CN111826353B - Methods of modulating T cell function and response - Google Patents

Abstract

The present disclosure relates to methods of producing T cells exhibiting an enhanced memory T cell phenotype, the method comprising: the T cell population is modulated to enhance HMGY expression and/or function. In embodiments, the method may include introducing a polynucleotide encoding HMGY into a population of T cells that has higher expression of HMGY than T cells that do not include the polynucleotide, and the memory T cell phenotype of the population of T cells is enhanced as compared to T cells that do not include the polynucleotide. In embodiments, the method may include introducing a polynucleotide encoding one or more genes associated with HMGY, e.g., a gene upstream or downstream of a signaling pathway associated with HMGY and/or a transcription factor associated with HMGY.

Description

Methods of modulating T cell function and response

Technical Field

The present disclosure relates to compositions and methods for expanding and maintaining genetically modified cells, and their use in the treatment of diseases including cancer.

Background

T cells that are genetically targeted to certain malignancies have shown tremendous clinical success. During CAR-T cell therapy, a physician will withdraw the patient's blood and obtain its cytotoxic T cells. Cells were engineered in the laboratory to attack specific cancers. Recent advances in genome editing technology have enabled scientists to regulate gene expression in T cells to enhance effector function or bypass tumor microenvironments where tumor immunosuppression and metabolism are unfavorable. Thus, there is a need to modulate T cells to improve immunotherapy.

Disclosure of Invention

Embodiments relate to a method of producing a T cell exhibiting an enhanced memory T cell phenotype, the method comprising: the T cell population is modulated to enhance HMGY expression and/or function. For example, the method can include introducing a polynucleotide encoding HMGY into a population of T cells that has higher expression of HMGY than T cells that do not include the polynucleotide, and the memory T cell phenotype of the population of T cells is enhanced as compared to T cells that do not include the polynucleotide. In embodiments, the method may include introducing a polynucleotide encoding one or more genes associated with HMGY, e.g., a gene upstream or downstream of a signaling pathway associated with HMGY and/or a transcription factor associated with HMGY.

Some embodiments relate to a method of producing a T cell exhibiting an enhanced memory T cell phenotype, the method comprising: the polynucleotide encoding HMGY is introduced into a population of T cells, wherein expression of HMGY is higher compared to T cells that do not comprise the polynucleotide. The memory T cell phenotype of the T cell population is enhanced compared to T cells that do not comprise the polynucleotide. In embodiments, the level of CD62L and/or CCR7 gene expression is increased as compared to a T cell that does not comprise the polynucleotide. In some embodiments, the method further comprises culturing and measuring expansion of the T cell population. In some embodiments, expansion of the population of T cells is enhanced as compared to T cells that do not comprise the polynucleotide.

This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

Drawings

The specific embodiments are described with reference to the accompanying drawings. The use of the same reference numbers in different figures indicates similar or identical items.

FIG. 1 shows the expression of HMGY in various cells.

FIGS. 2 and 3 are flow cytometry results showing the expression of markers CD62L and CCR7 in various cells.

Figures 4 and 5 are flow cytometry results showing the expression of the markers KLRG and CD137 for various cells.

FIGS. 6 and 7 are flow cytometry results showing the expansion of various cells.

Detailed Description

Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. For purposes of this disclosure, the following terms are defined as follows.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

By "about" is meant that the number, level, value, quantity, frequency, percentage, dimension, size, quantity, weight, or length varies by up to 20%,15%,10%,9%,8%,7%,6%,4%,3%,2%, or 1% relative to a reference number, level, value, quantity, frequency, percentage, dimension, size, quantity, weight, or length.

As used herein, the term "activate" or "activation" refers to the state of a cell that has been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated/activated T cells" particularly refers to T cells that are undergoing cell division.

The term "antibody" is used in its broadest senseBy sense is meant monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function. Antibodies in the present disclosure may exist in a variety of forms, including, for example, polyclonal antibodies; polyclonal antibodies, monoclonal antibodies, fv, fab, fab 'and F (ab') ₂ Fragments; and single chain and humanized antibodies (Harlow et al, 1999,In:Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,NY;Harlow et al, 1989,In:Antibodies:A Laboratory Manual,Cold Spring Harbor,New York;Houston et al; 1988,Proc.Natl.Acad.Sci.USA 85:5879-5883; bird et al, 1988, science 242:423-426).

The term "antibody fragment" refers to a portion of a full-length antibody, e.g., the antigen-binding or variable region of an antibody. Other examples of antibody fragments include Fab, fab ', F (ab') ₂ And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; multispecific antibodies formed from antibody fragments.

The term "Fv" refers to the smallest antibody fragment that contains the complete antigen recognition and binding site. The fragment consists of a dimer of tightly bound, non-covalently bound, one heavy and one light chain variable region domain. By folding of these two domains, six hypervariable loops are created (3 loops from the H chain and 3 loops from the L chain), which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three Complementarity Determining Regions (CDRs) that are specific for an antigen) has the ability to recognize and bind antigen, although with less affinity than the entire binding site (dimer).

As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains that are present in a naturally occurring configuration in all antibody molecules. As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains that are present in a naturally occurring configuration in all antibody molecules. Kappa and lambda light chains refer to two major antibody light chain isotypes.

The term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as an antibody expressed by phage. The term also includes antibodies produced by synthesizing DNA molecules encoding the antibodies and expression of the DNA molecules to obtain the antibodies or to obtain amino acids encoding the antibodies. Synthetic DNA is obtained using techniques available and well known in the art.

The term "antigen" refers to a molecule that elicits an immune response, which may involve antibody production or activation of specific immunocompetent cells, or both. Antigens include any macromolecule, including all proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein or peptide that elicits an immune response, and thus encodes the term "antigen" as used herein. The antigen need not be encoded solely by the full-length nucleotide sequence of the gene. Antigens may be produced, synthesized or derived from biological samples including tissue samples, tumor samples, cells or biological fluids.

As used herein, the term "anti-tumor effect" refers to a biological effect associated with reduced tumor volume, reduced tumor cell number, reduced metastasis number, reduced tumor cell proliferation, reduced tumor cell number. Survival of tumor cells, an increase in life expectancy of a subject with tumor cells, or an improvement in various physiological symptoms associated with a cancer condition. First, the "anti-tumor effect" can also be demonstrated by the ability of peptides, polynucleotides, cells and antibodies to prevent tumorigenesis.

The term "autoantigen" refers to an endogenous antigen that is incorrectly recognized by the immune system as foreign. Autoantigens include cellular proteins, phosphoproteins, cell surface proteins, cell lipids, nucleic acids, glycoproteins, including cell surface receptors.

The term "autologous" is used to describe material from the same subject that is later reintroduced into the subject.

The term "allograft" is used to describe grafts derived from different subjects of the same species. For example, the donor subject may or may not be related to the recipient subject, but the donor subject has an immune system marker similar to the recipient subject.

The term "xenogeneic" is used to describe grafts derived from subjects of different species. For example, a donor subject is from a different species than a recipient subject, and the donor subject and recipient subject may be genetically and immunologically incompatible.

The term "cancer" is used to refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread to other parts of the body locally or through the blood stream and lymphatic system. Examples of the various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The phrase "consisting of" is meant to include and be limited to anything after the phrase "consisting of. Thus, the phrase "consisting of" means that the listed elements are necessary or mandatory and that no other elements may be present.

The phrase "consisting essentially of" is meant to include any element listed after the phrase, and may include other elements that do not interfere with or contribute to the activities or actions specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present, depending on whether they affect the activity or effect of the listed elements.

The terms "complementary" and "complementarity" refer to polynucleotides (i.e., nucleotide sequences) related by the base pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA". Complementarity may be "portions" in which only certain nucleobases are matched according to the base pairing rules, or there may be "complete" or "full" complementarity between "nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

The term "corresponding to" or "corresponding to" refers to (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or that encodes an amino acid sequence that is identical to an amino acid sequence in a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence substantially identical to the amino acid sequence in the reference peptide or protein.

The term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal in addition to the primary signal provided by the binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule that mediates T cell responses including at least one of proliferation, activation, differentiation and other cellular responses. Co-stimulatory ligands may include B7-1 (CD 80), B7-2 (CD 86), PD-L1, PD-L2,4-1BBL, OX40L, an inducible co-stimulatory ligand (ICOS-L), an intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, ligands for CD7, agonists or antibodies that bind Toll ligand receptors and ligands that specifically bind to B7-H3. Costimulatory ligands include, inter alia, agonists or antibodies that specifically bind to costimulatory molecules present on T cells, such as CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specifically bind CD 83.

The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response, such as proliferation, of the T cell. Costimulatory molecules include MHC class I molecules, BTLA and Toll-like receptors.

The term "costimulatory signal" refers to a signal that, in combination with a primary signal (e.g., TCR/CD3 linkage), results in up-or down-regulation of T cell proliferation and/or a key molecule.

The terms "disease" and "condition" may be used interchangeably or may be different in that a particular disease or condition may not have a known causative agent (and therefore the cause has not been resolved) and thus has not been recognized as a disease, but merely as an adverse condition or syndrome, wherein a clinician has established a more or less specific set of symptoms. The term "disease" is a state of health of a subject, wherein the subject is unable to maintain homeostasis, and wherein the subject's health continues to deteriorate if the disease is not improved. In contrast, a "disorder" in a subject is a state of health in which an animal is able to maintain homeostasis, but in which the animal's state of health is less than in the absence of the disorder. If untreated, the disease does not necessarily lead to a further decline in the health of the animal.

The term "effective" means sufficient to achieve a desired, expected, or intended result. For example, an "effective amount" in a treatment may be an amount of a compound sufficient to produce a therapeutic or prophylactic benefit.

The term "coding" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, to be used as a template for the synthesis of other polymers and macromolecules having a particular structure in a biological process. Nucleotide sequences (i.e., rRNA, tRNA and mRNA) or defined amino acid sequences, and biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. Coding strands whose nucleotide sequences are identical to the mRNA sequences (except for the replacement of "T" with "U") are generally provided in the sequence listing, and "proteins" in which non-coding strands serve as templates for transcription of a gene or cDNA may be referred to as proteins or other products encoding the gene or cDNA.

The term "exogenous" refers to a molecule that does not occur naturally in a wild-type cell or organism but is typically introduced into the cell by molecular biology techniques. Examples of exogenous polynucleotides include vectors, plasmids, and/or artificial nucleic acid constructs encoding the desired protein. With respect to polynucleotides and proteins, the term "endogenous" or "native" refers to naturally occurring polynucleotide or amino acid sequences that can be found in a given wild-type cell or organism. Furthermore, a particular polynucleotide sequence that is isolated from a first organism and transferred to a second organism by molecular biological techniques is generally considered to be an "exogenous" polynucleotide or amino acid sequence with respect to the second organism. In particular embodiments, polynucleotide sequences may be "introduced" by molecular biological techniques into microorganisms that already contain such polynucleotide sequences, for example, to produce one or more additional copies of additional naturally occurring polynucleotide sequences, and thereby facilitate overexpression of the encoded polypeptide.

The term "expression or overexpression" refers to the transcription and/or translation of a particular nucleotide sequence into a precursor or mature protein, e.g., driven by its promoter. By "overexpression" is meant that the yield of a gene product in a transgenic organism or cell exceeds that in a normal or non-transformed organism or cell. As defined herein, the term "expression" refers to expression or overexpression.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control (regulatory) sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

Viruses can be used to deliver nucleic acids into cells in vitro and in vivo (in a subject). Examples of viruses that can be used to deliver nucleic acids into cells include retroviruses, adenoviruses, herpes simplex viruses, vaccinia viruses, and adeno-associated viruses.

Non-viral methods for delivering nucleic acids into cells also exist, such as electroporation, gene gun, acoustic electroporation, magnetic transfection, and the use of oligonucleotides, lipid complexes, dendrimers and inorganic nanoparticles.

