Modified T cells and methods of using the same

Abstract

The technology described herein relates to modified T cells and their use in immunotherapy. In various examples, the T cells are modified to reduce or eliminate CD3ζ, TRAC, and / or TRBC expression. [Selection diagram] None

Description

Technical Field

[0001] The technology described herein relates to engineered T cells and their use in immunotherapy.

Background Art

[0002] Adoptive T cell therapy involves the administration of antigen-specific T cells to treat diseases including cancer, infectious diseases, and autoimmune diseases. The T cells used in this therapy can be isolated from a subject and selected for a desired existing specificity. As an example, tumor-infiltrating T lymphocytes can be isolated from a subject, expanded ex vivo, and then administered to treat cancer in the subject. In other approaches, T cells can be engineered ex vivo to have new specificities. In one example of such an approach, T cells are genetically engineered ex vivo to express a chimeric antigen receptor (CAR). The CAR provides a way to direct a cytotoxic T cell response to target cells that express a selected target antigen, most often a tumor antigen or tumor-associated antigen. The CAR is a modified T cell receptor in which the antigen-binding domain has been replaced with the antigen-binding domain of an antibody that specifically binds the target antigen. Binding of the target antigen on the surface of the target cell by the CAR expressed on the T cell ("CAR T cell" or "CAR-T") promotes killing of the target cell. In another example, T cells are genetically engineered ex vivo to express a new T cell receptor.

[0003] Current approaches to adoptive T cell therapy generally utilize autologous cells, i.e., cells isolated from the subject to whom they are to be later administered. This approach can be beneficial with respect to minimizing the possibility of rejection of the administered cells by the recipient. However, drawbacks of this approach include the need for specialized staff and equipment, as well as the complexity associated with obtaining cells from a patient who may be critically ill and then making the patient wait for the cells to be processed. In view of these issues, it is desirable to use allogeneic cells obtained from a donor of the same species but genetically non-identical to the recipient in adoptive T cell therapy. This enables the generation, storage, and validation of "universal" T cells for use when needed. However, this approach presents its own challenges, arising from the recipient's immune response to the donated cells, which can lead to issues including low persistence of the cells, the host-versus-graft effect in immunocompetent subjects, and the graft-versus-host effect in immunodeficient subjects.

[0004] There is a need for a novel approach to solve the problems posed by existing methods of adoptive T cell therapy, such as those presented above. SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention provides an isolated T lymphocyte modified to have a reduced or eliminated expression of the T cell receptor (TCR) due to, for example, a reduced or eliminated expression of the CD3ζ, T cell receptor alpha chain (TRAC), and / or T cell receptor beta chain (TRBC) genes.

[0006] In one embodiment, the isolated T lymphocyte comprises a genome in which the CD3ζ, TRAC, and / or TRBC genes, regulatory sequences, coding sequences, exons, or portions thereof are mutated, reduced, null, or non-functional, resulting in reduced CD3ζ, CD3 eta, CD3 theta, TRAC, and / or TRBC expression (e.g., including deletions such as frameshift mutations).

[0007] In one embodiment, the mutation disrupts the assembly of the T cell receptor or CD3ζ signaling.

[0008] In one embodiment, the isolated T lymphocyte has a genome in which the CD3ζ, TRAC, and / or TRBC genes are deleted.

[0009] In one embodiment, the isolated T lymphocyte has a genome in which two alleles of the CD3ζ, TRAC, and / or TRBC genes are deleted.

[0010] In one embodiment, the reduced expression of the CD3ζ, TRAC, and / or TRBC genes is null expression.

[0011] In one embodiment, the isolated T lymphocyte further has reduced expression of CD3 epsilon or CD3 theta.

[0012] In one embodiment, the isolated T lymphocyte further has a deletion or mutation of the HLA locus (e.g., in humans, e.g., the HLA locus on chromosome 6) or a portion thereof.

[0013] In one embodiment, the isolated T lymphocyte further has reduced HLA class I expression.

[0014] In one embodiment, the isolated T lymphocyte is further modified to express HLA-G.

[0015] In one embodiment, the isolated T lymphocyte further contains a gene encoding a heterologous protein that promotes the T lymphocyte in the avoidance of an immune attack (e.g., T cell or NK-mediated rejection) from the host to which the T lymphocyte is administered.

[0016] In one embodiment, the heterologous protein is a viral protein derived from a virus selected from the group consisting of, for example, cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), and bovine herpesvirus-1 (BoHV-1).

[0017] In one embodiment, the viral protein is derived from CMV and is selected from the group consisting of US6, UL40, and UL18.

[0018] In one embodiment, the viral protein inhibits the transporter associated with antigen processing (TAP) and is optionally selected from the group consisting of CMV US6, HSV ICP47, BoHV-1 UL49.5, and EBV BNLF2a.

[0019] In one embodiment, the isolated T lymphocyte further comprises a gene encoding a reporter gene, such as a truncated epidermal growth factor receptor (EGFR) gene, truncated prostate-specific membrane antigen (PSMA), truncated low-affinity nerve growth factor receptor (LNGFR), truncated CD19.

[0020] In one embodiment, the isolated T lymphocyte further comprises a gene encoding a therapeutic protein (e.g., an antigen receptor that optionally confers specificity for a selected target antigen or ligand).

[0021] In one embodiment, the antigen receptor is a chimeric antigen receptor (CAR), which optionally comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0022] In one embodiment, the extracellular domain comprises a single-chain antibody and, optionally, the intracellular domain comprises a T cell activation domain.

[0023] In one embodiment, the isolated T lymphocytes further comprise a gene that induces cell death, such as a gene that is an activatable suicide gene (e.g., a suicide gene activated by a drug).

[0024] In one embodiment, the suicide gene expresses an FK506 binding domain fused to a caspase 9 apoptosis-promoting molecule.

[0025] In another aspect, the present invention provides a method for generating modified T lymphocytes (e.g., T lymphocytes lacking or having reduced expression of a functional TCR), the method comprising inactivating the CD3ζ, TRAC, and / or TRBC genes in the T lymphocytes.

[0026] In one embodiment, inactivation of the CD3ζ, TRAC, and / or TRBC genes is performed using a nuclease or system selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR / Cas9 system).

[0027] In one embodiment, the modified T lymphocytes are the modified T lymphocytes described in any of the embodiments listed above.

[0028] In another aspect, the present invention provides a method for treating a subject for a disease, the method comprising administering to the subject one or more of the isolated T lymphocytes of any of the embodiments listed above.

[0029] In one embodiment, the disease is selected from the group consisting of cancer, infectious diseases, and symptoms resulting from transplantation procedures.

[0030] In another aspect, the present invention provides a method for reducing an immunogenic response in a subject, the method comprising administering to the subject T lymphocytes according to any one or more of the embodiments listed above.

[0031] In one embodiment, the T lymphocytes express the transgene.

[0032] In one embodiment, the T lymphocytes have reduced competition with endogenous T cell signaling molecules.

[0033] In one embodiment, the T lymphocytes are autologous with respect to the subject.

[0034] In one embodiment, the T lymphocytes are allogeneic with respect to the subject.

[0035] In one embodiment, the modified T lymphocytes are expanded in vivo.

[0036] In one embodiment, the modified T lymphocytes are expanded in the subject's blood.

[0037] In one embodiment, the modified T lymphocytes are expanded in vitro prior to administration.

[0038] In another aspect, the present invention provides a vector comprising a gene encoding a therapeutic protein that promotes immune system evasion and a heterologous protein.

[0039] In one aspect, the heterologous protein is, for example, a viral protein derived from a virus selected from the group consisting of cytomegalovirus (CMV), Epstein - Barr virus (EBV), herpes simplex virus (HSV), and bovine herpesvirus - 1 (BoHV - 1).

[0040] In one embodiment, the viral protein is derived from CMV and is selected from the group consisting of US6, UL40, and UL18.

[0041] In one embodiment, the viral protein inhibits the transporter associated with antigen processing (TAP) and is selected from the group consisting of, for example, CMV US6, HSV ICP47, BoHV-1 UL49.5, and EBV BNLF2a.

[0042] In one embodiment, the therapeutic protein is a CAR.

[0043] In another aspect, the present invention provides a method of transducing one or more of the vectors of one or more of the embodiments described above into T lymphocytes.

[0044] In another aspect, the present invention provides a modified T lymphocyte or cell line produced by any one of the methods of the embodiments described above, or a subculture thereof.

[0045] In another aspect, the present invention provides a pharmaceutical composition comprising at least one modified T lymphocyte of one or more of the embodiments described above.

[0046] In another aspect, the present invention provides a method of treating a subject comprising: (a) preparing a population of modified T lymphocytes by any one or more of the methods of the embodiments described above; and (b) administering the modified T lymphocytes to the subject.

[0047] In one embodiment, the T lymphocytes are from the subject to be treated.

[0048] In one embodiment, the T lymphocytes are from a healthy donor.

[0049] The present invention also includes the use of the modified T lymphocytes described herein in a method, for example, a method comprising the methods described herein (e.g., a treatment method), and the use of the modified T lymphocytes for the preparation of a medicament for use, for example, in the methods described herein.

[0050] The various embodiments recited above may be combined with each other in any combination determined by one of ordinary skill in the art to be appropriate.

[0051] Definitions For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise indicated or implicitly indicated from the context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular 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 meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the use of a term in the art and its definition herein, the definition provided in the specification shall control.

[0052] Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition (ISBN 978-0-911910-19-3), published by Merck Sharp & Dohme Corp. in 2011; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine (ISBN 9783527600908), published by Blackwell Science Ltd. from 1999 to 2012; Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (ISBN 1-56081-569-8), published by VCH Publishers, Inc. in 1995; Immunology by Werner Luttmann, published by Elsevier in 2006; Janeway’s Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin’s Genes XI (ISBN - 1449659055), published by Jones & Bartlett Publishers in 2014; Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.), Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe (eds.), John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are hereby incorporated by reference in their entirety into this specification.

[0053] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease in a statistically significant amount. In some embodiments, "reduce", "reduction" or "decrease" or "inhibit" typically means a decrease of at least 10% compared to a reference level (e.g., the absence of a defined treatment, agent, mutation, or defect), e.g., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 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 decrease may be included. As used herein, "reduction" or "inhibition" does not include complete inhibition or reduction compared to the reference level. "Complete inhibition" is 100% inhibition compared to the reference level. Where applicable, the decrease can preferably be reduced to a level that is acceptable within the normal range for an individual without the defined disorder.

[0054] 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" mean an increase of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% increase compared to the reference level, or an increase of 100% or less or any increase from 10 - 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold compared to the reference level, or any increase from 2-fold to 10-fold or more. In the context of a marker or symptom, "increase" is a statistically significant increase at such level.

[0055] As used herein, "subject" means a human or an animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, rhesus monkeys, and macaques, such as the rhesus monkey. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic animals or game animals include, for example, cows, horses, pigs, deer, bison, buffalo, cat species, such as house cats, dog species, such as dogs, foxes, wolves, bird species, such as chickens, emus, ostriches, and fish, such as trout, catfish and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual", "patient" and "subject" are used interchangeably herein.

[0056] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Non-human mammals can be advantageously used as subjects representing animal models of diseases, such as cancer. The subject can be male or female, and can be pediatric or adult.

[0057] The subject has already been diagnosed or identified as having a condition in need of treatment (such as leukemia or another type of cancer, especially, for example, an infectious disease, an autoimmune disease, or the effect of transplantation) or one or more complications associated with such a condition, and optionally, has already experienced treatment for the condition or one or more complications associated with the condition. Alternatively, the subject can also be one that has not been previously diagnosed as having such a condition or associated complications. For example, the subject can be one that exhibits one or more risk factors for a condition or one or more complications associated with the condition, or a subject that does not exhibit risk factors.

[0058] A "subject in need of treatment" for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0059] A "disease" is the health state of an animal, e.g., a human, in which the animal is unable to maintain homeostasis and, if the disease is not ameliorated, will ultimately continue to deteriorate in health. In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the health state of the animal is less favorable than in the absence of the disorder. Left untreated, a disorder will not necessarily cause a further decrease in the health state of the animal.

