640 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 645 650 655 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 660 665 670 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 675 680 685 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 690 695 700 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 705 710 715 720 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 725 730 735 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 740 745 750 Pro Pro Arg 755 <210> 16 <211> 755 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 16 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Ser Phe Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln 50 55 60 Gly Leu Glu Trp Met Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr 65 70 75 80 Tyr Asn Arg Lys Phe Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 145 150 155 160 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 165 170 175 Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr Leu Ala Trp 180 185 190 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val Ser Ser Ala 195 200 205 Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 210 215 220 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 225 230 235 240 Ala Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp Thr Phe Gly 245 250 255 Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly 260 265 270 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val 275 280 285 Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala 290 295 300 Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp 305 310 315 320 Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr 325 330 335 Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser 340 345 350 Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 355 360 365 Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly 370 375 380 Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly 385 390 395 400 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 405 410 415 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu 420 425 430 Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp 435 440 445 Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp 450 455 460 Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr 465 470 475 480 Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser 485 490 495 Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr 500 505 510 Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 515 520 525 Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 530 535 540 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 545 550 555 560 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 565 570 575 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 580 585 590 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 595 600 605 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 610 615 620 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 625 630 635 640 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 645 650 655 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 660 665 670 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 675 680 685 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 690 695 700 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 705 710 715 720 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 725 730 735 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 740 745 750 Pro Pro Arg 755 <210> 17 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 17 Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 145 150 155 160 Thr Ser Glu Asn Val Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Val Ser Ser Ala Lys Thr Leu Ala Glu 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys 210 215 220 Gln His His Ser Asp Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 245 250 255 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 260 265 270 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 275 280 285 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 290 295 300 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 305 310 315 320 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 325 330 335 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 340 345 350 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 355 360 365 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 370 375 380 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 385 390 395 400 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 405 410 415 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 420 425 430 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 435 440 445 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 450 455 460 Pro Pro Arg 465 <210> 18 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45 Ser Ser Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 130 135 140 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 145 150 155 160 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 165 170 175 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 180 185 190 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 195 200 205 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 245 250 255 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 260 265 270 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 275 280 285 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 290 295 300 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 305 310 315 320 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 325 330 335 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 340 345 350 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 355 360 365 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 370 375 380 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 385 390 395 400 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 405 410 415 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 420 425 430 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 435 440 445 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 450 455 460 Pro Pro Arg 465 <210> 19 <211> 734 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 19 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr 130 135 140 Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 145 150 155 160 Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 165 170 175 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser 180 185 190 Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys 195 200 205 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys 210 215 220 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 225 230 235 240 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Val Gln Leu Val 260 265 270 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser 275 280 285 Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Asn Met Asn Trp Val 290 295 300 Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Asn Ile Asp Pro 305 310 315 320 Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe Lys Gly Arg Val Thr 325 330 335 Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser 340 345 350 Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Val Gly 355 360 365 Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 370 375 380 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 385 390 395 400 Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 405 410 415 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val 420 425 430 Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 435 440 445 Leu Leu Val Ser Ser Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg 450 455 460 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 465 470 475 480 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln His His Ser Asp 485 490 495 Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 500 505 510 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 515 520 525 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 530 535 540 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 545 550 555 560 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 565 570 575 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 580 585 590 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 595 600 605 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 610 615 620 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 625 630 635 640 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 645 650 655 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 660 665 670 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 675 680 685 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 690 695 700 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 705 710 715 720 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 725 730 <210> 20 <211> 734 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Ala Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 145 150 155 160 Thr Ser Glu Asn Val Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Val Ser Ser Ala Lys Thr Leu Ala Glu 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys 210 215 220 Gln His His Ser Asp Asn Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 260 265 270 Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 275 280 285 Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 290 295 300 Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser 305 310 315 320 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 325 330 335 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys 340 345 350 Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu 355 360 365 Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 370 375 380 Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro 385 390 395 400 Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser 405 410 415 Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro 420 425 430 Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr 435 440 445 Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn 450 455 460 Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp 465 470 475 480 Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr 485 490 495 Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr 500 505 510 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 515 520 525 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 530 535 540 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 545 550 555 560 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 565 570 575 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 580 585 590 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 595 600 605 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 610 615 620 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 625 630 635 640 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 645 650 655 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 660 665 670 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 675 680 685 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 690 695 700 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 705 710 715 720 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 725 730