The term "homologous" refers to sequence similarity or sequence identity between two polypeptides or between two polynucleotides when positions in two compared sequences are occupied by the same base or amino acid monomer subunit, e.g., if positions in each of the two polypeptides. The DNA molecule is occupied by adenine and then the molecule is homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, if 6 of the 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. When two sequences are aligned, they are compared to yield the greatest homology.

The term "immunoglobulin" or "Ig" refers to a class of proteins that function as antibodies. Five members included in this class of proteins are IgA, igG, igM, igD and IgE. IgA is a primary antibody present in secretions in the body, such as saliva, tears, breast milk, gastrointestinal secretions and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the primary immunoglobulin produced by most subjects in the primary immune response. It is the most potent immunoglobulin in agglutination, complement fixation and other antibody reactions, and is important in protecting against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by releasing mediators from mast cells and basophils upon exposure to allergens.

The term "isolated" refers to a material that is substantially or essentially free of components that normally accompany the material in its natural state. The material may be a cell or a macromolecule, such as a protein or a nucleic acid. For example, an "isolated polynucleotide" as used herein refers to a polynucleotide that has been purified from flanking sequences in a naturally-occurring state, such as a DNA fragment that has been removed from a normally normal sequence, adjacent to the fragment. Alternatively, "isolated peptide" or "isolated polypeptide" and the like as used herein refers to the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, as well as from other components of the cell.

The term "substantially purified" refers to a material that is substantially free of components normally associated with its natural state. For example, a substantially purified cell refers to a cell that has been isolated from other cell types with which it is normally associated in its naturally occurring or native state. In some cases, a substantially purified cell population refers to a homogenous cell population. In other cases, the term refers only to cells that have been isolated from cells naturally associated in nature. In embodiments, the cells are cultured in vitro. In embodiments, the cells are not cultured in vitro.

In the context of the present disclosure, the following abbreviations for the ubiquitous nucleobases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding a protein may comprise the intron(s) in some versions.

The term "lentivirus" refers to a genus of the retrovirus family. Lentiviruses are unique among retroviruses in being able to infect non-dividing cells. They can transfer a large amount of genetic information into the DNA of host cells, and thus they are one of the most effective methods in gene transfer vectors. In addition, the use of lentiviruses enables integration of genetic information into host chromosomes, thereby stably transducing the genetic information. HIV, SIV and FIV are all examples of lentiviruses. Lentiviral derived vectors provide a means to achieve significant levels of gene transfer in vivo.

The term "modulate" refers to a detectable increase or decrease in the level of a response in a subject compared to the level of a response in a subject in the absence of a treatment or compound, and/or compared to the level of a response in an otherwise identical but untreated subject. The term includes interfering with and/or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if the DNA of the pre-sequence or secretion leader is expressed as a pre-protein involved in the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site is positioned for translation.

The term "under transcriptional control" refers to a promoter operably linked to a polynucleotide and in the correct position and orientation relative to the polynucleotide to control (regulate) the initiation of transcription of an RNA polymerase and the expression of the polynucleotide.

The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to mean an abnormal level of expression of a tumor antigen in cells from a disease area, such as a solid tumor within a specific tissue or organ associated with a patient. Reaching the expression level in normal cells of the tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

Solid tumors are abnormal masses of tissue that typically do not contain cysts or areas of fluid. Solid tumors may be benign or malignant. Different types of solid tumors are named for the cell types that they form (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid cancer, papillary thyroid cancer, pheochromocytoma sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchus carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma and central nervous system tumors (such as brain stem glioma and hybrid glioma), glioblastoma (also known as glioblastoma multiforme), central nervous system lymphoma, adenoma, neuroblastoma, schwannoma, angioma, angioblastoma, and neuroblastoma.

The solid tumor antigen is an antigen expressed on a solid tumor. In embodiments, the solid tumor antigen is also expressed at low levels on healthy tissue. Examples of solid tumor antigens and their associated disease tumors are provided in table 1.

Table 1

The term "parenteral administration" of a composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intrasternal injection or infusion techniques.

The terms "patient," "subject," and "individual" and the like are used interchangeably herein and refer to any person or animal suitable for use in the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human or animal. In embodiments, the term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response may be elicited. Examples of subjects include humans and animals, such as dogs, cats, mice, rats, and transgenic species thereof.

A subject in need of treatment or in need thereof includes a subject suffering from a disease, disorder or condition in need of treatment. Subjects in need thereof also include subjects in need of treatment to prevent a disease, disorder, or condition.

The term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term generally refers to polymeric forms of nucleotides, ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide that are at least 10 bases in length. The term includes all forms of nucleic acid, including single-stranded and double-stranded forms of nucleic acid.

The terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity with a reference polynucleotide sequence or that hybridize to a reference sequence under stringent conditions as defined below. These terms also include polynucleotides that differ from the reference polynucleotide by the addition, deletion, or substitution of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted or replaced with a different nucleotide. In this regard, it is well known in the art that certain alterations, including mutations, additions, deletions and substitutions, may be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide or has increased activity (i.e., optimized) relative to the reference polynucleotide. The polynucleotide variants described herein include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages, e.g., 90%,95% or 98%) sequence identity to a reference polynucleotide sequence. The terms "polynucleotide variants" and "variants" also include naturally occurring allelic variants and orthologs.

The terms "polypeptide", "polypeptide fragment", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. In certain aspects, a polypeptide may include an enzymatic polypeptide or "enzyme" that generally catalyzes (i.e., increases the rate of) various chemical reactions.

The term "polypeptide variant" refers to a polypeptide that is distinguished from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In certain embodiments, the polypeptide variants are distinguished from the reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, polypeptide variants comprise conservative substitutions, and in this regard, it is well known in the art that certain amino acids may be changed to amino acids having broadly similar properties without altering the nature of the polypeptide activity. Polypeptide variants also include polypeptides in which one or more amino acids have been added or deleted or replaced with a different amino acid residue.

The term "promoter" refers to a DNA sequence recognized by a cellular synthesis machinery or an introduced synthesis machinery required to initiate specific transcription of a polynucleotide sequence. The term "expression control (regulatory) sequence" refers to a DNA sequence necessary for expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optional operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

The term "bind" or "interact with" refers to a molecule that recognizes and binds to a second molecule in a sample or organism but does not substantially recognize or bind to other structurally unrelated molecules in the sample. The term "specifically binds" as used herein with respect to an antibody refers to an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in the sample. For example, an antibody that specifically binds an antigen from one species may also bind antigens from one or more species. However, this cross-species reactivity does not itself alter the specific classification of antibodies. In another example, antibodies that specifically bind to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity does not itself change the classification of antibodies to specificity. In some cases, the term "specific binding" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that the interaction depends on a particular structure (e.g., an epitope or epitope) on the chemical species; for example, antibodies recognize and bind to a particular protein structure but not any protein. If the antibody is specific for epitope "A", the presence of the molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in the reaction containing labeled "A" and antibody.

By "statistically significant" is meant that the result is unlikely to happen by chance. Statistical significance may be determined by any method known in the art. Common important metrics include a p-value, which is the frequency or probability of an observed event occurring if the null hypothesis is true. If the obtained p-value is smaller than the significance level, the null hypothesis is rejected. In a simple case, the significance level is defined as a p value of 0.05 or less. The "reducing" or "weakening" amount is typically a "statistically significant" or physiologically significant amount and may include about 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2,2.5,3,3.5,4,4.5,5,6,7,8,9,10,15,20,30,40 or 50 or more times (e.g., 100,500,1000) (including all integers and decimal points between 1 and 1, e.g., 1.5,1.6,1.7.1.8, etc.) the amount or level reduction described herein.

The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as signaling via the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as the down regulation of TGF- β and/or recombination of cytoskeletal structures.

The term "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. For example, the functional signaling domain derived from a stimulatory molecule is a zeta chain associated with the T cell receptor complex. The stimulatory molecules include domains responsible for signal transduction.

The term "stimulatory ligand" refers to a ligand that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a cell, such as a T cell, thereby mediating a primary response of the T cell, including activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well known in the art and include, inter alia, MHC class I molecules loaded with peptides, anti-CD 3 antibodies, super agonist anti-CD 28 antibodies and super agonist anti-CD 2 antibodies.

The term "therapeutic" refers to treatment and/or prophylaxis. Therapeutic effects may be obtained by inhibiting, alleviating or eliminating the disease state or alleviating symptoms of the disease state.

The term "therapeutically effective amount" refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the development of or to alleviate to some extent one or more symptoms or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound of the subject to be treated, the disease and its severity and age, weight, etc.

The term "treating a disease" refers to reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. Cells include the primary test cells and their progeny.

The term "vector" refers to a polynucleotide that comprises an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term also includes non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like. For example, lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env. Lentiviral vectors are well known in the art. Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2 and simian immunodeficiency virus: SIV. Lentiviral vectors are generated by attenuating HIV virulence genes multiple times, e.g., genes env, vif, vpr, vpu and nef are deleted, making the vector biologically safe.

The range is as follows: throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have all possible subranges as specifically disclosed, as well as individual numerical values within that range. For example, descriptions of ranges such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual values within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

A "chimeric antigen receptor" (CAR) molecule is a recombinant polypeptide comprising at least an extracellular domain, a transmembrane domain, and a cytoplasmic domain or an intracellular domain. In embodiments, the domains of the CAR are on the same polypeptide chain, e.g., a chimeric fusion protein. In embodiments, the domains are on different polypeptide chains, e.g., the domains are discontinuous.

The extracellular domain of the CAR molecule includes an antigen binding domain. The antigen binding domain is used to expand and/or maintain modified cells, such as CAR T cells, or to kill tumor cells, such as solid tumors. In embodiments, the antigen binding domain used to amplify and/or maintain the modified cells binds to an antigen on the surface of WBCs, such as a cell surface molecule or a marker. In embodiments, the WBCs are granulocytes, monocytes and/or lymphocytes. In embodiments, the WBCs are lymphocytes, such as B cells. In some embodiments, the WBCs are B cells. In some embodiments, the cell surface molecule of the B cell comprises CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11B, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. In some embodiments, the cell surface molecule of the B cell is CD19, CD20, CD22 or BCMA. In some embodiments, the cell surface molecule of the B cell is CD19.

Modified cells (e.g., CAR cells and T cells) can be derived from stem cells. The stem cells may be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. The modified cells may also be dendritic cells, NK cells, B cells or T cells selected from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes. In another embodiment, the modified cells may be derived from cd4+ T lymphocytes and cd8+ T lymphocytes. Prior to expansion and genetic modification of the cells of the invention, a cell source may be obtained from a subject by a variety of non-limiting methods. T cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the invention, any number of T cell lines known to those of skill in the art may be used. In embodiments, the modified cells may be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with infection. In embodiments, the modified cells are part of a mixed population of cells exhibiting different phenotypic characteristics.

A cell population refers to a group of two or more cells. The cells of the population may be identical, such that the population is a homogenous population of cells. The cells of a population may be different such that the population is a mixed population or heterogeneous population of cells. For example, the mixed population of cells can include a modified cell comprising a first CAR and a cell comprising a second CAR, wherein the first CAR and the second CAR bind different antigens.

The term "stem cells" refers to certain cells that have the ability to self-renew and differentiate into other cell types. For example, a stem cell may produce two daughter stem cells (e.g., embryonic stem cells cultured in vitro) or one stem cell and cells undergoing differentiation (e.g., hematopoietic stem cells, up to blood cells). Different classes of stem cells can be distinguished based on their origin and/or their ability to differentiate into other types of cells. For example, stem cells may include Embryonic Stem (ES) cells (i.e., pluripotent stem cells), somatic stem cells, induced pluripotent stem cells, and any other type of stem cells.

Pluripotent embryonic stem cells exist in the inner cell mass of the blastocyst and have an innate differentiation capacity. For example, pluripotent embryonic stem cells may form any type of cell in vivo. When grown in vitro for prolonged periods, ES cells remain pluripotent because daughter cells retain the potential for multilineage differentiation.