[0060] As used herein, the terms "tumor antigen" and "cancer antigen" are used interchangeably to refer to antigens that are differentially expressed by cancer cells and can thereby be utilized to target cancer cells. Cancer antigens are antigens that can potentially elicit a tumor-specific immune response. Some of these antigens are not necessarily expressed by normal cells but are encoded by normal cells. These antigens can be characterized as those that are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation, and those that are transiently expressed, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as cancer genes (e.g., activated ras cancer gene), tumor suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Many tumor antigens have been identified with respect to multiple solid tumors: MAGE1, 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 prostate acid phosphatase (PAP). In addition, some viral proteins, such as those encoded by hepatitis B (HBV), Epstein-Barr (EBV), and human papillomavirus (HPV), have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively.

[0061] As used herein, the term "chimeric" refers to a product of the fusion of portions of at least two or more different polynucleotide molecules. In one embodiment, the term "chimeric" refers to a gene expression element produced through the manipulation of known elements or other polynucleotide molecules.

[0062] In some embodiments, "activation" can refer to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. In some embodiments, activation can refer to induced cytokine production. In other embodiments, activation can refer to detectable effector function. At a minimum, "activated T cells" as used herein are proliferative T cells.

[0063] As used herein, the terms "specific binding" and "specifically binds" refer to the physical interaction between two molecules, compounds, cells, and / or particles in which a first entity binds to a second, target, entity with a higher specificity and affinity than it binds to a third, non-target, entity. In some embodiments, specific binding can refer to an affinity of a first entity for a second, target, entity that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more higher than the affinity for a third non-target entity under the same conditions. A reagent specific for a defined target is one that exhibits specific binding for that target under the assay conditions utilized. Non-limiting examples include antibodies, or ligands that recognize and bind cognate binding partner (e.g., stimulatory and / or co-stimulatory molecules present on T cells) proteins.

[0064] As used herein, a "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (APC, e.g., macrophage, dendritic cell, B cell, artificial APC, etc.), specifically binds to a cognate binding partner (referred to herein as a "stimulatory molecule" or "co-stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including but not limited to proliferation, activation, initiation of an immune response, etc. Stimulatory ligands are well known in the art and include, inter alia, peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and MHC class I molecules loaded with superagonist anti-CD2 antibodies.

[0065] As used herein, the term "stimulatory molecule" means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0066] As used herein, the term "costimulatory ligand" refers to a molecule on an APC that specifically binds to a cognate costimulatory molecule on a T cell and thereby mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of a peptide-loaded MHC molecule of the TCR / CD3 complex. Costimulatory ligands include, but are not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll-like receptor, and a ligand that specifically binds to B7-H3. Costimulatory ligands also include, without limitation, antibodies that specifically bind to costimulatory molecules present on T cells, such as ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0067] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the T cell, including, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll-like receptors, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0068] In one embodiment, as used herein, the terms "modified," "engineered," and their grammatical equivalents can refer to one or more human-designed changes to a nucleic acid, e.g., a nucleic acid within an organism's genome. In another embodiment, engineered can refer to genetic changes, additions, and / or deletions. A "modified cell" or "engineered cell" can refer to a cell having added, deleted, and / or changed genes. As used herein, the terms "cell," "modified cell," or "engineered cell" and their grammatical equivalents can refer to cells of human or non-human animal origin.

[0069] As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, that is associated with a second transcribable polynucleotide molecule, such as a gene of interest, where the polynucleotide molecule is arranged so as to affect the function of the second polynucleotide molecule. The 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 controls or mediates the transcription of a gene of interest in a cell, the promoter is operably linked to the gene of interest.

[0070] In various embodiments described herein, it is further contemplated that any variant (naturally occurring or otherwise) of a particular polypeptide described, alleles, homologs, conservatively modified variants, and / or conservative substitution variants are included. With respect to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a low percentage of amino acids in the encoded sequence are "conservatively modified variants" where the change results in substitution of an amino acid with a chemically similar amino acid and maintains 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 disclosure.

[0071] The defined amino acids can be substituted by residues having similar physiochemical characteristics, for example, substitution of one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala for each other) or substitution of one polar residue for another (e.g., Lys for Arg; Glu for Asp; or Gln for Asn). Other such conservative substitutions, such as substitution of entire regions having similar hydrophobic characteristics, are well known. A polypeptide containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm retention of the desired activity of the native or reference polypeptide, e.g., receptor activity and specificity mediated by a ligand.

[0072] Amino acids can be grouped according to similarities in the properties of their side chains (as in A. L. Lehninger, Biochemistry, Second Edition, p73 - 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, the naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve the exchange of one member of these types for another. Specific conservative substitutions include, for example; Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0073] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of the wild-type reference polypeptide, as known in the art or according to the assays described herein below. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0074] In some embodiments, the polypeptides described herein can be variants of the polypeptides or molecules described herein. In some embodiments, the variant is a conservatively modified variant. Conservatively substituted variants can be obtained by mutations such as those in the native nucleotide sequence. As referred to herein, a "variant" is a polypeptide that is substantially homologous to the native or reference polypeptide, but has an amino acid sequence different from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide includes one or more additions, deletions, or substitutions of nucleotides when compared to the native or reference DNA sequence, but includes sequences encoding variant proteins or fragments thereof that retain the activity of the non-variant polypeptide. A variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0075] Variant amino acid or DNA sequences can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the native or reference sequence. The degree of homology (percent identity) between the native and variant sequences can be determined, for example, by comparing the two sequences using freely available computer programs commonly utilized for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0076] Changes to the native amino acid sequence can be accomplished by any of a number of techniques known to those of skill in the art. Mutations can be introduced, for example, by synthesizing oligonucleotides containing variant sequences adjacent to restriction enzyme sites that allow ligation of a fragment of the native sequence at a particular locus. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis techniques can be utilized to yield a changed nucleotide sequence having a particular codon changed by the required 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. Patent Nos. 4,518,584 and 4,737,462, which are hereby incorporated by reference in their entirety. Any cysteine residues not involved in maintaining the appropriate three-dimensional structure of the polypeptide can also generally be replaced with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, disulfide bonds can be added to the polypeptide to improve its stability or promote oligomer formation.

[0077] As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term "polynucleotide" is used interchangeably herein with "nucleic acid" to denote a polymer of nucleosides. Typically, a polynucleotide consists of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) that are naturally found in DNA or RNA linked by phosphodiester bonds. However, the term encompasses molecules containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to a polynucleotide, it is understood that both DNA and RNA are provided in both single-stranded and double-stranded forms (and the complementary strands of each single-stranded molecule). As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information that biochemically characterizes a particular nucleic acid (i.e., the sequence of letters used as abbreviations for bases). Polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise indicated.

[0078] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2 to 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, linkages, linkers for functionalization, etc. A polypeptide having a non-polypeptide moiety linked by a covalent or non-covalent bond is still considered a "polypeptide". Typical modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized through chemical means such as conventional solid-phase peptide synthesis. As used herein, the term "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide substance itself and / or the sequence information that biochemically characterizes the polypeptide (i.e., the sequence of letters or three-letter codes used as abbreviations for amino acid names). Polypeptide sequences presented herein are presented in the N-terminal to C-terminal direction unless otherwise indicated.

[0079] In some embodiments, the nucleic acids encoding the polypeptides described herein (e.g., proteins that promote immune surveillance evasion (e.g., TAP inhibitors or HLA homologs), markers, suicide proteins, or therapeutic proteins (e.g., CAR polypeptides)) are contained within a vector. In some of the embodiments described herein, the nucleic acid sequence encoding the defined polypeptides described herein, or any module thereof, is operably linked to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, the vector can be viral or non-viral. The term "vector" encompasses any genetic element that, when associated with appropriate control elements, is capable of replication and can transfer a gene sequence to a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virions, etc.

[0080] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or a polypeptide from a sequence that is operably linked to a transcriptional control sequence on the vector. The expressed sequence is often heterologous to the cell, but not necessarily so. An expression vector may contain additional elements. For example, an expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, e.g., in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the production of RNA and protein, and, where applicable, includes cellular processes involved as a secreted protein, including, but not limited to, transcription, transcriptional processing, translation, and protein folding, modification, and processing. An "expression product" includes RNA transcribed from a gene and a polypeptide obtained by translation of the mRNA transcribed from the gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA) to RNA in vitro or in vivo when operably linked to an appropriate regulatory sequence. A gene may or may not include sequences lying before and after the coding region, e.g., the 5' untranslated (5'UTR) or "leader" sequence and the 3'UTR or "trailer" sequence, and sequences (introns) between individual coding segments (exons).

[0081] 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. A viral vector may contain a nucleic acid encoding a polypeptide described herein in place of a non-essential viral gene. The vector and / or particle can be utilized for the purpose of transferring the nucleic acid into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0082] "Recombinant vector" means a vector containing a heterologous nucleic acid sequence or a "transgene" capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject as extrachromosomal DNA in a large copy number, thereby eliminating the potential for chromosomal integration effects.

[0083] Optionally, the vectors described herein can include a multicistronic construct containing multiple genes for expression. These constructs can include linkers that separate different coding sequences and facilitate cleavage of the resulting polyprotein. In various examples, the linker is or includes a viral 2A protein (e.g., T2A, P2A, E2A, and F2A).

[0084] As used herein, the terms "treating," "treatment," "treat," or "ameliorate" refer to a therapeutic treatment that has the purpose of reversing, alleviating, improving, inhibiting, delaying or stopping the progression or severity of a disease or disorder, such as acute lymphoblastic leukemia or other cancer, disease, or disorder related conditions. The term "treat" includes reducing or alleviating at least one side effect or symptom of a condition, disease or disorder. If one or more symptoms or clinical markers are reduced, the treatment is generally "effective." Alternatively, the treatment is "effective" if the progression of the disease is reduced or stopped. That is, "treatment" includes not only improvement of symptoms or markers, but also halting, or at least delaying, the progression or worsening of symptoms as compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of disease extent, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or alleviation of a disease state, remission (whether partial or total), and / or decreased mortality rate, whether detectable or undetectable. The term "treatment" of a disease also includes bringing about the relief (including palliative treatment) from the symptoms or side effects of the disease.

[0085] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier, such as carriers commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, the pharmaceutically acceptable carrier can be an artificial or engineered carrier, such as a carrier in which the active ingredient has not been found to occur naturally.

[0086] As used herein, the term "administering" refers to the placement of a therapeutic or pharmaceutical composition disclosed herein onto a subject by a method or route that results in at least partial delivery of the agent at a desired site. A pharmaceutical composition comprising an agent described herein can be administered by any suitable route that provides an effective treatment in a subject.

[0087] "T cells" or "T lymphocytes" are a type of lymphocyte (a subtype of white blood cells) that play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer (NK) cells, by the expression of cell surface T cell receptors. T cells include, for example, naive T cells, central memory T cells, and effector memory T cells. As used herein, the terms "modified T cells" or "modified T lymphocytes" (which are used interchangeably herein) are modified (e.g., genetically modified) T cells that have reduced or ablated (i.e., null) TCR expression or activity due to, for example, a deletion or mutation (e.g., a frameshift mutation) in CD3ζ, TRAC, and / or TRBC, or other knockdown or knockout. "Modified T cells" can be further modified to express a therapeutic protein, such as a chimeric antigen receptor (CAR), such that the "modified T cells" are given as CAR-T cells, including CD3ζ, TRAC, and / or TRBC-related modifications. "Modified T cells" can also optionally be modified to express one or more proteins that promote host immune surveillance avoidance, such as inhibitors of TAP or HLA homologs, as further described below. Additional, optional modifications include mutations or deletions that affect HLA expression, as well as the expression of HLA-G and / or HLA-E (e.g., mutations or deletions in the HLA locus on chromosome 6).

[0088] The terms "statistically significant" or "significantly" refer to statistical significance and generally mean a difference of two standard deviations (2SD) or more.

[0089] Unless otherwise indicated or used in an operating example, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

[0090] As used herein, the term "comprising" means that other elements may also be present in addition to the recited desired elements. The use of "comprising" indicates inclusion rather than limitation.

[0091] The term "consisting of" refers to the compositions, methods, and respective components thereof described herein, which exclude any element not recited in the description of that embodiment.

[0092] As used herein, the term "consisting essentially of" refers to those elements required for the specified embodiments. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the technology.

[0093] The singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but suitable methods and materials are described below. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0094] In some embodiments of any of the aspects, the disclosure described herein is not related to the process of cloning a human, the process of modifying the genetic identity of the human germline, the use of a human embryo for industrial or commercial purposes, or the process of modifying the genetic identity of an animal that is likely to afflict them without substantial medical benefit to a human or animal, and animals obtained from such processes.