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, in, The CD37 binding domain comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprises VH-CDR1, VH-CDR2 and VH-CDR3 contained in SEQ ID NO: 1, and the light chain variable domain comprises VL-CDR1, VL-CDR2 and VL-CDR3 contained in SEQ ID NO: 2, The CD19 binding domain comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprises VH-CDR1, VH-CDR2 and VH-CDR3 contained in SEQ ID NO:12, and the light chain variable domain comprises VL-CDR1, VL-CDR2 and VL-CDR3 contained in SEQ ID NO:

13.

2. The CAR of claim 1, wherein the CD19 binding domain is located at the N-terminus of the CD37 binding domain.

3. The CAR of claim 1, wherein the CD37 binding domain is located at the N-terminus of the CD19 binding domain.

4. The CAR of claim 1, wherein the CAR further comprises (iv) one or more co-stimulatory domains.

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

6. The CAR of claim 5, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of CD8 or 4-1BB.

7. The CAR of claim 6, wherein the hinge / transmembrane domain comprises the hinge / transmembrane domain of CD8.

8. 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.

9. The CAR of claim 8, wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ.

10. The CAR of claim 4, wherein the co-stimulatory domain comprises a co-stimulatory domain of 4-1BB, CD28, or OX-40.

11. The CAR of claim 10, wherein the co-stimulatory domain comprises the co-stimulatory domain of 4-1BB.

12. The CAR of claim 1, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

20.

13. The CAR according to claim 1, wherein: (1) the heavy chain variable domain of the CD37 binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 and retains the CD37 binding activity of the VH comprising the amino acid sequence of SEQ ID NO: 1; and (2) The light chain variable domain of the CD37 binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2 and retains the CD37 binding activity of the VL comprising the amino acid sequence of SEQ ID NO:

2.

14. The CAR of claim 1, wherein the heavy chain variable domain of the CD37 binding domain comprises the amino acid sequence of SEQ ID NO: 1, and the light chain variable domain of the CD37 binding domain comprises the amino acid sequence of SEQ ID NO:

2.

15. The CAR of claim 1, wherein the heavy chain variable domain of the CD37 binding domain is located at the N-terminus of the light chain variable domain of the CD37 binding domain.

16. The CAR of claim 1, wherein the light chain variable domain of the CD37 binding domain is located at the N-terminus of the heavy chain variable domain of the CD37 binding domain.

17. The CAR of claim 1, wherein the CD37 binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4 or 5.

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

19. The CAR according to claim 1, wherein: (1) the heavy chain variable domain of the CD19 binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12 and retains the CD19 binding activity of the VH comprising the amino acid sequence of SEQ ID NO: 12; and (2) The light chain variable domain of the CD19 binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 13 and retains the CD19 binding activity of the VL comprising the amino acid sequence of SEQ ID NO:

13.

20. The CAR of claim 1, wherein the heavy chain variable domain of the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable domain of the CD19 binding domain comprises the amino acid sequence of SEQ ID NO:

13.

21. The CAR of claim 1, wherein the CD19 binding domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 and retains the CD19 binding activity of the CD19 binding domain comprising the amino acid sequence of SEQ ID NO:

14.

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 according to claim 1.

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

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

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

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

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

29. A pharmaceutical composition comprising the immune cell according to claim 26 and a pharmaceutically acceptable carrier.

30. Use of the immune cell or pharmaceutical composition thereof according to claim 26 for the preparation of a medicament for treating cancer in a subject in need thereof, wherein the cancer comprises cells expressing CD37.

31. The use according to claim 30, wherein the cancer is B-cell non-Hodgkin's lymphoma, T-cell lymphoma or leukemia.

32. The use according to claim 31, wherein the B-cell non-Hodgkin lymphoma is mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) or Burkitt's lymphoma.

33. The use according to claim 31, 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).

34. The use according to claim 31, wherein the leukemia is chronic lymphocytic leukemia (CLL).

35. The use according to claim 30, wherein the subject is unresponsive to anti-CD19 therapy.

36. The use according to claim 30, wherein anti-CD19 therapy is co-administered to the subject.

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