Somatic stem cells may include fetal stem cells (from the fetus) and adult stem cells (present in various tissues, such as bone marrow). These cells are believed to have a lower capacity for differentiation than pluripotent ES cells-the capacity of fetal stem cells is greater than that of adult stem cells; they obviously differentiate into a limited range of cells and are described as pluripotent stem cells. "tissue-specific" stem cells generally produce only one type of cell. For example, embryonic stem cells can differentiate into blood stem cells (e.g., hematopoietic Stem Cells (HSCs)), which can further differentiate into various blood cells (e.g., erythrocytes, platelets, leukocytes, etc.).

The induced pluripotent stem cells (i.e., iPS cells or ipscs) may include one that is artificially derived from non-pluripotent cells (e.g., adult somatic cells) by inducing expression of specific genes. Induced pluripotent stem cells are similar in many ways to natural pluripotent stem cells, such as Embryonic Stem (ES) cells, e.g., expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling times, embryoid body formation, teratoma formation, viable chimerism formation, and potency and differentiation. The induced pluripotent cells may be made from adult stomach, liver, skin cells and blood cells.

In embodiments, the antigen binding domain for killing a tumor binds an antigen on the surface of the tumor, e.g., a tumor antigen or tumor marker. Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly T cell mediated immune responses. Tumor antigens are well known in the art and include, for example, tumor-associated MUC1 (tMUC 1), glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotrophin, alpha Fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyesterase, mut hsp70-2, M-CSF, prostanase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostatein, PSMA, her2/neu, survivin, telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin Growth Factor (IGF) -I, IGF-II, IGF-I receptor, CD19 and mesothelin. For example, when the tumor antigen is CD19, its CAR may be referred to as a CD19CAR or 19CAR, which is a CAR molecule comprising an antigen binding domain that binds CD 19.

In embodiments, the extracellular antigen-binding domain of the CAR comprises at least one scFv or at least a single domain antibody. For example, there may be two scfvs on the CAR. The scFv comprises a light chain variable region (VL) and a heavy chain variable region (VH) of a target antigen-specific monoclonal antibody linked by a flexible linker. Single chain variable region fragments can be prepared by ligating light and/or heavy chain variable regions using short connecting peptides (Bird et al, science 242:423-426, 1988). An example of a linker peptide is a GS linker with the amino acid sequence (GGGGS) 3 bridging between the carboxy terminus of one variable region and the amino terminus of the other variable region by about 3.5nm. Other sequence linkers have been designed and used (Bird et al, 1988, supra). In general, the linker may be a short flexible polypeptide and preferably comprises about 20 or fewer amino acid residues. Single-chain variants may be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer may be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv may be introduced into a suitable host cell, which is eukaryotic, such as a yeast, plant, insect or mammalian cell, or prokaryotic, such as E.coli. Polynucleotides encoding the scFv of interest can be prepared by conventional procedures such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

The cytoplasmic domains of the CAR molecules described herein include one or more co-stimulatory domains and one or more signaling domains. The co-stimulatory and signaling domains are used to transmit signals and activate molecules, such as T cells, in response to antigen binding. One or more co-stimulatory domains is/are derived from the stimulatory molecule and/or co-stimulatory molecule, and the signaling domain is derived from a primary signaling domain, e.g. a cd3ζ domain. In embodiments, the signaling domain further comprises one or more functional signaling domains derived from a co-stimulatory molecule. In embodiments, the costimulatory molecule is a cell surface molecule (other than the antigen receptor or ligand thereof) required to activate a cellular response to an antigen.

In embodiments, the costimulatory domain comprises the intracellular domain of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7 LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, or any combination thereof. In embodiments, the signaling domain comprises a cd3ζ domain derived from a T cell receptor.

The CAR molecules described herein also include a transmembrane domain. Incorporation of the transmembrane domain in the CAR molecule stabilizes the molecule. In embodiments, the transmembrane domain of the CAR molecule is the transmembrane domain of a CD28 or 4-1BB molecule.

Between the extracellular domain and the transmembrane domain of the CAR, a spacer domain may be incorporated. As used herein, the term "spacer domain" generally refers to any oligomer or polypeptide used to attach a transmembrane domain to an extracellular domain and/or cytoplasmic domain on a polypeptide chain. The spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids.

In embodiments, the modified cell comprises a binding molecule, which is a CAR. In embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain binds to a tumor antigen. In embodiments, the intracellular domain comprising a costimulatory domain comprises an intracellular domain selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated costimulatory molecules, antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof. In embodiments, the intracellular domain comprises a CD3 zeta signaling domain. In embodiments, the CAR is a bispecific CAR or a Tan CAR.

In embodiments, the binding molecule is a TCR. In embodiments, the T cell comprises a modified T Cell Receptor (TCR). In embodiments, the TCR is derived from a tumor-specific T cell that occurs spontaneously in the patient. In embodiments, the TCR binds a tumor antigen. In embodiments, the tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1. In embodiments, the TCRs comprise TCR gamma and TCR delta chains or TCR alpha and TCR beta chains, or a combination thereof.

In embodiments, the modified cells are derived from Tumor Infiltrating Lymphocytes (TILs). In embodiments, T cell clones expressing TCRs with high affinity for the target antigen may be isolated. Tumor Infiltrating Lymphocytes (TILs) or Peripheral Blood Mononuclear Cells (PBMCs) can be cultured in the presence of Antigen Presenting Cells (APCs) loaded with polypeptides that represent epitopes known to be useful in eliciting a dominant T cell response that is a response when present in the context of a particular HLA allele; high affinity clones can then be selected based on MHC-peptide tetramer staining and/or the ability to recognize and lyse target cells loaded with cognate peptide antigen at low titers. After selection of clones, TCR alpha and TCR beta chains or TCR gamma and TCR delta chains were identified and isolated by molecular cloning. For example, for TCR α and TCR β chains, the TCR α and TCR β gene sequences are then used to generate expression constructs that desirably promote stable, high level expression of both TCR chains in human TC cells. Transduction vectors, such as gamma retrovirus or lentivirus, can then be generated and tested for functionality (antigen specificity and functional affinity) and used to produce clinically significant quantities of the vector. An aliquot of the final product may then be used to transduce a target T cell population (typically purified from patient PBMCs) and then expanded prior to infusion into a patient.

Various methods can be performed to obtain a gene encoding a tumor-reactive TCR. More information was found in Kershaw et al, clin Transl Immunology, 5, 2014; 3 (5): e 16. In embodiments, the specific TCR may be derived from tumor-specific T cells that spontaneously occur in the patient. Antigens included in this class include the melanocyte differentiation antigens MART-1 and gp100, as well as the MAGE antigen and NY-ESO-1, which are expressed in a wider range of cancers. TCR specific for virus-related malignancies can also be isolated as long as the viral proteins are expressed by the transformed cells. Such malignancies include liver and cervical cancers associated with hepatitis and papilloma viruses and malignancies associated with epstein-barr virus. In embodiments, target antigens for TCRs may include CEA (e.g., for colorectal cancer), gp100, MART-1, p53 (e.g., for melanoma), MAGE-A3 (e.g., melanoma, esophageal and synovial sarcoma), NY-ESO-1 (e.g., for melanoma and sarcoma, and multiple myeloma).

In embodiments, the preparation and transfusion of tumor-infiltrating lymphocytes (TILs) may be performed in the following manner. For example, tumor tissue from a surgical or biopsy specimen may be obtained under sterile conditions and transported to a cell culture chamber in a refrigerator. Necrotic tissue and adipose tissue may be removed. Tumor tissue may be cut into small pieces of about 1-3 cubic millimeters. Collagenase, hyaluronidase and dnase may be added and digested overnight at 4 ℃. The cells were isolated and collected by centrifugation of lymphocyte isolates at 1500rpm for 5 minutes by filtration through a 0.2um filter. Cells can be expanded in medium containing PHA, 2-mercaptoethanol and CD3 monoclonal antibodies, and small doses of IL-2 (10-20 IU/ml) can be added to induce activation and proliferation. Cell density can be carefully measured and at 37℃5% CO ₂ At a temperature of 0.5 to 2x10 ⁶ The concentration of the catalyst is kept in the range of/ml for 7-14 days. TIL positive cells with the ability to kill homologous cancer cells can be selected by co-culture. TIL positive cells can be expanded in serum-free medium containing high doses of IL-2 (5000-6000 IU/ml) until greater than 1X10 is obtained ¹¹ Is not limited to TIL. For TIL administration, it was first collected in saline using continuous flow centrifugation, then filtered through platelet administration to a volume of 200-300ml of IL-2 containing 5% albumin and 450000 IU. TIL may be injected into the patient through the central venous catheter over a period of 30-60 minutes. In embodiments, the TIL may be infused into two to four separate bags, and each infusion may be separated by several hours.

A bispecific CAR (or tandem CAR (tanCAR)) may comprise two binding domains: scFv1 and scFv2. In embodiments, scFv1 binds to an antigen of a leukocyte (e.g., CD 19), and scFv2 binds to a solid tumor antigen (e.g., tMUC 1). In embodiments, scFv1 binds to a solid tumor antigen and scFv2 binds to another solid tumor antigen (e.g., tMUC1 and CLDN 18.2). Claudin18.2 (CLDN 18.2) is a stomach-specific isomer of Claudin-18. CLDN 18.2 is highly expressed in stomach and pancreatic adenocarcinomas. In embodiments, scFv1 binds to an antigen (e.g., tMUC 1) that is expressed on tumor cells but not on normal tissue; scFv2 binds to an antigen expressed on non-essential tissues associated with solid tumors, and killing of normal cells of the tissues does not cause life threatening events (e.g., complications) to the subject (e.g., TSHR, GUCY 2C). Examples of non-essential tissues include organs such as the prostate, breast or melanocytes. In embodiments, scFv1 and scFv2 bind to different antigens expressed on the same non-essential tissue (e.g., ACPP and SLC45A3 for prostate cancer, SIGLEC15 and UPK2 for urinary tract cancer). Table 2 lists the sequences of bispecific CARs and their compositions.

TABLE 2

/>

Note that: 3 (GGGGS) is (GGGGS) ₃ 4 (GGGGS) is (GGGGS) ₄ .

Furthermore, the invention describes modified cells comprising the nucleic acids or vectors described herein. Cells have been introduced with the nucleic acids or vectors described herein and express at least one or more different antigen binding domains. In embodiments, the cell expresses an antigen binding domain. In embodiments, the cell comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain binds to a cell surface molecule of the WBC and the second antigen binding domain binds to a different antigen than the cell surface molecule of the WBC. In embodiments, the second antigen binding domain binds a tumor antigen. In embodiments, the cell is a modified T cell. In embodiments, the modified T cell is a CAR T cell comprising one or more nucleic acids encoding the first antigen binding domain and/or the second antigen binding domain. In embodiments, the modified cell comprises a T cell comprising a TCR comprising a second antigen-binding domain.

The methods described herein relate to lymphocytes expressing an amplification molecule and a functional molecule. In embodiments, the amplification molecule amplifies and/or maintains lymphocytes in the subject, and the functional molecule inhibits growth of or kills tumor cells in the subject. In embodiments, the amplification molecule and the functional molecule are on the same CAR molecule, e.g., a bispecific CAR molecule. In embodiments, the amplification molecule and the functional molecule are on separate molecules, e.g., CAR and TCR or two different CARs. The amplification molecule can include a CAR that binds to an antigen associated with blood (e.g., blood cells and plasma) or non-essential tissue, and the functional molecule can include a CAR or TCR that targets an antigen associated with tumor cells.

Lymphocyte or T cell response in a subject refers to cell-mediated immunity associated with helper, killer, regulatory and other types of T cells. For example, T cell responses may include activities such as assisting other WBCs in the immune process as well as identifying and destroying virus-infected cells and tumor cells. T cell responses in a subject can be measured by a variety of indicators, such as a number of virus-infected cells and/or tumor cells killed by the T cells, the amount of cytokines released when the T cells are co-cultured with the virus-infected cells and/or tumor cells, the level of proliferation of the T cells in the subject, such as a change in the phenotype of the T cells, a change in memory T cells, and the life span or lifespan level of the T cells in the subject.