[0095] Other terms are defined within the description of the various aspects of the technology and the embodiments described herein below and in other places.

[0096] The present invention provides several advantages. For example, without CD3ζ, T cell receptor signaling cannot occur, so removal of CD3ζ expression according to certain embodiments of the present invention eliminates the risk of GvH disease. Since it eliminates competition between the endogenous T cell receptor signaling molecule and the novel receptor molecule, this is also advantageous in the context of T cells modified to express a novel receptor molecule (e.g., CAR).

[0097] In addition, in previous methods (e.g., methods involving deletion of the TRAC sequence), modified T cells continue to express their allogeneic HLA alleles and are thus rapidly rejected by the recipient. This problem is addressed by the present invention, which includes the option of expressing a heterologous protein (e.g., a viral protein) that can reduce the incidence of rejection. Thus, the present invention facilitates adoptive T cell therapy by providing modified T cells that lack natural T cell receptor expression and can avoid rejection mediated by the recipient's immune system. Thus, the resulting cells are safer to use and longer lasting.

[0098] Additional features and advantages of the present invention will be apparent from the following detailed description, claims, and drawings.

Brief Description of the Drawings

[0099]

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Mode for Carrying Out the Invention

[0100] The present invention provides T cells modified to have reduced expression of the T cell receptor (TCR) (e.g., partial reduction in expression or complete inhibition of expression) due to mutations or deletions in the CD3ζ, T cell receptor alpha chain (TRAC), and / or T cell receptor beta chain (TRBC) sequences. In one example, partial or complete inhibition of the expression of the CD3ζ gene is achieved, for example, due to mutations or deletions in the CD3ζ sequence. In the absence of CD3ζ, the T cell receptor complex cannot be formed.

[0101] Accordingly, the modified T cells of the present invention can be used as "universal" T cells, for example, in the context of adoptive T cell therapy. In one example, further described below, the modified T cells are transduced with a sequence encoding a chimeric antigen receptor (CAR) directed against a cancer-related antigen and are then administered to a patient, for example, to treat cancer. In other examples, the modified cells are used in the context of infectious diseases or organ transplantation.

[0102] Optionally, the modified T cells are further modified to avoid or reduce the occurrence of cell rejection when administered to a patient. These further modifications are particularly advantageous in the context of allogeneic T cell therapy but may also be useful in autologous approaches, for example, when heterologous proteins are expressed by autologous modified T cells.

[0103] In addition to the modified T cells, the present invention also provides methods of using the modified T cells, as well as related compositions and kits. The modified T cells, methods, compositions, and kits of the present invention are described in more detail below in a typical manner.

[0104] T cell The T cells (e.g., human T cells) that can be used in the present invention include autologous cells obtained from a subject to be administered later after ex vivo modification and expansion. For example, T cells can be obtained from an individual having or diagnosed with cancer, an infectious disease, an autoimmune disease, or a plasma cell disorder. T cells can also be obtained from an allogeneic donor, which is an individual of the same species but genetically non-identical to the intended recipient of the cells. T cells are typically obtained from peripheral blood collected from a subject, for example, by venipuncture or collection through an implanted port or catheter. Optionally, the blood can be obtained by a process including leukapheresis, where leukocytes are obtained from the subject's blood while the other blood components are returned to the subject. The blood or leukapheresis product (fresh or cryopreserved) is processed to enrich for T cells using methods known in the art. Thus, for example, density gradient centrifugation (e.g., using Ficoll) and / or counterflow centrifugal elutriation can be performed to enrich for mononuclear cells (including T cells). For example, a T cell stimulation step utilizing CD3 / CD28 antibodies coated on magnetic beads or artificial antigen-presenting cells (aAPCs) (see below) expressing, for example, anti-CD3 and anti-CD28 antibody fragments bound to the cell surface can be further performed to stimulate the T cells and deplete other cells, such as B cells. Next, the T cells of the enriched T cell preparation can be subjected to genetic modification. As an alternative to peripheral blood, tissues including bone marrow, lymph nodes, spleen, and tumors can be used as a source for T cells. T cells can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, although any other mammalian cells may be used. In certain embodiments of any aspect, the T cells are human.

[0105] Modification of T Cells T cells can be modified in several ways according to the present invention to enhance their use in therapeutic methods (e.g., adoptive T cell therapy). These modifications include (i) reduced CD3ζ, TRAC, and / or TRBC expression, for example, due to deletion / mutation (e.g., frameshift mutation) of the CD3ζ, TRAC, and / or TRBC sequences; (ii) expression of one or more proteins that promote immune surveillance evasion (e.g., TAP inhibitors or HLA homologs) (or deletion of HLA class I and expression of HLA-G); (iii) expression of markers and / or suicide genes; and / or (iv) expression of therapeutic proteins such as chimeric antigen receptors (CARs) or heterologous T cell receptors. Examples of each of these types of modifications are provided below.

[0106] CD3ζ, TRAC, or TRBC mutations and deletions The T cell receptor complex includes variable T cell receptor α and β chains, and three dimeric signaling molecules: CD3δ / ε, CD3γ / ε, and CD3ζ / ζ. According to the present invention, the expression of CD3ζ (also referred to herein as "CD3z" or "CD3 zeta"), TRAC, and / or TRBC genes is reduced or removed. This can be achieved using any of a number of methods known in the art. In one example, the CD3ζ sequence (e.g., the coding or control sequence; e.g., see ENSEMBL ID ENSG00000198821 of January 10, 2018), the TRAC sequence (e.g., the coding or control sequence), and / or the TRBC (e.g., the coding or control sequence) are mutated or deleted from the genome of T cells, for example, using gene editing methods. Thus, approaches that utilize, for example, RNA / DNA-guided endonucleases (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9, Cpf1, and Argonaute), transcription activator-like effector (TALE) nucleases, zinc finger nucleases (ZFNs), or meganucleases are suitable for use in the present invention. Furthermore, methods of manipulating nucleases that can be used in the present invention to achieve the desired sequence specificity are described, for example, in Kim (2014); Kim (2012); Belhaj et al. (2013); Urnov et al. (2010); Bogdanove et al. (2011); Jinek et al. (2012), Silva et al. (2011); Ran et al. (2013); Carlson et al. (2012); Guerts et al. (2009); Taksu et al. (2010); and Watanabe et al. (2012), each of which is hereby incorporated by reference in its entirety.

[0107] In various examples, insertions or deletions are made by gene editing that causes a frameshift mutation leading to gene knockout (i.e., lack of expression of a functional gene product). In certain examples, such mutations are made to target an early coding sequence, near the N-terminus of the protein, to maximize disruption and minimize the possibility of low-level protein expression. In various examples, any exon can be targeted for frameshift generation (e.g., exon coding sequences). As a particular example, more proximal exons may be targeted.

[0108] Particular examples of protocols used in the present invention in the context of CD3ζ include (i) electroporation of a guide RNA targeting CD3ζ having mRNA encoding Cas9 endonuclease, (ii) electroporation of a ribonucleoprotein (RNP) that makes up a precomplexed guide RNA and Cas9 endonuclease protein, and (iii) expression of a guide RNA using a human RNA polymerase promoter encoded in a CAR lentiviral backbone by electroporation of Cas9 protein or mRNA.

[0109] Alternatively, reduction or elimination of CD3ζ, TRAC, and / or TRBC expression can be achieved by use of inhibitory nucleic acids. As used herein, "inhibitory nucleic acids" refers to nucleic acid molecules that can inhibit the expression of a target gene or mRNA, and includes, for example, double-stranded RNA (dsRNA), inhibitory RNA (iRNA), and the like. Inhibitory nucleic acid technologies are more fully described, for example, in Wilson, RC, and Doudna, JA, (2013), Annual Review of Biophysics, 42(217-239) and references cited therein.

[0110] Expression of proteins that promote avoidance of immune surveillance Modified T cells characterized by reduced or ablated expression of a functional TCR (e.g., due to CD3ζ, TRAC, and / or TRBC gene deficiencies, such as frameshift mutations) can be advantageously used in the context of adoptive T cell therapy, such as those described herein. As further described below, these modified T cells can be further modified to express a chimeric antigen receptor (CAR) in order to direct the modified T cells to target cells (e.g., tumor cells). However, the function of the modified T cells can be further improved by one or more additional gene modifications, particularly in the context of allogeneic gene transfer methods. More specifically, gene-modified T cells lacking expression of the endogenous T cell receptor (e.g., due to CD3ζ, TRAC, and / or TRBC gene deficiencies or mutations described herein) are susceptible to attack by the immune system (T cell- and NK-mediated rejection) of the subject to which they are administered. Avoidance of this attack can be achieved by expression of certain heterologous proteins in the modified T cells. Thus, expression of these proteins can be used to increase the persistence of the modified T cells administered. Heterologous proteins that can be used in this context include, for example, viral proteins that promote immune evasion. Examples of these proteins are described below and include, for example, transporters associated with antigen processing (TAP) inhibitors and HLA homologs. Expression of these proteins may also be applicable in autologous settings where a reduced immune response to the expressed transgene may be desirable.

[0111] Thus, in one example, a viral inhibitor of TAP can be expressed in the modified T cells of the present invention. TAP plays a central role in antigen processing, particularly in the transport to the endoplasmic reticulum (ER) for MHC presentation. Without effective TAP transport, cells cannot load and express HLA class I molecules, and thus, inhibition of TAP can promote immune evasion. Viral inhibitors of TAP that can be used in the present invention include, for example, the viral protein US6 of cytomegalovirus (CMV), the viral protein ICP47 of herpes simplex virus (HSV), the bovine herpesvirus-1 (BoHV-1) protein UL49.5, and the Epstein-Barr virus (EBV) protein BNLF2a.

[0112] In another example, the full or partial (signal peptide) CMV UL40 protein is expressed in the modified T cells of the present invention. UL40 has homology to HLA class I and does not require TAP-dependent presentation for transport to the ER. After transport, the UL40 peptide can bind to non-classical HLA-E and promote surface expression. When expressed, HLA-E inhibits NK-mediated rejection via the inhibitory receptor CD94 / NKG2A. In this way, expression of UL40 provides protection to the modified T cells from host immune system attack.

[0113] In a further example, CMV UL18 is expressed in the modified T cells of the present invention. UL18 is a viral HLA homolog that, when expressed, can inhibit LIR+NK-mediated rejection. UL18 requires beta-2 microglobulin for cell surface expression and can be increased by co-expression of UL40. Another example of an HLA homolog that can be used in the present invention is UL142.

[0114] As an alternative to (or in addition to) adding a gene encoding a protein that promotes immune surveillance evasion, the modified T cells of the invention can be further modified to lack the endogenous HLA locus on chromosome 6. In another example, the B2M locus is targeted (Ensembl number ENSG00000166710 as of January 10, 2018). If such a deletion is made, the T cells are susceptible to NK-mediated lysis. In contrast, the cell can be transduced to express the universal HLA molecule HLA-G or a protein that increases the expression of HLA-E on the cell surface such as full or partial UL40, or UL18, or a portion thereof.

[0115] In certain embodiments, the individual genes that express a protein that promotes immune surveillance evasion are expressed in the modified T cells of the invention. In other embodiments, combinations of such proteins are expressed in such cells. In a particular example, UL40, US6, and UL18 are expressed together, for example, using the multicistronic vectors described herein.

[0116] Typical protein sequences that can be expressed in connection with promoting immune surveillance evasion include those listed below, as well as functional variants thereof. TIFF0007717439000001.tif207170

[0117] Expression of a reporter or suicide gene Additional modifications that can be made to the modified T cells of the invention include, for example, the expression of one or more reporter genes. For example, a truncated epidermal growth factor receptor (EGFR) lacking an intracellular signaling domain can be used with an anti-EGFR monoclonal antibody and used, for example, for in vivo depletion in the case of toxicity.

[0118] Another typical modification involves the expression of a suicide gene in the modified T cells of the present invention. By doing so, control mediated by drugs external to the administered cells can be promoted. For example, through the use of a suicide gene, modified cells can be depleted from a patient, for example, in the case of adverse events. In one example, the FK506 binding domain is fused to a caspase 9 apoptosis-promoting molecule. T cells engineered in this way are rendered 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 can be activated for conditional ablation by the administration of a specific molecule (e.g., ganciclovir for TK+ cells) or an antibody or a conjugate of an antibody and a drug.

[0119] Expression of a therapeutic protein - chimeric antigen receptor (CAR) In addition to the CD3ζ, TRAC, and / or TRBC genes modified above, and the additional optional modifications described above, the T cells of the present invention can be further optionally modified to express a therapeutic protein, such as a chimeric antigen receptor (CAR). As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs (plural)" refers to an engineered T cell receptor that confers ligand or antigen specificity to a T cell (e.g., naive T cells, central memory T cells, effector memory T cells, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0120] A CAR comprises a chimeric extracellular target-binding domain that binds specifically to a target, for example, a polypeptide expressed on the surface of a cell to be targeted for a T cell response, including a transmembrane domain, and an intracellular domain of a T cell receptor molecule is disposed on the construct. In one embodiment, the chimeric extracellular target-binding domain comprises an antigen-binding domain (e.g., a single-chain antibody) of an antibody that binds specifically to an antigen expressed on a cell to be targeted for a T cell response. The properties of the intracellular signaling domain of a CAR are known in the art and can vary as disclosed herein, but the chimeric target / antigen-binding domain can confer receptor sensitivity to signal transduction activation when the chimeric target / antigen-binding domain binds to a target / antigen on the surface of a target cell.

[0121] Regarding the intracellular signaling domain, so-called "first-generation" CARs include those that solely provide CD3ζ upon antigen binding. So-called "second-generation" CARs include those that provide both a co-stimulatory (e.g., CD28 or CD137) and an activation (CD3ζ) domain, and so-called "third-generation" CARs include those that provide multiple co-stimulatory (e.g., CD28 and CD137) domains as well as an activation domain (e.g., CD3ζ). In various embodiments, a CAR having a high affinity or binding activity for a target / antigen is selected. For example, an antibody-derived target or antigen-binding domain generally has a higher affinity and / or binding activity for a target antigen than a naturally occurring T cell receptor. This property, combined with the high specificity that a person can select for an antibody, results in highly specific T cell targeting by CAR T cells.

[0122] As used herein, "CAR T cell" or "CAR-T" refers to a T cell that expresses a CAR. When expressed in a T cell, the CAR has the ability to redirect T cell specificity and reactivity towards a target selected in a non-MHC-restricted manner and to utilize the antigen-binding properties of a monoclonal antibody. Non-MHC-restricted antigen recognition confers upon CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing a major mechanism of tumor avoidance. The CAR T cells of the present invention, in addition to the CAR, include further modifications described herein (i.e., modifications resulting in reduced or eliminated TCR expression due to mutations or deletions in the CD3ζ, TRAC, and / or TRBC sequences described herein). These modifications optionally include, for example, the expression of one or more additional proteins promoting immune surveillance avoidance (e.g., TAP inhibitors or HLA homologs), suicide genes, and / or marker genes, as shown above, in combination with one or more further modifications. As an alternative to the expression of proteins promoting immune surveillance avoidance, the cells can be depleted for expression of the HLA locus on chromosome 6 and can further optionally express HLA-G, as described above.

[0123] 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 specifically binds to its binding partner, i.e., the target. By way of non-limiting example, the extracellular target binding domain can include the antigen-binding domain of an antibody or a ligand that recognizes and binds to a cognate binding partner (e.g., CD19, BCMA, or CD37) protein. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or receptor. Cognate binding partners for ligands useful in the methods and compositions described herein can generally be found on the surface of a cell. Binding of a ligand to a cognate partner can result in a change in the receptor caused by the ligand or can activate a physiological response, e.g., a signaling pathway. In one embodiment, the ligand can be non-native to the genome. Optionally, the ligand has a function conserved across at least two species. In one embodiment, the extracellular target binding domain includes a non-antibody ligand (e.g., a ligand that induces proliferation (APRIL)).

[0124] Antibody reagent In various embodiments, the CARs described herein include an antibody reagent or its antigen-binding domain as an extracellular target binding domain.

[0125] As used herein, the term "antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a defined antigen. The antibody reagent can include an antibody or polypeptide that includes the antigen-binding domain of an antibody. In some embodiments of any of the aspects, the antibody reagent can include a monoclonal antibody or a polypeptide that includes the antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as V H as used herein) and a light (L) chain variable region (abbreviated herein as V Lmay include (abbreviated as). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" includes antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab’)2, Fd fragments, Fv fragments, scFv, CDRs, and domain antibody (dAb) fragments (e.g., de Wildt et al., Eur J. Immunol, 1996;26(3):629-39; which is hereby incorporated by reference in its entirety), as well as full antibodies. Antibodies may have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (as well as their subtypes and combinations). Antibodies are derived from any source, including mice, rabbits, pigs, rats, and primates (human and non-human primates), and may be primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, etc. A fully human antibody binding domain can be selected, for example, from a phage display library using methods known to those skilled in the art.

[0126] V H and V L regions can be further divided into regions of high-frequency variability called complementarity determining regions ("CDRs") and regions where more conserved regions called framework regions ("FRs") are dispersed. The extent of the framework regions and CDRs is precisely defined (see Kabat, E. A. et al., (1991), Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al., (1987), J. Mol. Biol. 196:901-917, which are hereby incorporated by reference in their entirety). Each V H and V L typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0127] In one embodiment, the antibody or antibody reagent is not a human antibody or antibody reagent (e.g., the antibody or antibody reagent is murine), but is humanized. A "humanized antibody or antibody reagent" refers to a non-human antibody or antibody reagent that has been modified at the protein sequence level to increase its similarity to antibodies or antibody reagent variants that are naturally produced in humans. One approach to humanizing an antibody utilizes the transplantation of murine or other non-human CDRs into a human antibody framework.

[0128] In one embodiment, the extracellular target binding domain of the CAR is fused to, or consists essentially of, a single-chain Fv (scFv) fragment produced by fusing the V H and V L domains of an antibody, typically a monoclonal antibody, via a flexible linker peptide. In various embodiments, the scFv is fused to a transmembrane domain and a T cell receptor intracellular signaling domain, such as the engineered intracellular signaling domains described herein.

[0129] In one embodiment, a CAR useful in the technologies described herein includes at least two antigen-specific target regions, extracellular domains, transmembrane domains, and intracellular signaling domains. In such embodiments, the two or more antigen-specific target regions target at least two different antigens, are arranged in tandem, and may be separated by a linker sequence. In another embodiment, the CAR is a bispecific CAR. A bispecific CAR is specific for two different antigens.

[0130] Target / antigen Any cell surface moiety can be targeted by a CAR. Most frequently, the target is a cell surface polypeptide that is differentially or preferentially expressed on the cells that a person desires to target for a T cell response. In this regard, tumor antigens or tumor-associated antigens provide attractive targets, which provide a means of targeting tumor cells while avoiding or at least limiting collateral damage to non-tumor cells or tissues. Non-limiting examples of tumor antigens or tumor-associated antigens include CD19, BCMA, CD37, CEA, immature laminin receptor, TAG-72, HPV E6 and E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, BRCA1 / 2, CDK4, MART-2, p53, Ras, MUC1, and TGF-βRII.

[0131] In one embodiment, the target is B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17). BCMA is a cell surface receptor that is preferentially expressed on mature B lymphocytes that specifically recognize B-cell activating factor (BAFF). BCMA sequences are known for a number of species, for example, human BCMA (NCBI Gene ID: 608) polypeptide (e.g., NCBI Reference Sequence NP_001183.2) and mRNA (e.g., NCBI Reference Sequence NM_001192.2). BCMA can refer to human BCMA, including its naturally occurring variants, molecules, and alleles. In some embodiments of any of the aspects, for example, in veterinary applications, BCMA can refer to BCMA of, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human BCMA can be readily identified by those skilled in the art for such species, for example, using the NCBI ortholog search function or by searching available sequence data for the specified species for sequence similarity to the reference BCMA sequence.

[0132] In one embodiment, the BCMA binding sequence comprises a ligand of BCMA or an antibody reagent that specifically binds to BCMA. In one embodiment, the antibody reagent that specifically binds to BCMA is an scFv derived from a humanized anti-BCMA m mouse antibody. The orientation of the single-chain variable fragment derived from the humanized mouse antibody is V L -V H or V H -V L and can be.

[0133] In one embodiment, the target is CD37. CD37 is a cell surface protein containing four hydrophobic transmembrane domains. CD37 is expressed exclusively on immune cells, highly expressed on mature B cells, and moderately expressed on T cells and myeloid cells. CD37 sequences are known for a number of species, for example, human CD37 (NCBI Gene ID: 951) polypeptide (e.g., NCBI Reference Sequence NP_001035120.1) and mRNA (e.g., NCBI Reference Sequence NM_NM_001040031.1). CD37 can refer to human CD37, including its naturally occurring variants, molecules, and alleles. In some embodiments of any of the aspects, for example, in veterinary applications, CD37 can refer to CD37 of, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human CD37 can be readily identified by those skilled in the art for such species, for example, using the NCBI ortholog search function or by searching available sequence data for the specified species for sequence similarity to the reference CD37 sequence. In one embodiment, the CD37 binding sequence includes a ligand of CD37 or an antibody reagent that specifically binds to CD37.

[0134] Transmembrane domain Each CAR described herein necessarily includes a transmembrane domain that links an extracellular target binding domain to an intracellular signaling domain.

[0135] As used herein, the "transmembrane domain" (TM domain) refers to the common hydrophobic region of the CAR that spans the cell membrane of a cell. The TM domain can be a transmembrane region or a fragment thereof of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Specific examples are provided herein, while other transmembrane domains will be apparent to those skilled in the art and can be used in connection with alternative embodiments of the technology. The selected transmembrane region or fragment thereof preferably does not interfere with the intended function of the CAR. The "fragment thereof" as used in connection with the transmembrane domain of a protein or polypeptide refers to a portion of the transmembrane domain that is sufficient to anchor or bind the protein to the cell surface.

[0136] In one embodiment, the transmembrane domain of the CAR or a fragment thereof is or comprises the transmembrane domain of CD8. In an alternative embodiment of any aspect, the transmembrane domain of the CAR or a fragment thereof described herein is the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or the transmembrane domain selected from the transmembrane domains of NKG2C.

[0137] CD8 is an antigen that is preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-to-cell interactions within the immune system and functions as a T cell coreceptor. CD8 consists of alpha (CD8α) and beta (CD8β) chains. CD8a sequences are known for a number of species, for example, human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBI Reference Sequence NP_001139345.1) and mRNA (e.g., NCBI Reference Sequence NM_000002.12). CD8 can refer to human CD8, including its naturally occurring variants, molecules, and alleles. In some embodiments of any of the aspects, for example, in veterinary applications, CD8 can refer to CD8 of, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human CD8 can be readily identified by those skilled in the art for such species, for example, using the NCBI ortholog search function or by searching available sequence data for the specified species for sequence similarity to a reference CD8 sequence.

[0138] Costimulatory domain Each CAR described herein may include an intracellular domain of a costimulatory molecule, or a costimulatory domain. As used herein, the term "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In one embodiment, the intracellular domain is the intracellular domain of 4-1BB.

[0139] 4-1BBL is a type II transmembrane glycoprotein belonging to the TNF superfamily. 4-1BBL is expressed on activated T lymphocytes. 4-1BBL sequences are known for a number of species, for example, human 4-1BBL, also known as TNFSF9 (NCBI gene number: 8744) polypeptide (e.g., NCBI reference sequence NP_003802.1) and mRNA (e.g., NCBI reference sequence NM_003811.3). 4-1BBL can refer to human 4-1BBL, including its naturally occurring variants, molecules, and alleles. In some embodiments of any of the aspects, for example, in veterinary applications, 4-1BBL can refer to 4-1BBL of, for example, dogs, cats, cows, horses, pigs, etc. Homologs and / or orthologs of human 4-1BBL can be readily identified by those skilled in the art for such species, for example, using the NCBI ortholog search function or by searching available sequence data for a defined species for sequence similarity to the reference 4-1BBL sequence.

[0140] Intracellular signaling domain The CARs described herein include an intracellular signaling domain. The "intracellular signaling domain" refers to a part of the CAR polypeptide that is involved in inducing effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to target cells to which the CAR is bound, when a message of effective CAR binding to a target antigen is transferred inside the immune effector cell, or other cellular responses induced after binding of the antigen to the extracellular CAR domain.