In embodiments, the method of enhancing a T cell response comprises treating a subject in need thereof, e.g., a subject diagnosed with a tumor. The term tumor refers to a tumor, which may be a collection of fluids such as blood, or a solid mass. Tumors may be malignant (cancerous) or benign. Examples of hematological cancers include chronic lymphocytic leukemia, acute myelogenous leukemia, acute lymphocytic leukemia, and multiple myeloma.

Solid tumors typically do not contain cysts or areas of fluid. The major types of malignant solid tumors include sarcomas and carcinomas. Sarcomas are tumors that develop in soft tissue cells called mesenchymal cells, which can be found in blood vessels, bones, adipose tissue, ligament lymphatic vessels, nerves, cartilage, muscles, ligaments or tendons, while carcinomas are tumors that form in epithelial cells, which can be found in skin and mucous membranes. The most common types of sarcomas include undifferentiated polymorphous sarcomas, involving soft tissues and bone cells. Smooth muscle sarcomas, which involve smooth muscle cells lining the blood vessels, gastrointestinal tract and uterus; osteosarcoma involving bone cells and liposarcoma involving adipocytes. Some examples of sarcomas include ewing's sarcoma, rhabdomyosarcoma, chondrosarcoma, mesothelioma, fibrosarcoma, and glioma.

Five of the most common cancers include adrenal cancer, which involves organs that produce fluid or mucus, such as the breast and prostate. Basal cell carcinoma, involving the outermost cells of the skin, such as skin carcinoma; squamous cell carcinoma, involving basal cells of the skin; transitional cell carcinoma affects transitional cells in the urinary tract including the bladder, kidneys and ureters. Examples of the cancer include thyroid cancer, breast cancer, prostate cancer, lung cancer, intestinal cancer, skin cancer, pancreatic cancer, liver cancer, kidney cancer and bladder cancer, and bile duct cancer.

The methods described herein can be used to treat a subject diagnosed with cancer. The cancer may be a blood cancer or may be a solid tumor, such as a sarcoma or carcinoma. The method of treatment comprises administering to the subject an effective amount of T cells comprising a first antigen binding domain that binds to a cell surface molecule of WBCs and a second antigen binding domain that binds to an antigen that is different from the cell surface molecule of WBCs to provide a T cell response. In embodiments, enhancing a T cell response in a subject comprises selectively enhancing proliferation of T cells expressing a first antigen binding domain and a second antigen binding domain in vivo.

The present disclosure describes pharmaceutical compositions. The pharmaceutical composition comprises one or more of the following: CAR molecules, TCR molecules, modified CAR T cells, modified cells comprising a CAR or TCR, a mixed population of modified cells, nucleic acids, and vectors described herein. The pharmaceutical composition is administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

The term "pharmaceutically acceptable" refers to the pharmacopoeias approved by a regulatory agency or EMA (european medicines agency) of the federal or state government or listed in the U.S. pharmacopoeia (U.S. pharmacopoeia-33/national formulary-28 reissue, U.S. pharmacopoeia convention, rockville Md., date of publication: month 4 2010) or other generally accepted pharmacopoeias for animals, particularly humans.

The term "carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient or vehicle with which the therapeutic agent is administered. The pharmaceutical carrier may be a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solutions as well as aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

The present disclosure also describes pharmaceutical compositions comprising the first and second cell populations described herein. The pharmaceutical compositions described herein are suitable for cancer treatment, comprising a first population of cells having a first antigen binding molecule and a second population of cells comprising a second antigen binding domain. For example, binding of the first antigen binding molecule to the antigen enhances expansion of cells suitable for cancer treatment.

When "immunologically effective amount", "antitumor effective amount", "tumor inhibiting effective amount" or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered is determined by a doctor in consideration of the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition (subject) of the patient. It can be said that the pharmaceutical composition comprising the T cells described herein can be 10 ⁴ To 10 ⁹ The individual cells/kg body weight are dosed, preferably at 10 ⁵ To 10 ⁶ Individual cells/kg body weight, including all integer values within those ranges. The modified cell composition may also be administered in multiple doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Med.319:1676, 1988). By monitoring patient symptoms and adjusting the treatment accordingly, one of skill in the medical arts can readily determine the optimal dosage and treatment regimen for a particular patient. In certain embodiments, it may be desirable to administer activated T cells to a subject, then draw blood (or collect blood), collect the activated and expanded T cells, and inject the activated and expanded T cells into the patient. This process may be performed several times per several weeks. In certain embodiments, the T cells may be activated from 10cc to 400cc of blood draw. In certain embodiments, the T cells are activated from 20cc,30cc,40cc,50cc,60cc,70cc,80cc,90cc or 100cc of blood withdrawal. Without being bound by theory, certain T cell populations may be selected using this multiple blood draw/multiple reinfusion protocol.

Administration of the pharmaceutical compositions described herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient by intravenous (iv) injection or subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, transdermal (iv) injection. In embodiments, the modified cell compositions described herein are administered to a subject by intradermal or subcutaneous injection. In embodiments, the T cell compositions of the invention are administered by intravenous injection. The composition of the modified cells may be injected directly into a tumor, lymph node or infection site. In embodiments, cells activated and expanded using the methods described herein or other methods known in the art of expanding T cells to therapeutic levels are co-administered to a patient (e.g., before, simultaneously or after) in combination (e.g., before, simultaneously or after), e.g., as a combination therapy, including, but not limited to, treatment with antiviral therapies, cidofovir and interleukin 2, cytarabine (also known as ARA-C); or natalizumab treatment of MS patients; patients with psoriasis use efalizumab therapy or PML patients use other therapies. In further embodiments, T cells described herein may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies or other immune depleting agents such as CAM PATH, anti-CD 3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. . These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporin and FK 506) or inhibit p70S6 kinase (rapamycin) important for growth factor-induced signaling. (Liu et al, cell 66:807-815, 1991; henderson et al, immun 73:316-321, 1991; bierer et al, curr. Opin. Immun 5:763-773, 1993; isoniemi (supra)). In embodiments, the cell compositions described herein are administered to a subject in combination (e.g., before, concurrently with, or after) with bone marrow transplantation, T cell ablation therapy using any chemotherapeutic agent, such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies, such as OKT3 or CAMPATH. In embodiments, the cell compositions described herein are administered after B cell ablation therapy. For example, an agent that reacts with CD20, such as rituximab, may be administered to a patient. In embodiments, the subject may receive standard treatment for high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present disclosure. In embodiments, the expanded cells are administered before or after surgery. The dosage of the above treatments administered to a subject in need thereof will vary with the exact nature of the disease being treated and the treatment recipient. The dosage for human administration may be scaled by a physician according to a variety of factors and practices recognized in the art. Additional information regarding methods of cancer treatment using modified cells is provided in U.S. patent No. US8,906,682, which is incorporated by reference in its entirety.

Embodiments described herein relate to in vitro methods for preparing modified cells. The method may comprise obtaining a cell sample from a subject. For example, the sample may comprise T cells or T cell progenitors. The method may further comprise transfecting the cell sample with DNA encoding at least one CAR, and culturing the cell sample ex vivo in a medium that selectively enhances proliferation of T cells expressing the CAR. The cell sample may be a mixed population of modified cells as described herein.

In embodiments, the sample is a cryopreserved sample. In embodiments, the cell sample is from umbilical cord blood or a peripheral blood sample from the subject. In embodiments, the cell sample is obtained by apheresis or venipuncture. In embodiments, the cell sample is a subpopulation of T cells.

Embodiments relate to modified cells engineered to express an antigen binding molecule, wherein the expression and/or function of one or more genes in the modified cells has been enhanced. In embodiments, the one or more genes comprise BATF, HMGA1, STAT5A, ZNF580, GLMP, JAZF1, RUNX1, ZGPAT, ZNF511, GTF2IRD2B, ATF4, MBD4, TBPL1, GTF2B, RBCK1, ZBTB38, PIN1, DRAP1, HSF1, PRDM1, ZNF428, NFYC, and ZNF706. In embodiments, one or more genes is HMGA1 and/or ZBTB38.

Some embodiments relate to modified cells engineered to express an antigen binding molecule, wherein the expression and/or function of one or more genes in the modified cells has been reduced or eliminated. In embodiments, the one or more genes comprise GTF3A, JUN, IRF1, JUNB, TMF1, ELF1, AKNA, BCL11B, KLF2, ZNF292, RORA, HMGN3, KDM2A, ASCL2, SP140L, NFATC2, RUNX3, NFE2L2, KLF6, MTERF4, PHF20, RELB, MAZ, ARID A, REL, ZEB2, ARID5B, KLF3, CREM, ZNF207, IRF7, DR1, SP140, BBX, MECP2, STAT4, ZBTB1, CREBZF, NFATC3, GPBP1, IKZF1, SON, ZNF800, STAT6, CGGBP1, FOXN2, DNATC 1, KM100, GAMT 3, EOMES, YY1, SP110, SAFB, REST, NR3C1, FON 3, BRF 7, DR1, SP140, BBX, MECP2, STAT4, ZBTF 1, CREBZF 3, NFATC3, NFF 1, XK 1, KXZNF 1, ZTK 1, ZNF148, CDC5L, CREB1, HBP1, ZNF721, KAT7, SP4, ZC3H8, AKAP8L, ZNF326, ZNF451, ZNF131, CEBPZ, TOPORS, ZNF a, ncap 3, STAT2, DDIT3, ZNF217, KLF9, CSRNP1, ncaa 1, SAFB2, ZNF107, ZFX, E2F4, HIF1A, ZNF480, CTCF, ZBTB44, NCOA2, ZHX1, ZNF644, ASH1L, STAT5B, AEBP2, MYSM1, ZNF91, CEBPB, MXD4, YBX, RLF, JUND, ZNF600, SMAD4, TET2, ZNF267, PRDM2, ZBTB7A, THAP12, ETV3, nf2, KLF13, sant 1, ZNF791, 35507, fox 2, fox 8, irk 8, thin 1L, STAT4, AEBP2, MYSM1, ZNF4, and at least one of skf 1, ZNF4, and skf 1, n 4. In embodiments, one or more genes is AKNA.

In embodiments, overexpression of HMGA1 can increase T cell expansion and inhibit T cell death and differentiation. Meanwhile, HMGA1 may promote T cells to secrete IL2 and release ifnγ. HMGA1 may inhibit autophagy and enhance mitochondrial function, thereby promoting phosphorylation and providing more energy to T cells. Thus, overexpression of this gene can enhance the function of CAR-T. In embodiments, a decrease in AKNA gene expression may promote release of immune cytokines and enhance inflammatory responses and enhance the killing capacity of CAR-T.

In embodiments, overexpression of one or more genes in a modified cell can be achieved by introducing a polynucleotide encoding the one or more genes. In embodiments, overexpression of one or more genes in the modified cell can be modulated by a transcriptional regulator that is or includes Hif1a, NFAT, FOXP3, and/or NFkB. A promoter comprising a binding site for one or more NFAT responsive elements (e.g., NFAT1, NFAT2, NFAT3, and/or NFAT 4). "NFAT promoter" refers to one or more NFAT responsive elements linked to the minimal promoter of one or more genes expressed by T cells. In embodiments, the minimal promoter of a gene expressed by a T cell is the minimal human IL-2 promoter. The NFAT response element may include, for example, NFAT1, NFAT2, NFAT3, and/or NFAT4 response elements. The NFAT promoter (or functional portion or functional variant thereof) can comprise any number of binding motifs, such as at least two, at least three, at least four, at least five or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs. In embodiments, the NFAT promoter comprises six NFAT binding motifs. In a particularly preferred embodiment, the NFAT promoter nucleotide sequence comprises SEQ ID NO:63 or a functional part or functional variant thereof. The NFAT promoter (or a functional portion or functional variant thereof) is operably linked to a nucleotide sequence encoding one or more genes (or a functional portion or functional variant thereof). By "operably bound to" is meant that when the NFAT protein binds to the NFAT promoter sequence (or functional portion thereof), a nucleotide sequence encoding one or more genes (or functional portions or functional variants thereof) is transcribed into one or more gene mrnas. Without being bound by a particular theory, NFAT is believed to be regulated by a calcium signaling pathway. In particular, TCR stimulation (e.g., by an antigen) and/or stimulation of the cellular calcium signaling pathway (e.g., by PMA/ionomycin) is thought to increase intracellular calcium concentration and activate calcium channels. The NFAT protein is then dephosphorylated by calmodulin and transferred into the nucleus where it binds to the NFAT promoter sequence (or a functional portion or functional variant thereof) and activates downstream gene expression. By providing an NFAT promoter (or functional part or functional variant thereof) operably linked to a nucleotide sequence encoding one or more genes (or functional parts or functional variants thereof), the nucleic acids of the invention advantageously make it possible to express one or more genes (or functional parts or functional variants thereof) only when a host cell comprising the nucleic acid is stimulated, for example by PMA/inomycin and/or antigen. Can be found in U.S. patent nos.: 8,556,882, which is incorporated by reference.

In embodiments, the antigen binding molecule is a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In embodiments, the antigen binding domain that binds to a tumor antigen is selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1,CD33,EGFRvIII,GD2,GD3,BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her 2/neu), MUC1, EGFR, NCAM, protease, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, upk2, havcr1, adrb3, panx3, gpr20, ly6k, or51e2, tarp, wt1, NY-ESO-1, age-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17,XAGE1,Tie 2,MAD-CT-1, MAD-CT-2, fos associated antigen 1, p53, p53 mutants, proline, survivin and telomerase, PCTA-1/Galectin 8, melana/MART1, ras mutants, hTERT, SAR coma translocation breakpoints, ML-IAP, ERG (TMPRSS 2 ETS fusion genes), NA17, PAX3, androgen receptor, cyclin B1, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, enterocarboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1 in embodiments, the intracellular signaling domain comprises ase:Sub>A costimulatory signaling domain, or ase:Sub>A primary signaling domain and ase:Sub>A costimulatory signaling domain, wherein the costimulatory signaling domain comprises ase:Sub>A signaling domain selected from the group consisting of CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, one and CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF 1), CD160, CD19, CD4, CD8 alphase:Sub>A, CD8 betase:Sub>A, IL2R betase:Sub>A, IL2 Rgammase:Sub>A, IL7R alphase:Sub>A, ITGA4, VLA1, CD49 ase:Sub>A, ITGA4, IA4, the functional signaling domain of the ligand binding protein of CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11 ase:Sub>A, LFA-1, ITGAM, CD11B, ITGAX, CD11C, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (tactile), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLASME (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, p30, NKG 46 and NKG 2D.

In embodiments, the antigen binding molecule is a modified TCR. In embodiments, the TCR is derived from a tumor-specific T cell that occurs spontaneously in the patient. In embodiments, the TCR binds a tumor antigen. In embodiments, the tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1. In embodiments, the TCRs comprise TCR gamma and TCR delta chains or TCR alpha and TCR beta chains, or a combination thereof.

In embodiments, the cell is an immune effector cell (e.g., a population of immune effector cells). In embodiments, the immune effector cell is a T cell or NK cell. In embodiments, the immune effector cell is a T cell. In embodiments, the T cells are cd4+ T cells, cd8+ T cells, or a combination thereof. In embodiments, the cell is a human cell.

In embodiments, the modified cell comprises an inhibitor of the expression or function of one or more genes. In embodiments, the inhibitor is (1) a gene editing system that targets one or more sites within a gene encoding one or more genes or corresponding regulatory elements; (2) Nucleic acids encoding one or more components of a gene editing system of one or more genes; or (3) combinations thereof.

Some embodiments relate to pharmaceutical compositions comprising the cell populations described above. Some embodiments relate to a method of eliciting a T cell response and/or treating a tumor in a subject in need thereof, the method comprising administering an effective amount of the composition.

Embodiments relate to a method of producing a T cell exhibiting an enhanced memory T cell phenotype, the method comprising: the T cell population is modulated to enhance HMGY expression and/or function. For example, the method can comprise introducing a polynucleotide encoding HMGY into a population of T cells, wherein HMGY is expressed more than a T cell that does not comprise the polynucleotide, and the memory T cell phenotype of the population of T cells is enhanced as compared to a T cell that does not comprise the polynucleotide. In embodiments, the method may include introducing a polynucleotide encoding one or more genes associated with HMGY, e.g., upstream or downstream of a signaling pathway associated with HMGY and/or a transcription factor associated with HMGY.

Embodiments relate to a method of producing a T cell exhibiting an enhanced memory T cell phenotype, the method comprising: the polynucleotide encoding HMGY is introduced into a population of T cells, wherein HMGY is expressed more than a T cell comprising no polynucleotide and wherein the memory T cell phenotype of the population of T cells is enhanced as compared to a T cell comprising no polynucleotide.

In embodiments, a population of T cells exhibiting increased levels of gene expression in CD62L and/or CCR7 as compared to T cells not comprising the polynucleotide.

In some embodiments, the method further comprises culturing the population; and measuring cell expansion of the T cell population. In embodiments, expansion of the T cell population is enhanced as compared to T cells that do not comprise the polynucleotide.

In embodiments, the polynucleotide comprises SEQ ID NO:61, and HMGY is overexpressed.

The method further comprises contacting the population of T cells with an antigen to which the population of T cells binds. In embodiments, a population of T cells exhibiting reduced levels of gene expression in CD137 and/or KLRG as compared to T cells not comprising the polynucleotide.

In embodiments, the enhanced memory T cell phenotype comprises an increase in the level of gene expression in CD62L and/or CCR 7. In embodiments, the enhanced memory T cell phenotype includes reduced levels of gene expression in CD137 and/or KLRG.

As used herein, the term "memory T cells" or TCM refers to a subpopulation or subpopulation of T cells that express higher levels of genes associated with trafficking to secondary lymphoid organs including CD62L and/or CCR 7. In embodiments, the memory T cells express lower levels of genes, including CD137 and/or KLRG.

HMGY, HMGA1 or HMG-I/Y are used interchangeably and refer to a small groove-like structure that preferentially binds to the A+T rich region of double stranded DNA. It was shown that these proteins could play a role in phasing of nucleosomes and processing of the 3' end of mRNA transcription. They are also involved in transcriptional regulation of genes containing or in close proximity to the a+t-rich region. Three known members of the High Mobility Group (HMG) mammalian non-histone nucleoprotein HMGI (Y) family (HMG-1, HMG-Y and HMGI-C) are thought to be involved in many biological processes (transcription, replication, retroviral integration, genetic recombination, etc.) because of their ability to recognize and alter DNA and chromatin substrate structures. More information about HMGY can be found in U.S. patent publication No.: found in US2015315589, which is incorporated herein by reference.

In embodiments, the T cell population may comprise antigen binding molecules. In embodiments, the cell is a human cell.

In embodiments, the antigen binding molecule is a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In embodiments, the antigen binding domain that binds to a tumor antigen is selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1,CD33,EGFRvIII,GD2,GD3,BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her 2/neu), MUC1, EGFR, NCAM, protease, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephA2, fucosyl GM1, sLe, GM3, TGS5, MAA, O-acetyl GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, poly-acid, PLAC1, gloH, NY-BR-1, UPKK 2, VCADK 1, VCR-abl 3, PAGE 1, R-four, R-1, W-R-Y-1, W-Y-R-Y-W1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos-associated antigen 1, p53, p53 mutant, proline, survivin and telomerase, PCTA-1/Galectin 8, melana/MART1, ras mutant, hTERT, SAR coma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyesterase, mut hsp70-2, CD79a, CD79B, 72, CDIR1, AR, CDLILF 2, FCLR 2, FCRLC 1, and FCRLC 2. In embodiments, the intracellular signaling domain comprises ase:Sub>A costimulatory signaling domain, or ase:Sub>A primary signaling domain and ase:Sub>A costimulatory signaling domain, wherein the costimulatory signaling domain comprises ase:Sub>A signaling domain selected from the group consisting of CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT), SLAMF7, NKp80 (KLRF 1), CD160, CD19, CD4, CD8 alphase:Sub>A, CD8 betase:Sub>A, IL2R betase:Sub>A, IL2R gammase:Sub>A, IL7R alphase:Sub>A, ITGA4, VLA1, CD49 ase:Sub>A, specific signaling domains of ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11 ase:Sub>A, LFA-1, ITGAM, CD11B, ITGAX, CD11C, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244,2B 4), CD84, CD96 (tactile), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BL AME (SLAMF 8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKG 30 and NKG 2D. .

In embodiments, the antigen binding molecule is a modified TCR. In embodiments, the TCR is derived from a tumor-specific T cell that occurs spontaneously in the patient. In embodiments, the TCR binds a tumor antigen. In embodiments, the tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1. In embodiments, the TCRs comprise TCR gamma and TCR delta chains or TCR alpha and TCR beta chains, or a combination thereof.

The present disclosure is further described by reference to the following exemplary embodiments and examples. These exemplary embodiments and examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present disclosure should in no way be construed as limited to the following exemplary embodiments and examples, but rather should be construed to encompass any and all variations that become evident as a result of the teachings provided herein.

Exemplary embodiments of the invention

The following are exemplary embodiments:

1. a modified cell engineered to express an antigen binding molecule, wherein expression and/or function of one or more genes in the modified cell has been enhanced.

2. The modified cell of embodiment 1, wherein the one or more genes comprise BATF, HMGA1, STAT5A, ZNF580, GLMP, JAZF1, RUNX1, ZGPAT, ZNF511, GTF2IRD2B, ATF4, MBD4, TBPL1, GTF2B, RBCK1, ZBTB38, PIN1, DRAP1, THYN1, HSF1, PRDM1, ZNF428, NFYC, and ZNF706.

3. The modified cell of embodiment 1, wherein the one or more genes is HMGA1 and/or ZBTB38.

4. A modified cell engineered to express an antigen binding molecule, wherein the expression and/or function of one or more genes in the modified cell has been reduced or eliminated.

5. The modified cell of example 4, wherein the one or more genes comprise GTF3A, JUN, IRF1, JUNB, TMF1, ELF1, AKNA, BCL11B, KLF2, ZNF292, RORA, HMGN3, KDM2A, ASCL2, SP140L, NFATC2, RUNX3, NFE2L2, KLF6, MTERF4, PHF20, 625A, REL, ZEB2, ARID5B, KLF3, CREM, ZNF207, IRF7, DR1, SP140, BBX, MECP2, STAT4, ZBTB1, CREBZF, NFATC3, GPBP1 IKZF1, SON, ZNF800, STAT3, STAT6, CGGBP1, FOXN2, DNMT1, SP100, GATA3, EOMES, YY1, SP110, SAFB, REST, NR3C1, FOXN3, ELF2, GTF2I, BAZ2A, ZNF683, STAT1, BHLHE40, ZNF276, ETS1, NFAT5, BPTF, KMT2A, FOS, PA2G4, IKZF3, ZNF148, CDC5L, CREB1, HBP1, ZNF721, KAT7, SP4, ZC3H8, AKAP8L, ZNF326, ZNF451, ZNF131, CEBPZ, TOPORS, ZNF a, ncao 3, STAT2, DDIT3, ZNF217, KLF9, CSRNP1, NCOA1, SAFB2, ZNF107, ZFX, E2F4, HIF1A, ZNF480, CTCF, ZBTB44, nca 2, hx1, ZNF644, ASH1L, STAT5B, AEBP2, MYSM1, ZNF91, CEBPB, MXD4, YBX, RLF, jd, ZNF600, SMAD4, TET2, ZNF 7A, PRDM2, znb 7A, THAP12, ETV3, nff 13, SAFB 1, ZNF13, ZNF 35, ZNF1, ZNF 35, ZNF4, ZNF1, ZNF4, and ZNF1, ZNF 4.