[0141] As shown above, CD3 is a T cell co-receptor that promotes T lymphocyte activation when involved simultaneously with appropriate co-stimulation (e.g., binding of co-stimulatory molecules). The CD3 complex consists of four different chains, and mammalian CD3 consists of the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains are associated with molecules known as the T cell receptor (TCR) and CD3ζ to generate activation signals in T lymphocytes. The complete TCR complex includes the TCR, CD3ζ, and the complete CD3 complex.

[0142] In some embodiments of any aspect, the CAR polypeptides described herein include an intracellular signaling domain that includes an immunoreceptor tyrosine-based activation motif or ITAM derived from CD3 zeta (CD3ζ). In some embodiments of any aspect, the ITAM includes three motifs of the ITAM of CD3ζ (ITAM3). In some embodiments of any aspect, the three motifs of the ITAM of CD3ζ are mutated.

[0143] ITAM is known as a primary signaling domain that regulates the primary activation of the TCR complex either in a stimulatory or an inhibitory manner. A primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Non-limiting examples of ITAMs containing an intracellular signaling domain for specific uses in technology include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0144] One of ordinary skill in the art has the ability to introduce mutations into a gene or gene product, such as the nucleic acid sequence of an ITAM, using standard techniques. For example, point mutations can be introduced via site-directed mutagenesis, PCR techniques. Site-directed mutagenesis kits are commercially available, for example, through New England Biolabs, Ipswich, MA. Non-limiting examples of alternative methods for introducing point mutations into the nucleic acid sequence of a gene or gene product include cassette mutagenesis or whole plasmid mutagenesis.

[0145] In one embodiment, the ITAM utilized in the CAR comprises an alternative to CD3ζ that contains a mutated ITAM derived from CD3ζ (containing three ITAM motifs), CD3ζ, and CD3ε, truncations of alternative splice variants known as CD3θ, and artificial constructs engineered to express fusions between CD3ε or CD3θ and CD3ζ.

[0146] In one embodiment, the CD3ζ ITAM3 sequence corresponds to the sequence of SEQ ID NO: 9, or comprises the sequence of SEQ ID NO: 9, or comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequence of SEQ ID NO: 9.

[0147] In one embodiment, the intracellular signaling domain comprises a CD3 ITAM3 selected from SEQ ID NO: 9, 10, 11, or 12. In one embodiment, the tyrosine residue is mutated to a phenylalanine residue, thereby inhibiting phosphorylation of the native tyrosine residue. It is contemplated that typical tyrosine residues that can be mutated include any residue where the tyrosine residue results in inhibition of tyrosine phosphorylation. In another aspect of any aspect, tyrosine is mutated in at least 1, at least 2, or all 3 ITAMs (e.g., ITAM I, II, and III).

[0148] In one embodiment, the T cell intracellular signaling domain comprises CD3 epsilon (CD3ε), CD3 theta (CD3θ), or the ITAM of CD3ζ. In one embodiment, the T cell intracellular signaling domain is CD3 epsilon (CD3ε), CD3 theta (CD3θ), or the ITAM of CD3ζ.

[0149] In one embodiment, the intracellular signaling domain comprises a CD3 ITAM3 sequence that contains a deletion related to the CD3 ITAM3 sequence of SEQ ID NO: 9.

[0150] The sequence of SEQ ID NO:9 is provided below, followed by additional information regarding SEQ ID NOs:10, 11, and 12. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:9)

[0151] CD3ζ-mutITAM1 (SEQ ID NO:10). Certain residues described herein are mutated in CD3ζ-ITAM3 to inhibit the function of CD3ζ-ITAM3, namely Y21 and Y32. The positions of these residues are depicted in bold below. RVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:10)

[0152] CD3ζ-mutITAM1 and mutITAM2 (SEQ ID NO:11). Certain residues described herein are mutated in CD3ζ-ITAM3 to inhibit the function of CD3ζ-ITAM3, namely Y21, Y32, Y59, and Y71. The positions of these residues are depicted in bold below. RVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:11)

[0153] CD3ζ-mutITAM1 and mutITAM3 (SEQ ID NO:12). Certain residues described herein are mutated in CD3ζ-ITAM3 to inhibit the function of CD3ζ-ITAM3, namely Y21, Y32, Y90, and Y100. The positions of these residues are depicted in bold below. RVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 12)

[0154] Deficiencies related to the CD3ζ ITAM3 sequence can be made using techniques well known in the art, such as CRISPR, TALEN, or ZFN technology (see also above). Methods of engineering nucleases to achieve the desired sequence specificity are known in the art and are described, for example, in the references cited above.

[0155] More detailed descriptions of CARs and CAR T cells that may be adapted for use in the present invention can be found, for example, in Maus et al., Blood, 2014, 123:2624-35; Reardon et al., Neuro-Oncology, 2014, 16:1441-1458; Hoyos et al., Haematologica, 2012, 97:1622; Byrd et al., J Clin Oncol, 2014, 32:3039-47; Maher et al., Cancer Res, 2009, 69:4559-4562; and Tamada et al., Clin Cancer Res, 2012, 18:6436-6445, each of which is hereby incorporated by reference in its entirety.

[0156] In one embodiment, the CAR further includes a linker domain. As used herein, "linker domain" refers to an oligopeptide or polypeptide region about 2 to 100 amino acids in length that binds together with any of the domains / regions of the CAR described herein. In some embodiments, the linker includes or consists of flexible residues such as glycine and serine so that adjacent, linked protein domains are free to move relative to each other. Longer linkers can be used when it is desired to ensure two adjacent domains that do not sterically interfere with each other. The linker may or may not be cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In one embodiment, the linker region is T2A from the virus Thosea asigna. Non-limiting examples of linkers that can be used in this technology include P2A and F2A. In addition to use in the context of CARs, these cleavable linkers can also be used in the multicistronic vectors described herein.

[0157] In one embodiment, the CARs described herein further include, for example, a reporter molecule to enable non-invasive imaging (e.g., positron emission tomography PET scan). In a bispecific CAR comprising a reporter molecule, the first extracellular binding domain and the second extracellular binding domain may contain different or the same reporter molecule. In bispecific CAR T cells, the first CAR and the second CAR may express different or the same reporter molecule. In another embodiment, the CARs described herein are imaged alone or in combination with a substrate or chemical (e.g., 9-[4- 18 F]fluoro-3-(hydroxymethyl)butyl]guanine( 18 F]FHBG)) and may further include a reporter molecule (e.g., hygromycin phosphotransferase (hph)). In another embodiment, the CARs described herein are non-invasive techniques (e.g., 64 Cu2+ It further includes nanoparticles that can be easily imaged using gold nanoparticles (GNPs) functionalized with 2+ . The labeling of CAR T cells for non-invasive imaging is reviewed, for example, in Bhatnagar P et al., Integr Biol, (Camb), January 2013; 5(1):231-238, and Keu KV et al., Sci Transl Med, January 2017, 18; 9(373), which are hereby incorporated by reference in their entirety.

[0158] GFP and mCherry are shown herein as fluorescent tags useful for imaging CARs expressed on T cells (e.g., CAR T cells). It is predicted that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, the CAR need not include a fluorescent tag or fluorescent protein.

[0159] Constructs, vectors, and expression The present invention also provides constructs and vectors for use in the generation of the modified T cells described herein. In various examples, the present invention provides a construct comprising separate coding sequences for a plurality of proteins to be expressed in the modified T cells of the present invention. These separate coding sequences can be separated from each other by a cleavable linker sequence as described herein. For example, sequences encoding viral 2A proteins (e.g., T2A, P2A, E2A, and F2A) can be placed between separate genes and, when transcribed, can direct cleavage of the resulting polyprotein. As shown above, the constructs and vectors of the present invention can include any of a number of different combinations of sequences. For example, the construct or vector of the present invention can optionally include, in combination with a CAR, a sequence encoding one or more full-length or partial sequences (e.g., each) of UL40, US6, UL18, HLA-E, and HLA-G.

[0160] A construct comprising a sequence encoding a protein for expression in a modified T cell of the invention is contained within a vector, which is also provided by the present invention. In various examples, the vector is a retroviral vector. Retroviruses such as lentiviruses provide a suitable platform for the delivery of nucleic acid sequences encoding a gene of interest, or a chimeric gene. The selected nucleic acid sequence is inserted into the vector and can be packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, for example, in vitro or ex vivo. Retroviral systems are well known in the art and are described, for example, in U.S. Patent No. 5,219,740; Kurth and Bannert, (2010), "Retroviruses: Molecular Biology, Genomics and Pathogenesis", Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu and Pathak, Pharmacological Reviews, 2000, 52:493-512, which are hereby incorporated by reference in their entirety. A lentiviral system for efficient DNA delivery can be purchased from OriGene; Rockville, MD. In various embodiments, the protein is expressed in T cells by transfection or electroporation of an expression vector containing a nucleic acid encoding the protein using vectors and methods known in the art.

[0161] Efficient expression of the proteins in the modified T cells described herein can be evaluated using standard assays for detecting mRNA, DNA, or gene products of the nucleic acids encoding the proteins. For example, RT-PCR, FACS, Northern blotting, Western blotting, ELISA, or immunohistochemistry can be used. In various embodiments, the proteins described herein are constitutively expressed. In other embodiments, the proteins are encoded by recombinant nucleic acid sequences.

[0162] Treatment methods, compositions, and kits The present invention provides methods and compositions for use in the treatment and prevention of diseases and conditions, including, for example, cancer, infectious diseases, autoimmune diseases or disorders, plasma cell diseases or disorders, or transplant-related conditions, in a subject in need thereof (e.g., a subject having or diagnosed with a disease or condition). These methods include modifying T cells by the methods described herein and then administering the modified T cells to the subject. In some embodiments of any of the aspects, the modified T cells (e.g., CAR-T cells comprising one or more additional modifications described herein) are stimulated and / or activated prior to administration to the subject.

[0163] As used herein, "condition" includes cancer, infectious diseases, autoimmune diseases or disorders, plasma cell diseases or disorders, or transplant-related conditions. A subject having a disease or condition can be identified by a physician using current methods for diagnosing the disease or condition. Symptoms and / or complications of a disease or condition that characterize and aid in the diagnosis of these conditions are well known in the art and include, but are not limited to, fatigue, persistent infection, and persistent bleeding. For example, tests that can aid in the diagnosis of a disease or condition include, but are not limited to, blood screening and bone marrow tests, which are known in the art for a given condition. A family history of a disease or condition, or exposure to risk factors for a disease or condition, can also be useful in determining whether a subject is likely to have a disease or condition or in making a diagnosis of a disease or condition.

[0164] As used herein, "cancer" can refer to the uncontrolled growth of cells where the loss of their normal traits and cell control results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis, and can be leukemia, lymphoma, multiple myeloma, or solid tumors. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In one embodiment, the cancer is ALL or CLL. Non-limiting examples of lymphoma include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, hairy cell leukemia (HCL). In one embodiment, the cancer is DLBCL or follicular lymphoma. Non-limiting examples of solid tumors include adrenocortical tumors, alveolar soft part sarcoma, carcinoma, chondrosarcoma, colorectal cancer, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, endodermal sinus tumor, epitheloid hemangioendothelioma, Ewing sarcoma, germ cell tumor (solid tumor), giant cell tumor of bone and soft tissue, hepatoblastoma, hepatocellular cancer, melanoma, kidney tumor, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, paravertebral sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms tumor. Solid tumors can be found in bone, muscle, tissue, or organ and can be sarcoma or carcinoma. It is contemplated that any aspect of the technology described herein can be used to treat all types of cancer, including cancers not listed in the immediate application. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue, for example, of a malignant or benign type.

[0165] As used herein, an "autoimmune disease or disorder" is characterized by the inability of a person's immune system to distinguish foreign cells from healthy cells. This results in a person's immune system targeting the person's healthy cells for programmed cell death. Non-limiting examples of autoimmune diseases or disorders include inflammatory arthritis, type 1 diabetes, multiple sclerosis (MS), psoriasis, inflammatory bowel disease, systemic lupus erythematosus (SLE), vasculitis, allergic asthma, atopic dermatitis, and allergic inflammation such as contact hypersensitivity. Other examples of autoimmune-related diseases or disorders include rheumatoid arthritis, Graves' disease (hyperthyroidism), Hashimoto's thyroiditis (hypothyroidism), celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barré syndrome, primary biliary cirrhosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), autoimmune Addison's disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, opsoclonus-myoclonus ataxia, optic neuritis, autoimmune thyroiditis, pemphigus, pernicious anemia, polyarthritis in dogs, Reiter's syndrome, Takayasu arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, and fibromyalgia (FM), but are not limited thereto.

[0166] In one embodiment, mammalian T cells are obtained from a patient having an immune system disorder, or an immunodeficiency disorder, that results in an abnormally low-activity immune system that impairs the person's ability to fight foreign cells (i.e., viral or bacterial cells).

[0167] Plasma cells are white blood cells generated from B lymphocytes that function to produce and secrete antibodies necessary to fight infections. As used herein, "plasma cell disorder or disease" is characterized by abnormal proliferation of plasma cells. Abnormal plasma cells have the ability to "crowd out" healthy plasma cells, resulting in a reduced ability to fight foreign targets such as viral or bacterial cells. Non-limiting examples of plasma cell disorders include amyloidosis, Waldenström's macroglobulinemia, osteosclerotic myeloma (POEMS syndrome), monoclonal gammopathy of undetermined significance (MGUS), and plasma cell myeloma.

[0168] The compositions described herein (see below) can be administered to a subject having or diagnosed with a disease or condition. In some embodiments, the methods described herein include administering to the subject an effective amount of the modified T cells described herein (e.g., activated CAR T cells) to alleviate the symptoms of the condition. As used herein, "alleviating the symptoms of a condition" is improving any condition or symptoms associated with the condition. Such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique compared to an equivalent untreated control. Various means of administering the compositions described herein to a subject are known to those of skill in the art. In one embodiment, the compositions described herein are administered systemically or locally. In another embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the site of the tumor.

[0169] As used herein, the term "effective amount" refers to the amount of modified T cells (e.g., activated CAR T cells) required to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of a cell preparation or composition to produce a desired effect. The term, its "therapeutically effective amount", refers to the amount of modified T cells (e.g., activated CAR T cells) that is sufficient to produce a particular anti-disease or state effect when administered to a typical subject. In various contexts, the effective amount used herein also includes an amount sufficient to delay the onset of symptoms of a disease, alter the course of a symptomatic disease (e.g., without limitation, slow the progression of a disease or condition), or ameliorate the symptoms of a condition. Thus, it is generally not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0170] Effective amount, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical techniques in cell cultures or experimental animals. Dosage may vary depending upon the dosage form employed and the route of administration utilized. The dosage ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods showing a large therapeutic index are preferred. A therapeutically effective dosage can first be estimated from cell culture assays. Also, the dosage can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of modified T cells that achieves half-maximal inhibition of symptoms) determined in cell culture or in a suitable animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by suitable bioassays, such as, among others, assays for bone marrow tests. The dosage is determined by a physician and can be adjusted as needed to suit the observed effects of the treatment.

[0171] In one aspect, the technology described herein relates to a pharmaceutical composition comprising the modified T cells described herein and optionally a pharmaceutically acceptable carrier. The active ingredient of the pharmaceutical composition comprises at least the modified T cells described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of the modified T cells described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of the modified T cells described herein. Pharmaceutically acceptable carriers for cell-based therapeutic agents 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.

[0172] In some embodiments, the pharmaceutical composition comprising the modified T cells described herein can be in a parenteral administration form. Administration in parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, so components other than the modified T cells themselves are either sterile or capable of being sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. Any of these can be added to the modified T cell preparation prior to administration.

[0173] Suitable vehicles that can be used to provide the parenteral dosage form of the modified T cells disclosed herein are well known to those skilled in the art. Examples include, but are not limited to, aqueous solutions of sodium chloride; glucose solutions; aqueous vehicles including sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, coconut oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0174] The invention further includes a kit for use in practicing the methods of the invention. Thus, the kit can optionally include one or more reagents for generating the modified T cells (e.g., nucleic acid molecules and / or enzymes used to effect deletion and / or addition of genetic sequences). The kit further includes "universal" T cells generated by the methods of the invention and can be used as a "shelf" source of therapeutic material for patient treatment. The kit can further include instructions or devices for administering the modified T cells and, optionally, instructions for use of the compositions and methods of the invention.

[0175] Dosage As used herein, the term "unit dosage form" refers to a dosage amount for a suitable single administration. For illustrative purposes, a unit dosage form can be the amount of therapeutic agent disposed in a delivery device, e.g., a syringe or 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.

[0176] In some embodiments, the modified T cells described herein are administered as monotherapy, i.e., no other treatment for the condition is administered concurrently to the subject.

[0177] The pharmaceutical composition comprising the modified T cells described herein is generally administered at a dosage of from 10 4 to 10 9 cells per kg of body weight, and in some cases, at a dosage of from 10 5 to 10 6 cells per kg of body weight, including all integer values within that range. If necessary, the modified T cell composition can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med, 319:1676, 1988).

[0178] In certain embodiments, it may be desirable to administer the modified T cells to a subject, then subsequently withdraw blood again (or perform a depletion therapy), activate the T cells derived therefrom as described herein, and reinfuse the patient with these activated and expanded T cells. This process can be performed multiple times every few weeks. In certain embodiments, the T cells can be activated from 10 cc to 400 cc of withdrawn blood. In certain embodiments, the T cells are activated from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc of withdrawn blood.

[0179] The mode of administration can include, for example, intravenous (i.v.) injection or infusion. The compositions described herein can be administered to a patient by transarterial, intratumoral, interarticular, or intramedullary routes. In some embodiments, the composition of modified T cells may be directly injected into a tumor, lymph node, or site of infection. In one embodiment, the compositions described herein are administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0180] In certain exemplary embodiments, as described above, the subject may undergo leukapheresis to collect, concentrate, or ex vivo deplete leukocytes to select and / or isolate the desired cells, e.g., T cells. This process may include stimulation by standard methods, e.g., using magnetic beads coated with antibodies to CD3 and CD28. Alternatively, T cells may be expanded by contact with artificial antigen-presenting cells (aAPCs). These cells are engineered to express chimeric stimulatory receptors (CSRs), and optionally, the cells are modified by knockdown or inactivation of low density lipoprotein receptor (LDLR) expression. Each CSR comprises (i) a T cell co-stimulatory receptor (e.g., in particular, CD3, CD28, OX40, or 4-1BB) or an antibody reagent or natural ligand for a T cell receptor; (ii) a linker domain, and (iii) a transmembrane domain. In one example, aAPCs are engineered to express CSRs to CD3 and CD28. The antibody reagent, linker domain, and transmembrane domain are described elsewhere herein, for example. Cells that may be used to generate aAPCs include, for example, human cells such as erythroid myeloid cells (e.g., K562 cells), myeloid cells, or cells engineered to lack HLA expression or functional HLA. Thus, T cell isolates are expanded by contact with aAPCs described herein (e.g., aAPCs expressing anti-CD28 and anti-CD3 CDRs as described herein) and processed such that one or more CAR constructs of the technology can be introduced, thereby generating CAR T cells. The subject in need thereof may subsequently undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. After or simultaneously with transplantation, the subject can receive an infusion of the expanded CAR T cells. In one embodiment, the expanded cells are administered before or after surgery.

[0181] In some embodiments, lymphocyte depletion is performed in a subject prior to administration of one or more of the modified T cells described herein. In such embodiments, lymphocyte depletion may include administering one or more of melphalan, cyclophosphamide, and fludarabine.

[0182] The dosage of the above-described treatment to be administered to a patient will vary with the condition being treated and the exact nature of the recipient of the treatment. Dosage regimens for human administration can be made according to practices acceptable in the art.

[0183] In some embodiments, a single treatment plan is required. On the other hand, administration of one or more subsequent dosages or treatment plans can be done. For example, after treatment bi-weekly for 3 months, the treatment can be repeated monthly for 6 months or more than a year. In some embodiments, no additional treatment is administered after the initial treatment.

[0184] The dosage of the compositions described herein is determined by a physician and can be adjusted as needed to suit the observed effects of the treatment. With respect to the duration and frequency of treatment, it is typical for a skilled clinician to monitor the subject to determine when the treatment is having a therapeutic effect, to determine whether to administer additional cells, to discontinue treatment, to resume treatment, or to make other changes to the treatment plan. The dosage should not be so large as to cause adverse side effects such as cytokine release syndrome. Generally, the dosage will vary with the age, condition, and gender of the patient and can be determined by one of ordinary skill in the art. The dosage can also be adjusted by the individual physician in the event of any complications.

[0185] Combination Therapy The modified T cells (e.g., activated CAR T cells) described herein can be used in combination with other known agents and therapies. As used herein, “administered in combination” means that two (or more) different treatments are delivered to a subject during the course of the subject's suffering associated with a disorder (e.g., a disease or condition), e.g., two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or the treatment has been discontinued for some other reason. In some embodiments, the delivery of one treatment still occurs when the delivery of the second treatment begins such that there is an overlap with respect to the administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective for the combined administration. For example, the second treatment is more effective than would be observed if administered in the absence of the first treatment, e.g., an equivalent effect is observed with less of the second treatment, or the second treatment reduces symptoms to a greater degree, or a similar situation is observed with respect to the first treatment. In some embodiments, the delivery is such that the reduction in symptoms, or other parameter associated with the disorder, is greater than that observed with one of the treatments delivered in the absence of the other. The effects of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second is delivered. The modified T cells (e.g., activated CAR T cells) described herein and at least one additional therapeutic agent can be administered simultaneously or sequentially, in the same composition or in separate compositions. For sequential administration, the modified T cells (e.g., cells expressing a CAR) described herein can be administered first and the additional agent can be administered second, or the order of administration can be reversed.CAR T therapy and / or other therapeutic agents, methods or modalities can be administered during the period of an active disorder or during remission or a less active disease. Modified T cell therapy can be administered before, concurrently with, after another treatment, or during remission of the disorder.

[0186] When administered in combination, the modified T cells and additional agents (e.g., a second or third agent), or all, can be administered in an amount or dosage that is more, less, or the same as the amount or dosage of each agent used individually, e.g., as monotherapy. In certain embodiments, the amount or dosage of the modified T cells, additional agents (e.g., a second or third agent), or all administered is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually. In other embodiments, the amount or dosage of the modified T cells, additional agents (e.g., a second or third agent), or all that produces a desired effect (e.g., treatment of cancer) is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50% less) than the amount or dosage of each agent required individually to achieve the same therapeutic effect. In further embodiments, the modified T cells described herein can be used in treatment regimens in combination with surgery, chemotherapy, radiation, and immunosuppressive agents such as mTOR pathway inhibitors, cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunosuppressive 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 by Izumoto et al., J Neurosurg, 108:963-971, 2008.

[0187] In one embodiment, the modified T 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-CTLA4 inhibitors (ipilimumab and tremelimumab), anti-PDL1 inhibitors (atezolizumab, avelumab, MSB0010718C, MEDI4736, and MPDL3280A), and anti-TIM3 inhibitors.