6. The modified cell of embodiment 4, wherein the one or more genes is AKNA.

7. The modified cell of one of embodiments 1-6, wherein the antigen binding molecule is a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

8. The modified cell of embodiment 7, wherein the antigen binding domain binds to a tumor antigen selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1,CD33,EGFRvIII,GD2,GD3,BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her 2/neu), MUC1, EGFR, NCAM, protease, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17,XAGE1,Tie 2,MAD-CT-1, MAD-CT-2, fos associated antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, melana/MART1, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CDLF 300, ECA, 12, GLE 2, and GLFCLB 3, and GLC 1, GL 2, and GLC 1.

9. A modified cell of one of embodiments 7 and 8, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain, or a primary signaling domain and a co-stimulatory signaling domain, wherein the co-stimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of: CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF 1), CD160, CD19, CD4, CD8 alphase:Sub>A, CD8 betase:Sub>A, IL2R betase:Sub>A, IL2 Rgammase:Sub>A, IL7R alphase:Sub>A, ITGA4, VLA1, CD49 ase:Sub>A, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11 ase:Sub>A, LFA-1, ITGAM, CD11B, ITGAX, CD11C, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (Tatile), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLASME (SLAMF 8), SEL (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, p46 and G2D.

10. The modified cell of any one of embodiments 1-6, wherein the antigen binding molecule is a modified TCR.

11. The modified cell of embodiment 10, wherein the TCR is derived from a tumor-specific T cell that occurs spontaneously in the patient.

12. The modified cell of embodiment 10, wherein the TCR binds a tumor antigen.

13. The modified cell of embodiment 12, wherein the tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1.

14. The modified cell of embodiment 10, wherein the TCR comprises a TCR γ and TCR δ chain or a TCR α and TCR β chain, or a combination thereof.

15. The modified cell of any one of the preceding embodiments, wherein the cell is an immune effector cell (e.g., a population of immune effector cells).

16. The modified cell of embodiment 15, wherein the immune effector cell is a T cell or NK cell.

17. The modified cell of embodiment 15, wherein the immune effector cell is a T cell.

18. The modified cell of embodiment 15, wherein the T cell is a cd4+ T cell, a cd8+ T cell, or a combination thereof.

19. The modified cell of any one of the preceding embodiments, wherein the cell is a human cell.

20. The modified cell of any one of the preceding embodiments, wherein the modified cell comprises an inhibitor of expression or function of one or more genes.

21. The modified cell of embodiment 20, wherein the inhibitor is (1) a gene editing system that targets one or more sites in the gene encoding the one or more genes or corresponding regulatory elements; (2) Nucleic acids encoding one or more components of a gene editing system of one or more genes; or (3) combinations thereof.

22. A pharmaceutical composition comprising the population of cells of any one of embodiments 1-21.

23. A method of eliciting a T cell response and/or treating a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of embodiment 22.

24. A method of modulating T cell activity, the method comprising: the polynucleotide encoding HMGY is introduced into a T cell population.

25. A method of producing a T cell exhibiting an enhanced memory T cell phenotype, the method comprising: the polynucleotide encoding HMGY is introduced into a population of T cells compared to T cells that do not comprise the polynucleotide.

26. A method of producing a T cell exhibiting a reduced level of activation and/or reduced level of differentiation in the presence of an antigen to which the T cell binds, the method comprising: the polynucleotide encoding HMGY is introduced into a population of T cells compared to T cells that do not comprise the polynucleotide.

27. A method of enhancing T cell expansion in response to the presence of a T cell-bound antigen, the method comprising: the polynucleotide encoding HMGY is introduced into a T cell population.

28. A method of producing a T cell, the method comprising enhancing HMGY gene expression and/or function of the T cell as compared to a T cell that does not comprise enhanced HMGY gene expression and/or function.

29. The method of any one of the preceding embodiments, wherein the population of T cells that exhibit increased gene expression in CD62L and/or CCR7 as compared to T cells that do not comprise the polynucleotide or enhanced HMGY gene expression and/or function.

30. The method of any one of the preceding embodiments, wherein the population of T cells exhibits reduced gene expression in CD137 and/or KLRG as compared to T cells that do not comprise the polynucleotide or enhanced HMGY gene expression and/or function.

31. The method of any one of the preceding embodiments, further comprising: culturing a population of T cells; and measuring cell expansion of the T cell population.

32. The method of any one of the preceding embodiments, further comprising: contacting the population of T cells with an antigen to which the population of T cells binds.

33. The method of any suitable embodiment of the foregoing, wherein said enhanced memory T cell phenotype comprises reduced gene expression in CD137 and/or KLRG, or said enhanced memory T cell phenotype comprises increased gene expression in CD62L and/or CCR 7.

34. A population of T cells produced using the method of any of the preceding embodiments.

35. A modified cell engineered to express an antigen binding molecule, wherein expression and/or function of one or more genes in the modified cell has been enhanced.

36. The modified cell of embodiment 35, wherein the one or more genes is HMGA1 and/or ZBTB38 (SEQ ID NO: 62).

37. The modified cell of embodiment 35, wherein the modified cell exhibits increased gene expression in CD62L and/or CCR7 as compared to a cell that does not comprise increased HMGY gene expression and/or function.

38. The modified cell of embodiment 35, wherein the modified cell exhibits reduced gene expression in CD137 and/or KLRG as compared to a cell that does not comprise enhanced HMGY gene expression and/or function.

39. The method or modified cell of any of the preceding embodiments, wherein the modified cell or population of T cells is engineered to express an antigen binding molecule.

40. The modified cell or T cell population of any of the preceding embodiments, wherein the antigen binding molecule is a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

41. The modified cell or T cell population of embodiment 40, wherein the antigen binding domain binds to a tumor antigen selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, cll-1,CD33,EGFRvIII,GD2,GD3,BCMA,Tn Ag,PSMA,ROR1,FLT3,FAP,TAG72,CD38,CD44v6,CEA,EPCAM,B7H3,KIT,IL-13Ra2, mesothelin, IL-11Ra, psca, prss21, vegfr2, lewis y, CD24, pdgfr-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her 2/neu), MUC1, EGFR, NCAM, prosase, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephA2, fucosyl GM1, sLe, GM3, TGS5, mahmwa, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, cx61, CD97, CD179a, polysialic acid, ac1, gloh, NY-BR-1, kbr-2, upv 1, rbra-2, rbra 3, rbra 1, rbra-2, MAGE 1, MAGE-5, MAGE 1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAG 1, tie2, MAD-CT-1, MAD-CT-2, fos associated antigen 1, p53, p53 mutant, proline, survivin and telomerase, PCTA-1/Galectin 8, melana/MART1, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, rhoC, TRP-2, CYP1 GE, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAFCFCGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, CDAR 2, CDLRA 2, 300LRA, CYP1 GE1, BORRLYE 2, SSX 1, and RGFCLF 5.

42. The modified cell or T cell population of embodiment 40 of 19, wherein the intracellular signaling domain comprises ase:Sub>A costimulatory signaling domain, or ase:Sub>A primary signaling domain and ase:Sub>A costimulatory signaling domain, wherein the costimulatory signaling domain comprises ase:Sub>A signaling domain selected from the group consisting of CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, icos, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ase:Sub>A signaling domain that is complementary to CD83, CDs, ICAM-1, gitr, baffr, hvem (LIGHT), SLAMF7, NKp80 (KLRF 1), CD160, CD19, CD4, CD8 alphase:Sub>A, CD8 betase:Sub>A, IL2R betase:Sub>A, IL2 rgammase:Sub>A, IL7 ralphase:Sub>A, the binding domain of proteins specific to the NKp signal domain of ITGA4, VLA1, CD49 ase:Sub>A, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11 ase:Sub>A, LFA-1, ITGAM, CD11B, ITGAX, CD11C, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (Tactile), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SE MA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLASME (SLAMF 8), SELLPG (CD 162), SLC62-76 bp, PAG 44 and NKp, NKp 46.

43. The modified cell or T cell population of any suitable preceding embodiment, wherein the antigen binding molecule is a modified TCR.

44. The modified cell or T cell population of embodiment 43, wherein the TCR is derived from a tumor-specific T cell that occurs spontaneously in the patient.

45. The modified cell or T cell population of embodiment 43, wherein the TCR binds a tumor antigen.

46. The modified cell or T cell population of embodiment 45, wherein the tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3, or NY-ESO-1.

47. The modified cell or T cell population of embodiment 43, wherein the TCR comprises a TCR γ and TCR δ chain or a TCR α and TCR β chain, or a combination thereof.

48. The modified cell of any one of the preceding embodiments, wherein the modified cell is an immune effector cell (e.g., a population of immune effector cells).

49. The modified cell of embodiment 48, wherein the immune effector cell is a T cell or NK cell.

50. The modified cell of embodiment 49, wherein the immune effector cell is a T cell.

51. The modified cell of embodiment 49, wherein the T cell is a cd4+ T cell, a cd8+ T cell, or a combination thereof.

52. The modified cell or T cell population of any one of the preceding embodiments, wherein the cell is a human cell.

53. The modified cell or T cell population of the preceding embodiments, wherein the modified cell comprises an inhibitor of expression or function of one or more genes.

54. The modified cell or T cell population of embodiment 53, wherein the inhibitor is (1) a gene editing system targeted to one or more sites within a gene encoding the one or more genes or corresponding regulatory elements; (2) Nucleic acids encoding one or more components of a gene editing system of one or more genes; or (3) combinations thereof.

55. A pharmaceutical composition comprising the population of cells of any one of the preceding embodiments.

56. A method of administering a therapeutic agent, the method comprising administering to a subject an effective amount of the composition of embodiment 55, or a method of eliciting or eliciting a T cell response and/or treating a tumor in a subject in need thereof. The method comprises administering to the subject an effective amount of the composition of embodiment 55.

57. The modified cell, method, pharmaceutical composition, cell of any of embodiments 24-56, wherein the one or more polynucleotides are present in the modified cell as a recombinant DNA construct, mRNA, or viral vector.

58. The modified cell, method, pharmaceutical composition, cell of embodiment 57, wherein said nucleic acid sequence is an mRNA that is not integrated into the genome of said modified cell.

59. The modified cell, method, pharmaceutical composition, cell of one of embodiments 56-58, wherein the one or more polynucleotides are associated with an oxygen-sensitive polypeptide domain.

60. The modified cell, method, pharmaceutical composition, cell of embodiment 59, wherein said oxygen-sensitive polypeptide domain comprises a HIF VHL binding domain.

61. The modified cell, method, pharmaceutical composition, cell of one of embodiments 56-60, wherein expression of one or more polynucleotides is regulated by a promoter comprising a binding site for a transcriptional regulator that regulates the expression and/or secretion of a therapeutic agent in the cell.

62. The modified cell, method, pharmaceutical composition, cell of embodiment 61, wherein said transcriptional regulator is or comprises hif1α, NFAT, FOXP3 and/or NFkB.

63. The modified cell of any one of preceding embodiments (24-62), wherein expression of the one or more polynucleotides is modulated by NFAT such that the Extracellular Vesicles (EVs) assemble in response to activation of the modified cell.

64. A polynucleotide comprising a binding site for a transcriptional regulator (e.g., NFAT) and encoding one or more proteins that assemble an Extracellular Vesicle (EV) and a therapeutic agent.

65. The modified cell, method, pharmaceutical composition, cell of any of embodiments 24-64, wherein the one or more proteins are self-assembling proteins.

66. The modified cell, method, pharmaceutical composition, cell of any of embodiments 24-65, wherein the one or more proteins that direct their release through vesicles are luminal-binding proteins selected from the group consisting of: retroviral group specific antigen, retroviral group specific antigen variation, influenza M1 protein, ARRDC1 protein, ARC protein, ebola virus VP40 protein and vesicular stomatitis virus M protein.

67. The modified cell, method, pharmaceutical composition, cell of any of embodiments 24-66, wherein the one or more proteins comprise an Arc protein and the one or more polynucleotides comprise a nucleic acid encoding a therapeutic agent.

68. An EV comprising an Arc protein and a nucleic acid encoding or comprising a therapeutic agent, said nucleic acid being DNA or RNA encoding a therapeutic agent.

69. The modified cell, method, pharmaceutical composition, cell or EV of any one of embodiments 67 and 68, wherein said therapeutic agent is selected from the group consisting of siRNA, shRNA and RNAi.

70. The modified cell, method, pharmaceutical composition, cell or EV of any one of embodiments 67 and 68 wherein the nucleic acid encoding the therapeutic agent is linked to a 3' utr sequence.

71. The modified cell, method, pharmaceutical composition, cell or EV of embodiment 70 wherein the 3' utr sequence binds to the Arc protein.

72. The modified cell, method, pharmaceutical composition, cell or EV of embodiment 71 wherein the 3'utr sequence is an antisense mRNA 3' utr sequence.

73. The modified cell, method, pharmaceutical composition, cell or EV of any one of embodiments 67 and 72 wherein said nucleic acid further comprises a transcriptional regulator sequence.

74. The modified cell, method, pharmaceutical composition, cell or EV of any one of embodiments 24-73, wherein the therapeutic agent is a tumor antigen to which the scFv binds on or within the membrane of a tumor cell.

75. The modified cell, method, pharmaceutical composition, cell or EV of embodiment 74 wherein the tumor antigen is at least one tumor antigen of the embodiments listed in the present disclosure.

Examples of the invention

Expression of CAR and modified PD-1 on Primary T cells

Primary T cells are obtained from the patient. Primary T cells obtained were transduced with lentiviral vectors. Flow cytometry was performed and analyzed to determine the expression of CARs and various modified PD-1 in primary T cells. U.S. patent No. US9,572,837 (U.S. patent assigned to Innovative Cellular Therapeutics co., ltd.) provides techniques related to cell culture, construction of lentiviral vectors, flow cytometry, and other related techniques, which are incorporated by reference in their entirety. The sequences listed in this disclosure can be found in table 3. More information on the sequences listed in this disclosure can be found in PCT patent publication No.: WO2020106843, WO2019140100 and PCT patent application nos.: PCT/US20/13099, incorporated herein by reference.

Table 3 sequence listing

Expression of HMGY in CAR T cells

FIG. 1 shows the expression of HMGY in various cells. DAY0 was obtained from peripheral blood of healthy volunteers, and cd3+ T cells were sorted using pan T Kit, and stimulated with 100ul of tranact per 1000 ten thousand T cells. DAY1 infected T cells, 6922 (CAR-h 19-28 z) (sequence 64) infected 4×10 according to mio=20.79 ⁶ T cells, 7413 (H19-28 z-2 a-HMGY) (SEQ ID NO: 65) were infected with 4X 10 according to MOI=60.03 ⁶ T cells, leave 6X 10 ⁶ T cells act as NTs. DAY2 was changed, lentiviruses were removed, tranact was removed, and T cells were resuspended in fresh medium. DAY7 flow assay for CAR ratio and cell phenotype, human CAR antibodies were detected since both vectors were humanized antibodies. As shown in FIG. 1, CAR-H19-28z shares 27.49% hCAR expression, H19-28z-2a-HMGY shares19.89% hCAR expression, leveling according to 19.89% CAR after detection, performing experiments according to Table 5, co-culturing for 24 hours to obtain sample, performing flow-through CAR+ polychromic, and performing cell flow detection and amplification for 96 hours to obtain cell tracking markers. The cell lines 3T3 overexpressing 6922 and 7413 and T cells on day 5 after infection were collected and examined, RNA was extracted and reverse transcribed for QPCR detection, and relative quantification was performed by SYBR Green method, and the difference in the RNA expression level of HMGY relative to the expression level of the reference beta-actin was shown to result in high expression of both 3T3 overexpressing 7413 and HMGY RNA of T cells, and little expression of T cells themselves.

TABLE 4 Table 4

Table 5 experimental design and grouping

Figures 2 and 3 show the results of flow cytometry for the expression of markers CD62L and CCR7 in various cells. Flow-through detection of NT,6922, 7413 background and expression of the 24h cell surface marker day7 co-cultured with nalm6 cells. 6922. 7413 after 24h co-culture with or without nalm6 stimulation, the results of flow-testing memory-related markers CD62L, CCR7 showed that HMGY-overexpressing 7413 in both CD4 and CD 8T cell subsets maintained higher CD62L and CCR7 expression with or without nalm6 stimulation, allowing T cells to remain in a memory T cell phenotype. The background CD62L and CCR7 expression levels are up-regulated after the HMGY gene is over-expressed, and the down-regulation proportion is obviously reduced after the HMGY gene is stimulated.

Figures 4 and 5 show the results of flow cytometry for the expression of the markers KLRG and CD137 for the various cells. Flow assay 6922, 7413 background and expression of 24h cell surface marker day7 co-cultured with nalm6 cells. 6922. 7413 after 24h co-culture with or without nalm6 stimulation, differentiation related markers KLRG and activation markers CD137 were separately flow tested, and the results showed that in both CD4 and CD 8T cell subsets, the expression of CD137 was reduced in the presence of nalm6 stimulation by 7413 overexpressing HMGY, and KLRG expression was reduced, leaving the cells in a weakly activated and poorly differentiated state.

Fig. 6 and 7 show the results of flow cytometry for cell expansion of various cells. Flow detection of the cell tracking markers 6922, 7413 background and amplification after 96h co-culture with nalm6 cells. 6922. 7413 after co-culturing for 96h with or without nalm6 stimulation, the results of flow-testing the expansion of T cells with cell tracking markers showed that 7413 overexpressing HMGY in both CD4 and CD 8T cell subsets had more expansion algebra and absolute numbers in the presence of nalm6 stimulation, and thus 7413 also showed better expansion in the absence of nalm6 stimulation due to the P2A linked CAR and HMGY genes.

These data demonstrate that the H19-28z-2a-HMGY vector can effectively express both CAR and HMGY; after the HMGY gene is over-expressed, the CART background has higher expression of CD62L and CCR7, is more biased to a memory state, can effectively reduce the reduction of the expression of the CD62L and the CCR7 after being stimulated, and promotes the T cells to be in a better state; after the HMGY gene is over-expressed, the stimulated CART can express lower levels of CD137 and KLRG, so that the cells are in a weak activation and low differentiation state; after overexpression of the HMGY gene, the cells can expand more after receiving the stimulus.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, those skilled in the art will recognize that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof.

Sequence listing

<110> Shanghai upper phase of technology Co., ltd

<120> modulation of T cell function and response

<130> SDS1.0082US

<150> 201910670168.6

<151> 2019-07-24

<160> 6

<170> SIPOSequenceListing 1.0

<210> 60

<211> 96

<212> PRT

<213> HMGY aa(Homo sapiens)

<400> 60

Met Ser Glu Ser Ser Ser Lys Ser Ser Gln Pro Leu Ala Ser Lys Gln

1 5 10 15

Glu Lys Asp Gly Thr Glu Lys Arg Gly Arg Gly Arg Pro Arg Lys Gln

20 25 30

Pro Pro Lys Glu Pro Ser Glu Val Pro Thr Pro Lys Arg Pro Arg Gly

35 40 45

Arg Pro Lys Gly Ser Lys Asn Lys Gly Ala Ala Lys Thr Arg Lys Thr

50 55 60

Thr Thr Thr Pro Gly Arg Lys Pro Arg Gly Arg Pro Lys Lys Leu Glu

65 70 75 80

Lys Glu Glu Glu Glu Gly Ile Ser Gln Glu Ser Ser Glu Glu Glu Gln

85 90 95

<210> 61

<211> 291

<212> DNA

<213> HMGY nucleotide (homosapiens)

<400> 61

atgagtgagt cgagctcgaa gtccagccag cccttggcct ccaagcagga aaaggacggc 60

actgagaagc ggggccgggg caggccgcgc aagcagcctc cgaaggagcc cagcgaagtg 120

ccaacaccta agagacctcg gggccgacca aagggaagca aaaacaaggg tgctgccaag 180

acccggaaaa ccaccacaac tccaggaagg aaaccaaggg gcagacccaa aaaactggag 240

aaggaggaag aggagggcat ctcgcaggag tcctcggagg aggagcagtg a 291

<210> 62

<211> 1195

<212> PRT

<213> ZBTB38 aa(Homo sapiens)