[0188] In one embodiment, the modified T cells described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), antimetabolites (e.g., folic acid 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., actinomycin A, gliotoxin, or bortezomib), and immunomodulatory substances such as thalidomide or thalidomide derivatives (e.g., lenalidomide). General chemotherapeutic agents considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomal injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegen), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone,Docetaxel (Taxotere®), Doxorubicin Hydrochloride (Adriamycin®, Rubex®), Etoposide (Vepesid®), Fludarabine Phosphate (Fludara®), 5-Fluorouracil (Adrucil®, Efudex®), Flutamide (Eulexin®), Tiazofurin, Gemcitabine (Difluorodeoxycytidine), Hydroxyurea (Hydrea®), Idarubicin (Idamycin®), Ifosfamide (IFEX®), Irinotecan (Camptosar®), L-Asparaginase (ELSPAR®), Leucovorin Calcium, Melphalan (Alkeran®), 6-Mercaptopurine (Purinethol®), Methotrexate (Folex®), Mitoxantrone (Novantrone®), Myotarg, Paclitaxel (Taxol®), Phoenix (Yttrium90 / MX-DTPA), Pentostatin, Polifeprosan 20 with Carmustine Implant (Gliadel®), Tamoxifen Citrate (Nolvadex®), Teniposide (Vumon®), 6-Thioguanine, Thiotepa, Tirapazamine (Tirazone®), Topotecan Hydrochloride for Injection (Hycamptin®), Vinblastine (Velban®), Vincristine (Oncovin®), and Vinorelbine (Navelbine®). Typical alkylating agents include nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: Uracil Mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®,Uramustine (registered trademark), Chlorambucil (Mustargen (registered trademark)), Cyclophosphamide (Cytoxan (registered trademark), Neosar (registered trademark), Clafen (registered trademark), Endoxan (registered trademark), Procytox (registered trademark), Revimmune (trademark)), Ifosfamide (Mitoxana (registered trademark)), Melphalan (Alkeran (registered trademark)), Chlorambucil (Leukeran (registered trademark)), Pipobroman (Amedel (registered trademark), Vercyte (registered trademark)), Triethylenemelamine (Hemel (registered trademark), Hexalen (registered trademark), Hexastat (registered trademark)), Triethylenethiophosphoramide, Temozolomide (Temodar (registered trademark)), Thiotepa (Thioplex (registered trademark)), Busulfan (Busilvex (registered trademark), Myleran (registered trademark)), Carmustine (BiCNU (registered trademark)), Lomustine (CeeNU (registered trademark)), Streptozocin (Zanosar (registered trademark)), and Dacarbazine (DTIC-Dome (registered trademark)), including but not limited to these. Additional typical alkylating agents are Oxaliplatin (Eloxatin (registered trademark)); Temozolomide (Temodar (registered trademark) and Temodal (registered trademark)); Dactinomycin (also known as Actinomycin-D, Cosmegen (registered trademark)); Melphalan (also known as L-PAM, L-Sarcolysin, and Phenylalanine Mustard, Alkeran (registered trademark)); Altretamine (also known as Hexamethylmelamine (HMM), Hexalen (registered trademark)); Carmustine (BiCNU (registered trademark)); Bendamustine (Treanda (registered trademark)); Busulfan (Busulfex (registered trademark) and Myleran (registered trademark)); Carboplatin (Paraplatin (registered trademark)); Lomustine (also known as CCNU, CeeNU (registered trademark)); Cisplatin (also known as CDDP, Platinol (registered trademark) and Platinol (registered trademark)-AQ); Chlorambucil (Leukeran (registered trademark)); Cyclophosphamide (Cytoxan (registered trademark) and Neosar (registered trademark)); Dacarbazine (DTIC,DTIC-Dome (registered trademark), also known as DIC and imidazole carboxamide; altretamine (registered trademark Hexalen, also known as hexamethylamine (HMM)); ifosfamide (registered trademark Ifex); prednumastine; procarbazine (registered trademark Matulane); mechlorethamine (nitrogen mustard, also known as mustine and mechlorethamine hydrochloride, registered trademark Mustargen); streptozocin (registered trademark Zanosar); thiotepa (registered trademark Thioplex, also known as thiophosphoramide, TESPA and TSPA); cyclophosphamide (registered trademarks Endoxan, Cytoxan, Neosar, Procytox, Revimmune); and bendamustine HCl (registered trademark Treanda), including but not limited to. Typical mTOR inhibitors include, for example, temsirolimus; ridaforolimus (officially known as deforolimus, Also known as AP23573 and MK8669, described in PCT Publication No. 03 / 064383: (lR,2R,45)-4-[(2R)-2[(1R,95,125,15R,16E,18R,19R,21R,235,24E,26E,28Z,305,325,35R)-l,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-ll,36-dioxa-4-azatricyclo[30.3.1.04’9]hexatriaconta-16,24,26,28-tetraene-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate); Everolimus (Afinitor® or RAD001); Rapamycin (AY22989, Sirolimus®); Simapimod (CAS164301-51-3); Msirolimus, (5-{2,4-bis[(35,)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[irau5,-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-JJpyrimidin-7(8H)-one (PF04691502, CAS1013101-36-4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-l-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-a-aspartyl-L-serine, inner salt (SF1126, CAS936487-67-1), and XL765. Typical immunomodulatory substances include, for example, Aflibercept (available from Roche®); Pegfilgrastim (Neulasta®); Lenalidomide (CC-5013, Revlimid®); Thalidomide (Thalomid®), Actimid (CC4047); and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics).Typical anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; rubidomycin; and desacetylrubidomycin. Typical vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®); vinblastine (vinblastine sulfate, vincaleukoblastine and also known as VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). Typical proteasome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, (5)-4-methyl-N-((5)-l-(((5)-4-methyl-l-((R)-2-methyloxirane-2-yl)-l-oxopentan-2-yl)amino)-l-oxo-3-phenylpropan-2-yl)-2-((5,)-2-(2-morpholinoacetamido)-4-phenylbutanamide)-pentanamide); marizomib (NPT0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(llS’)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-l-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0189] One of ordinary skill in the art can readily identify chemotherapeutic agents for use (see, e.g., Physicians' Cancer Chemotherapy Drug Manual, 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Harrison's Principles of Internal Medicine, Chapter 85, Section XVIII; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Abeloff's Clinical Oncology, 2013, Elsevier, Chs. 28-29; and Fischer D S (ed.): The Cancer Chemotherapy Handbook, 4th ed., St. Louis, Mosby-Year Book, 2003).

[0190] In one example, the modified T cells described herein are administered to a subject in combination with a molecule that targets GITR and / or a molecule that modulates the level and / or activity of a molecule that modulates GITR function, a molecule that reduces regulatory T cell populations, an mTOR inhibitor, a GITR agonist, a kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a CDK4 inhibitor, and / or a BTK inhibitor.

[0191] Efficacy For example, in the treatment of the conditions described herein, or for inducing a response (e.g., reduction in cancer cells) described herein, the efficacy of a modified T cell (e.g., an activated CAR T cell) can be determined by a skilled clinician. However, if one or more of the signs or symptoms of the condition described herein are changed in a beneficial manner, or other clinically acceptable symptoms are improved or enhanced, or the desired response is induced, e.g., at least 10% after treatment by the methods described herein, the treatment is considered to be an "effective treatment" as the term is used herein. Efficacy can be evaluated, for example, by measuring markers, indicators, symptoms, and / or incidence of the condition being treated according to the methods described herein, or any other suitable measurable parameter. The methods described herein can reduce the level of a marker or the symptoms of a condition, e.g., 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.

[0192] Efficacy can also be evaluated by hospitalization or measured by the failure of an individual to deteriorate, i.e., the progression of the disease is halted, which is required for medical intervention. Methods for measuring these indicators are known to those of skill in the art and / or are described herein.

[0193] 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 the 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 the treatment of a disease means an amount that, when administered to a subject in need thereof, is sufficient to effect an effective treatment for that disease as the term is defined herein. The effectiveness of a drug can be determined by evaluating physical indicators of the condition or a desired response. Monitoring the effectiveness of administration and / or treatment by measuring any one of such parameters, or any combination of parameters, is well within the ability of one of ordinary skill in the art. The effectiveness of a given approach can be evaluated in an animal model of the condition described herein, e.g., in the treatment of ALL. When using an experimental animal model, the effectiveness of the treatment is demonstrated when a statistically significant change in a marker is observed.

[0194] All patents and other publications, including reference documents, issued patents, published patent applications, and pending patent applications cited through this application, may be used, for example, in connection with the technology described herein and are hereby expressly incorporated by reference into this specification for the purpose of describing and disclosing the methodologies described in such publications. These publications are provided solely for disclosures prior to the filing date of this application. In no way should any of this 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 oaths as to dates or statements as to the content of these documents are based on information available to the applicant and do not constitute an admission as to the accuracy of the dates or the content of these documents.

[0195] The description of embodiments of the present disclosure is not intended to be comprehensive or to limit the disclosure to the exact forms disclosed. Specific embodiments of the disclosure, and examples thereof, are described herein for illustrative purposes, while those skilled in the art will readily recognize that various equivalent modifications are possible within the scope of the disclosure. For example, the steps or functions of a method are presented in a defined order, while alternative embodiments may function in a different order or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein may be applied to other techniques or methods as needed. Combinations of the various embodiments described herein may provide further embodiments. The aspects of the disclosure may be modified, if necessary, to utilize the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the disclosure. Further, due to considerations of biological functional equivalence, some changes may be made to the protein structure without affecting the biological or chemical action in kind or amount. These and other changes may be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0196] Any particular component of any of the foregoing embodiments may be combined with or replaced by components in other embodiments. Further, while the advantages associated with certain embodiments of the disclosure are described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments are necessarily required to exhibit such advantages within the scope of the disclosure.

[0197] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limitations.

Example

[0198] Example 1: Knockout of CD3ζ in Jurkat T cells and primary T cells, and transduction of CD3ζ knockout cells with CAR To achieve gene knockout using CRISPR, various coding sequences of CD3ζ were targeted to develop guides for CRISPR-mediated gene disruption by generating insertions and / or deletions in the target genomic sequence that introduce frameshift mutations in the absence of expression.

[0199] Using CRISPR, CD3ζ or T cell receptor alpha chain (TRAC) was knocked out in Jurkat cells (Figure 1). Two specific guide sequences used for CD3z KO were (i) CAGTTGCCGATTACAGGTA and (ii) GTGGAAGGCGCTTTTCACCG. The resulting cells were analyzed by flow cytometry to show the effect of the knockout on the expression of the CD3 / T cell receptor complex. Both knockouts removed the expression of the CD3 / T cell receptor in these cells, as determined by the use of anti-CD3e antibody 7 days after electroporation (EP) compared to mock electroporated (EP) cells. CRISPR was also used to knock out CD3ζ or TRAC in primary T cells (Figure 2). Flow cytometry analysis using anti-CD3e antibody 8 days after EP / 12 days after stimulation showed that each knockout removed the expression of the CD3 / T cell receptor complex in these cells.

[0200] A T7E1 disruption assay using Streptococcus pyogenes Cas9 (SpCas9) was performed to screen for the percent gene disruption obtained using various gRNAs directed against CD3ζ in Jurkat T cell lines as well as primary human T cells (Figure 3). Maximum disruption was obtained using gRNA(2) directed against the exon / intron 1 site and an additional guide GTGGAAGGCGCTTTTCACCG targeting exon 1 of CD3z.

[0201] Negative selection of CD3e using magnetic beads was used to enrich jackett cells knocked out for CD3ζ by CRISPR using gRNA(2) (Figure 4). T cell receptor knockout was confirmed by staining with antibodies against CD3e and TCRa / b. By negative selection, it was enriched to approximately 50% TCR(-) cells.

[0202] Parental unmodified jackett cells and cells knocked out for CD3ζ using gRNA(2) and enriched by negative selection for CD3e were subjected to single cell sorting (Figure 5). The results obtained using antibodies against TCRa / b and CD3e indicate that T cell receptor expression was removed in these cells.

[0203] After sorting, single cell clones (n = 24) were expanded and evaluated for CD3ζ and CD3e staining (Figure 6). Clone 5, which showed removal of T cell receptor expression compared to the parental control, was selected for use in functional assays.

[0204] Vectors encoding various CARs directed against CD19 using multiple intracellular signaling domains were transduced into parental unmodified cells and cells knocked out for CD3ζ using CRISPR as described above. After transduction, cells were analyzed for T cell receptor expression by CD3e in combination with various CAR constructs (Figure 7).

[0205] T cells transduced with CD19CAR were stimulated overnight with either plate-bound OKT3 (anti-CD3) or Nalm6 (2:1) (Figure 8). NFAT luciferase activation was evaluated. CAR-T cells were specific for hCD19 and were >95% transduced. In the CD3ζ knockout jackett strain, there was a lack of NFAT activation in response to TCR-specific CD3e. However, the CD3ζ knockout jackett strain maintained CAR-specific activation against Nalm6. The experiment was repeated 3 times with N = 3 per condition.

[0206] Example 2: Modification of CD3ζ knockout T cells to reduce rejection A lentiviral vector was constructed for use in the transduction of T cells (Figure 9). Nunchaku (pMGH81) is a construct that expresses three full-length CMV proteins (UL40, US6, and UL18), corn earworm green luciferase (CBG; for use in cell killing assays and in vivo imaging), enhanced green fluorescent protein (EGFP; for downstream fluorescence sorting), and viral 2A proteins (T2a, P2A, E2A, and F2A; to direct cleavage of the encoded polyprotein). The CMV proteins function to promote avoidance of the immune attack of the recipient administered with T cells as described above.

[0207] Ninja (pMGH82) is a construct that expresses a modified anti-human CD19_BBz chimeric antigen receptor, as well as the CMV UL40 CMV viral protein and a signal peptide. The signal peptide, when expressed, loads non-classical HLA-E, which helps inhibit NK cell killing. CMV US6 and UL18 are also included, and mCherry is also included as a reporter gene for transgene expression. As shown above, it includes viral 2A elements (T2A, P2A, and E2A) to direct cleavage of the encoded polyprotein.

[0208] Viruses encoded by the ninja vector were transduced into Jurkat and primary human T cells (Figure 10). The data show that the encoded protein is expressed in the cells. Viruses encoded by the nunchaku vector (pMGH81) were transduced into large tumor cell lines (Figure 11). Transduced gene-positive large cells were labeled with GFP expressed from the construct 2A element. GFP(+) cells decreased the expression of HLA class I protein as shown in the GFP(+), HLA low / negative cell population. Non-transduced cells were used as controls. As discussed above, decreased HLA expression protects allogeneic T cell products from parental rejection after fusion of allogeneic T cell products. Figure 12 shows that sorted large tumor cells expressing pMGH81 decreased HLA class I expression.

[0209] Figure 13 shows the expression of TAP inhibitors (BoHV1 UL49.5, CMV US6, EBV BNLF2a, and HSV ICP47) in primary human T cells and the resulting HLA class I downregulation / knockout. Transduced gene (GFP) positive = HLA class I negative.

[0210] Example 3: Knockout of the cell receptor alpha chain (TRAC) Using a method similar to that described above in relation to CD3ζ, TRAC was targeted. The specific gRNA used for TRAC was AGAGTCTCTCAGCTGGTACA. As shown in Figure 14, knockout of TRAC (AGAGTCTCTCAGCTGGTACA) or CD3ζ (GTGGAAGGCGCTTTTCACCG) in primary T cells 8 days after electroporation / 12 days after stimulation causes knockout of cell surface expression of the TCR.

[0211] Various aspects of the present invention are described in the following numbered paragraphs. 1. An isolated T lymphocyte modified to have reduced or ablated expression of the T cell receptor (TCR) due to reduced or ablated expression of the CD3ζ, T cell receptor alpha chain (TRAC), and / or T cell receptor beta chain (TRBC) gene. 2. An isolated T lymphocyte of paragraph 1, comprising a genome in which the CD3ζ, TRAC, and / or TRBC gene, regulatory sequence, coding sequence, exon, or portion thereof is mutated to result in reduced, null, or non-functional CD3ζ, CD3 eta, CD3 theta, TRAC, and / or TRBC expression. 3. An isolated T lymphocyte of paragraph 2, wherein the mutation is a deletion. The mutation can optionally be a frameshift mutation or a deletion. 4. An isolated T lymphocyte of paragraph 2 or 3, wherein the mutation disrupts the assembly of the T cell receptor or CD3ζ signaling. 5. An isolated T lymphocyte of any one of paragraphs 1 to 4, comprising a genome in which the CD3ζ, TRAC, and / or TRBC gene is deleted. 6. An isolated T lymphocyte of paragraph 5, comprising a genome in which two alleles of the CD3ζ, TRAC, and / or TRBC gene are deleted. 7. An isolated T lymphocyte of any one of paragraphs 1 to 6, wherein the reduced expression of the CD3ζ, TRAC, and / or TRBC gene is null expression. 8. An isolated T lymphocyte of any one of paragraphs 1 to 7, having reduced expression of CD3 eta or CD3 theta. 9. An isolated T lymphocyte of any one of paragraphs 1 to 8, wherein the HLA locus, or a portion thereof, is deleted. 10. An isolated T lymphocyte of paragraph 9, wherein the HLA locus is on chromosome 6. 11. An isolated T lymphocyte of any one of paragraphs 1 to 10, further having reduced HLA class I expression. 12. An isolated T lymphocyte of any one of paragraphs 1 to 11, further modified such that the isolated T lymphocyte expresses HLA-G. 13. An isolated T lymphocyte according to any one of paragraphs 1 to 12, further comprising a gene encoding a heterologous protein that promotes T lymphocytes in avoiding immune attack from a host to which the T lymphocytes are administered. 14. The isolated T lymphocyte of paragraph 13, wherein the heterologous protein promotes avoidance of T cell- or NK-mediated rejection. 15. The isolated T lymphocyte of paragraph 13 or 14, wherein the heterologous protein is a viral protein. 16. The isolated T lymphocyte of paragraph 15, wherein the viral protein is derived from a virus selected from the group consisting of cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), and bovine herpesvirus-1 (BoHV-1). 17. The isolated T lymphocyte of paragraph 16, wherein the viral protein is derived from CMV and is selected from the group consisting of US6, UL40, and UL18. 18. The isolated T lymphocyte of paragraph 16, wherein the viral protein inhibits the transporter associated with antigen processing (TAP). 19. The isolated T lymphocyte of paragraph 18, wherein the viral protein is selected from the group consisting of CMV US6, HSV ICP47, BoHV-1 UL49.5, and EBV BNLF2a. 20. An isolated T lymphocyte according to any one of paragraphs 1 to 19, further comprising a gene encoding a reporter gene. 21. The isolated T lymphocyte of paragraph 20, wherein the reporter gene comprises a truncated epidermal growth factor receptor (EGFR) gene, a truncated prostate-specific membrane antigen (PSMA), a truncated low-affinity nerve growth factor receptor (LNGFR), and a truncated CD19. 22. An isolated T lymphocyte according to any one of paragraphs 1 to 21, further comprising a gene encoding a therapeutic protein. 23. The isolated T lymphocyte of paragraph 22, wherein the therapeutic protein comprises an antigen receptor. 24. The isolated T lymphocyte of paragraph 23, wherein the antigen receptor confers specificity for a selected target antigen. 25. An isolated T lymphocyte according to paragraph 23, wherein the antigen receptor confers specificity for a selected ligand. 26. An isolated T lymphocyte according to paragraph 23, wherein the antigen receptor is a chimeric antigen receptor (CAR). 27. An isolated T lymphocyte according to paragraph 26, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. 28. An isolated T lymphocyte according to paragraph 27, wherein the extracellular domain comprises a single-chain antibody and the intracellular domain comprises a T cell activation domain. 29. An isolated T lymphocyte according to any one of paragraphs 1 to 28, further comprising a gene that induces cell death. 30. An isolated T lymphocyte according to paragraph 29, wherein the gene is an activatable suicide gene. 31. An isolated T lymphocyte according to paragraph 30, wherein the suicide gene is activated by a drug. 32. An isolated T lymphocyte according to paragraph 31, wherein the suicide gene expresses a FK506 binding domain fused to a caspase 9 apoptosis-promoting molecule. 33. A method for generating a modified T lymphocyte, the method comprising inactivating the CD3ζ, TRAC, and / or TRBC genes in the T lymphocyte. 34. The method according to paragraph 33, wherein the inactivation of the CD3ζ, TRAC, and / or TRBC genes is performed using a nuclease or system selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR / Cas9 system). 35. The method according to paragraph 33 or 34, wherein the modified T lymphocyte is a modified T lymphocyte according to any one of paragraphs 1 to 32. 36. A method for treating a subject for a disease, the method comprising administering to the subject an isolated T lymphocyte according to any one of paragraphs 1 to 32. 37. The method according to paragraph 36, wherein the disease is selected from the group consisting of cancer, infectious diseases, and symptoms resulting from transplantation procedures. 38. A method for reducing an immunogenic response in a subject, the method comprising administering to the subject a T lymphocyte as described in any one of paragraphs 1 to 32. 39. The method of paragraph 38, wherein the T lymphocyte expresses a transgene. 40. The method of paragraph 38, wherein the T lymphocyte has a reduced competition with an endogenous T cell receptor signaling molecule. 41. The method of any one of paragraphs 36 to 40, wherein the T lymphocyte is autologous with respect to the subject. 42. The method of any one of paragraphs 36 to 40, wherein the T lymphocyte is allogeneic with respect to the subject. 43. The method of any one of paragraphs 36 to 42, wherein the modified T lymphocyte is expanded in vivo. 44. The method of any one of paragraphs 36 to 43, wherein the modified T lymphocyte is expanded in the blood of the subject. 45. The method of any one of paragraphs 36 to 44, wherein the modified T lymphocyte is expanded in vitro prior to administration. 46. A vector comprising a gene encoding a therapeutic protein and a heterologous protein that promotes immune system evasion. 47. The vector of paragraph 46, wherein the heterologous protein is a viral protein. 48. The vector of paragraph 47, wherein the viral protein is derived from a virus selected from the group consisting of cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), and bovine herpesvirus-1 (BoHV-1). 49. The vector of paragraph 48, wherein the viral protein is derived from CMV and is selected from the group consisting of US6, UL40, and UL18. 50. The vector of paragraph 48, wherein the viral protein inhibits the transporter associated with antigen processing (TAP). 51. The vector of paragraph 50, wherein the viral protein is selected from the group consisting of CMV US6, HSV ICP47, BoHV-1 UL49.5, and EBV BNLF2a. 52. A vector according to any one of paragraphs 46 to 51, wherein the therapeutic protein is a CAR. 53. A method of transducing one or more vectors according to paragraphs 46 to 52 into T lymphocytes. 54. A modified T lymphocyte or cell line, or a subculture thereof, produced by the method according to any one of paragraphs 33 to 35 or 53. 55. A pharmaceutical composition comprising at least one modified T lymphocyte according to any one of paragraphs 1 to 32. 56. A method of treating a subject, comprising: (a) preparing a population of T lymphocytes modified by the method according to any one of paragraphs 33 to 35 or 53; and (b) administering the modified T lymphocytes to the subject. 57. The method of paragraph 56, wherein the T lymphocytes are derived from the subject to be treated. 58. The method of paragraph 56, wherein the T lymphocytes are derived from a healthy donor.

Claims

1. An isolated T lymphocyte modified to have a reduced or eliminated expression of the T cell receptor (TCR) due to a reduced or eliminated expression of the CD3ζ, T cell receptor alpha chain (TRAC), and / or T cell receptor beta chain (TRBC) gene, wherein the isolated T lymphocyte expresses a viral protein selected from the group consisting of: (i) US6, UL40, and UL18, or (ii) BoHV-1 UL49.5 and EBV BNL F2a.

2. The isolated T lymphocyte according to claim 1, wherein the CD3ζ gene, the TRAC gene, and / or the TRBC gene, or at least one regulatory sequence, coding sequence, or exon of the CD3ζ gene, the TRAC gene, and / or the TRBC gene, or at least one portion of the CD3ζ gene, the TRAC gene, and / or the TRBC gene, and / or a portion of the regulatory sequence, coding sequence, or exon is mutated, reduced, null, or non-functional, resulting in reduced CD3ζ, CD3 eta, CD3 theta, TRAC, and / or TRBC expression, and comprising a genome.

3. The isolated T lymphocyte according to claim 2, wherein the mutation is a deletion and / or frameshift mutation, or the mutation disrupts the assembly of the T cell receptor or CD3ζ signal transduction.

4. The isolated T lymphocyte according to claim 1, comprising a genome in which the CD3ζ, TRAC, and / or TRBC gene is deleted.

5. The isolated T lymphocyte according to claim 4, comprising a genome in which two alleles of the CD3ζ, TRAC, and / or TRBC gene are deleted.

6. The isolated T lymphocyte according to claim 1, wherein the reduced expression of the CD3ζ, TRAC, and / or TRBC gene is null expression, or has reduced expression of CD3 eta or CD3 theta.

7. The isolated T lymphocyte according to claim 1, wherein the HLA locus, or a portion thereof, is deleted.

8. The isolated T lymphocyte according to claim 7, wherein the HLA locus is on chromosome 6.

9. The isolated T lymphocyte according to claim 1, further having reduced HLA class I expression, or further modified to express HLA-G.

10. The isolated T lymphocyte according to any one of claims 1 to 9, wherein the viral protein promotes avoidance of T cell- or NK-mediated rejection.

11. The isolated T lymphocyte according to claim 1, further comprising a gene that induces cell death.

12. The isolated T lymphocyte according to claim 1 for use in the treatment of a subject for a disease.

13. A pharmaceutical composition comprising at least one modified T lymphocyte according to any one of claims 1 to 12.

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