<400> 62

Met Thr Val Met Ser Leu Ser Arg Asp Leu Lys Asp Asp Phe His Ser

1 5 10 15

Asp Thr Val Leu Ser Ile Leu Asn Glu Gln Arg Ile Arg Gly Ile Leu

20 25 30

Cys Asp Val Thr Ile Ile Val Glu Asp Thr Lys Phe Lys Ala His Ser

35 40 45

Asn Val Leu Ala Ala Ser Ser Leu Tyr Phe Lys Asn Ile Phe Trp Ser

50 55 60

His Thr Ile Cys Ile Ser Ser His Val Leu Glu Leu Asp Asp Leu Lys

65 70 75 80

Ala Glu Val Phe Thr Glu Ile Leu Asn Tyr Ile Tyr Ser Ser Thr Val

85 90 95

Val Val Lys Arg Gln Glu Thr Val Thr Asp Leu Ala Ala Ala Gly Lys

100 105 110

Lys Leu Gly Ile Ser Phe Leu Glu Asp Leu Thr Asp Arg Asn Phe Ser

115 120 125

Asn Ser Pro Gly Pro Tyr Val Phe Cys Ile Thr Glu Lys Gly Val Val

130 135 140

Lys Glu Glu Lys Asn Glu Lys Arg His Glu Glu Pro Ala Ile Thr Asn

145 150 155 160

Gly Pro Arg Ile Thr Asn Ala Phe Ser Ile Ile Glu Thr Glu Asn Ser

165 170 175

Asn Asn Met Phe Ser Pro Leu Asp Leu Arg Ala Ser Phe Lys Lys Val

180 185 190

Ser Asp Ser Met Arg Thr Ala Ser Leu Cys Leu Glu Arg Thr Asp Val

195 200 205

Cys His Glu Ala Glu Pro Val Arg Thr Leu Ala Glu His Ser Tyr Ala

210 215 220

Val Ser Ser Val Ala Glu Ala Tyr Arg Ser Gln Pro Val Arg Glu His

225 230 235 240

Asp Gly Ser Ser Pro Gly Asn Thr Gly Lys Glu Asn Cys Glu Ala Leu

245 250 255

Ala Ala Lys Pro Lys Thr Cys Arg Lys Pro Lys Thr Phe Ser Ile Pro

260 265 270

Gln Asp Ser Asp Ser Ala Thr Glu Asn Ile Pro Pro Pro Pro Val Ser

275 280 285

Asn Leu Glu Val Asn Gln Glu Arg Ser Pro Gln Pro Ala Ala Val Leu

290 295 300

Thr Arg Ser Lys Ser Pro Asn Asn Glu Gly Asp Val His Phe Ser Arg

305 310 315 320

Glu Asp Glu Asn Gln Ser Ser Asp Val Pro Gly Pro Pro Ala Ala Glu

325 330 335

Val Pro Pro Leu Val Tyr Asn Cys Ser Cys Cys Ser Lys Ala Phe Asp

340 345 350

Ser Ser Thr Leu Leu Ser Ala His Met Gln Leu His Lys Pro Thr Gln

355 360 365

Glu Pro Leu Val Cys Lys Tyr Cys Asn Lys Gln Phe Thr Thr Leu Asn

370 375 380

Arg Leu Asp Arg His Glu Gln Ile Cys Met Arg Ser Ser His Met Pro

385 390 395 400

Ile Pro Gly Gly Asn Gln Arg Phe Leu Glu Asn Tyr Pro Thr Ile Gly

405 410 415

Gln Asn Gly Gly Ser Phe Thr Gly Pro Glu Pro Leu Leu Ser Glu Asn

420 425 430

Arg Ile Gly Glu Phe Ser Ser Thr Gly Ser Thr Leu Pro Asp Thr Asp

435 440 445

His Met Val Lys Phe Val Asn Gly Gln Met Leu Tyr Ser Cys Val Val

450 455 460

Cys Lys Arg Ser Tyr Val Thr Leu Ser Ser Leu Arg Arg His Ala Asn

465 470 475 480

Val His Ser Trp Arg Arg Thr Tyr Pro Cys His Tyr Cys Asn Lys Val

485 490 495

Phe Ala Leu Ala Glu Tyr Arg Thr Arg His Glu Ile Trp His Thr Gly

500 505 510

Glu Arg Arg Tyr Gln Cys Ile Phe Cys Leu Glu Thr Phe Met Thr Tyr

515 520 525

Tyr Ile Leu Lys Asn His Gln Lys Ser Phe His Ala Ile Asp His Arg

530 535 540

Leu Ser Ile Ser Lys Lys Thr Ala Asn Gly Gly Leu Lys Pro Ser Val

545 550 555 560

Tyr Pro Tyr Lys Leu Tyr Arg Leu Leu Pro Met Lys Cys Lys Arg Ala

565 570 575

Pro Tyr Lys Ser Tyr Arg Asn Ser Ser Tyr Glu Asn Ala Arg Glu Asn

580 585 590

Ser Gln Met Asn Glu Ser Ala Pro Gly Thr Tyr Val Val Gln Asn Pro

595 600 605

His Ser Ser Glu Leu Pro Thr Leu Asn Phe Gln Asp Thr Val Asn Thr

610 615 620

Leu Thr Asn Ser Pro Ala Ile Pro Leu Glu Thr Ser Ala Cys Gln Asp

625 630 635 640

Ile Pro Thr Ser Ala Asn Val Gln Asn Ala Glu Gly Thr Lys Trp Gly

645 650 655

Glu Glu Ala Leu Lys Met Asp Leu Asp Asn Asn Phe Tyr Ser Thr Glu

660 665 670

Val Ser Val Ser Ser Thr Glu Asn Ala Val Ser Ser Asp Leu Arg Ala

675 680 685

Gly Asp Val Pro Val Leu Ser Leu Ser Asn Ser Ser Glu Asn Ala Ala

690 695 700

Ser Val Ile Ser Tyr Ser Gly Ser Ala Pro Ser Val Ile Val His Ser

705 710 715 720

Ser Gln Phe Ser Ser Val Ile Met His Ser Asn Ala Ile Ala Ala Met

725 730 735

Thr Ser Ser Asn His Arg Ala Phe Ser Asp Pro Ala Val Ser Gln Ser

740 745 750

Leu Lys Asp Asp Ser Lys Pro Glu Pro Asp Lys Val Gly Arg Phe Ala

755 760 765

Ser Arg Pro Lys Ser Ile Lys Glu Lys Lys Lys Thr Thr Ser His Thr

770 775 780

Arg Gly Glu Ile Pro Glu Glu Ser Asn Tyr Val Ala Asp Pro Gly Gly

785 790 795 800

Ser Leu Ser Lys Thr Thr Asn Ile Ala Glu Glu Thr Ser Lys Ile Glu

805 810 815

Thr Tyr Ile Ala Lys Pro Ala Leu Pro Gly Thr Ser Thr Asn Ser Asn

820 825 830

Val Ala Pro Leu Cys Gln Ile Thr Val Lys Ile Gly Asn Glu Ala Ile

835 840 845

Val Lys Arg His Ile Leu Gly Ser Lys Leu Phe Tyr Lys Arg Gly Arg

850 855 860

Arg Pro Lys Tyr Gln Met Gln Glu Glu Pro Leu Pro Gln Gly Asn Asp

865 870 875 880

Pro Glu Pro Ser Gly Asp Ser Pro Leu Gly Leu Cys Gln Ser Glu Cys

885 890 895

Met Glu Met Ser Glu Val Phe Asp Asp Ala Ser Asp Gln Asp Ser Thr

900 905 910

Asp Lys Pro Trp Arg Pro Tyr Tyr Asn Tyr Lys Pro Lys Lys Lys Ser

915 920 925

Arg Gln Leu Lys Lys Met Arg Lys Val Asn Trp Arg Lys Glu His Gly

930 935 940

Asn Arg Ser Pro Ser His Lys Cys Lys Tyr Pro Ala Glu Leu Asp Cys

945 950 955 960

Ala Val Gly Lys Ala Pro Gln Asp Lys Pro Phe Glu Glu Glu Glu Thr

965 970 975

Lys Glu Met Pro Lys Leu Gln Cys Glu Leu Cys Asp Gly Asp Lys Ala

980 985 990

Val Gly Ala Gly Asn Gln Gly Arg Pro His Arg His Leu Thr Ser Arg

995 1000 1005

Pro Tyr Ala Cys Glu Leu Cys Ala Lys Gln Phe Gln Ser Pro Ser Thr

1010 1015 1020

Leu Lys Met His Met Arg Cys His Thr Gly Glu Lys Pro Tyr Gln Cys

1025 1030 1035 1040

Lys Thr Cys Gly Arg Cys Phe Ser Val Gln Gly Asn Leu Gln Lys His

1045 1050 1055

Glu Arg Ile His Leu Gly Leu Lys Glu Phe Val Cys Gln Tyr Cys Asn

1060 1065 1070

Lys Ala Phe Thr Leu Asn Glu Thr Leu Lys Ile His Glu Arg Ile His

1075 1080 1085

Thr Gly Glu Lys Arg Tyr His Cys Gln Phe Cys Phe Gln Arg Phe Leu

1090 1095 1100

Tyr Leu Ser Thr Lys Arg Asn His Glu Gln Arg His Ile Arg Glu His

1105 1110 1115 1120

Asn Gly Lys Gly Tyr Ala Cys Phe Gln Cys Pro Lys Ile Cys Lys Thr

1125 1130 1135

Ala Ala Ala Leu Gly Met His Gln Lys Lys His Leu Phe Lys Ser Pro

1140 1145 1150

Ser Gln Gln Glu Lys Ile Gly Asp Val Cys His Glu Asn Ser Asn Pro

1155 1160 1165

Leu Glu Asn Gln His Phe Ile Gly Ser Glu Asp Asn Asp Gln Lys Asp

1170 1175 1180

Asn Ile Gln Thr Gly Val Glu Asn Val Val Leu

1185 1190 1195

<210> 63

<211> 390

<212> DNA

<213> NFAT promoter (Artificial Sequence)

<400> 63

tcgaggtcga cggtatcgat aagcttgata tcgaattagg aggaaaaact gtttcataca 60

gaaggcgtca attaggagga aaaactgttt catacagaag gcgtcaatta ggaggaaaaa 120

ctgtttcata cagaaggcgt caattggtcc catcgaatta ggaggaaaaa ctgtttcata 180

cagaaggcgt caattaggag gaaaaactgt ttcatacaga aggcgtcaat taggaggaaa 240

aactgtttca tacagaaggc gtcaattggt cccgggacat tttgacaccc ccataatatt 300

tttccagaat taacagtata aattgcatct cttgttcaag agttccctat cactctcttt 360

aatcactact cacagtaacc tcaactcctg 390

<210> 64

<211> 1458

<212> DNA

<213> CAR-h19-28z(Artificial Sequence)

<400> 64

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggatatcc agatgaccca gagcccgagc agcctgagcg cgagcgtggg tgatcgcgtg 120

accattacct gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccgggtaaag cgccgaaact gttaatttat catacatcaa gattacactc aggcgtgccg 240

tcgcgtttta gcggctcggg ttcgggcacc gattttaccc tgaccatctc gagcttgcag 300

ccggaggact tcgccaccta ctattgccaa cagggtaata cgcttccgta cacgttcggt 360

cagggcacca aagtggagat caaaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgcagct ggtggagtct gggggaggct tggtacagcc tggggggtcc 480

ctgagactct cctgtgcagc ctctggagtg tccctgcctg attatggcgt gtcctgggtc 540

cgccaggctc cagggaaggg gctggagtgg gtttcagtga tctggggcag cgagacaacc 600

tactacaaca gcgccctgaa gtcccgattc accatctcca gagacaatgc caagaactca 660

ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ctgtgtatta ctgtgcgaag 720

cactactact acggcggcag ctacgctatg gactactggg gccaaggaac cctggtcacc 780

gtgtcctcag cggccgcaat tgaagttatg tatcctcctc cttacctaga caatgagaag 840

agcaatggaa ccattatcca tgtgaaaggg aaacaccttt gtccaagtcc cctatttccc 900

ggaccttcta agcccttttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 960

ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 1020

cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1080

ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1140

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200

cgaagagagg agtacgatgt tttggacaag aggcgtggcc gggaccctga gatgggggga 1260

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440

gccctgcccc ctcgctaa 1458

<210> 65

<211> 1812

<212> DNA

<213> H19-28z-2a-HMGY(Artificial Sequence)

<400> 65

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggatatcc agatgaccca gagcccgagc agcctgagcg cgagcgtggg tgatcgcgtg 120

accattacct gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccgggtaaag cgccgaaact gttaatttat catacatcaa gattacactc aggcgtgccg 240

tcgcgtttta gcggctcggg ttcgggcacc gattttaccc tgaccatctc gagcttgcag 300

ccggaggact tcgccaccta ctattgccaa cagggtaata cgcttccgta cacgttcggt 360

cagggcacca aagtggagat caaaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgcagct ggtggagtct gggggaggct tggtacagcc tggggggtcc 480

ctgagactct cctgtgcagc ctctggagtg tccctgcctg attatggcgt gtcctgggtc 540

cgccaggctc cagggaaggg gctggagtgg gtttcagtga tctggggcag cgagacaacc 600

tactacaaca gcgccctgaa gtcccgattc accatctcca gagacaatgc caagaactca 660

ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ctgtgtatta ctgtgcgaag 720

cactactact acggcggcag ctacgctatg gactactggg gccaaggaac cctggtcacc 780

gtgtcctcag cggccgcaat tgaagttatg tatcctcctc cttacctaga caatgagaag 840

agcaatggaa ccattatcca tgtgaaaggg aaacaccttt gtccaagtcc cctatttccc 900

ggaccttcta agcccttttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 960

ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 1020

cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1080

ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1140

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200

cgaagagagg agtacgatgt tttggacaag aggcgtggcc gggaccctga gatgggggga 1260

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440

gccctgcccc ctcgcggttc cggagccacg aacttctctc tgttaaagca agcaggagac 1500

gtggaagaaa accccggtcc tatgagtgag tcgagctcga agtccagcca gcccttggcc 1560

tccaagcagg aaaaggacgg cactgagaag cggggccggg gcaggccgcg caagcagcct 1620

ccgaaggagc ccagcgaagt gccaacacct aagagacctc ggggccgacc aaagggaagc 1680

aaaaacaagg gtgctgccaa gacccggaaa accaccacaa ctccaggaag gaaaccaagg 1740

ggcagaccca aaaaactgga gaaggaggaa gaggagggca tctcgcagga gtcctcggag 1800

gaggagcagt ga 1812

Claims (9)

1. A method of producing T cells exhibiting an enhanced memory T cell phenotype in vitro, the method comprising: introducing a polynucleotide encoding a High Mobility Group Y (HMGY) into a population of T cells, wherein HMGY is expressed more highly than T cells not comprising the polynucleotide and memory T cell phenotype of the population of T cells is enhanced;

the population of T cells comprises an antigen binding molecule, wherein the antigen binding molecule is a CAR;

the carrier structure of the CAR is H19-28z-2a-HMGY, and the polynucleotide is SEQ ID NO:65.

2. the method of claim 1, wherein the population of T cells comprising HMGY polynucleotides has an increased level of gene expression of CD62L and/or CCR7 as compared to T cells not comprising the polynucleotides.

3. The method of claim 1, wherein HMGY is overexpressed.

4. The method as recited in claim 1, further comprising: contacting the population of T cells with an antigen to which the population of T cells binds.

5. The method of claim 4, wherein the population of T cells comprising HMGY polynucleotides has reduced levels of gene expression of CD137 and/or KLRG as compared to T cells not comprising HMGY polynucleotides.

6. The method of claim 1, wherein the enhanced memory T cell phenotype comprises an increase in the gene expression level of CD62L and/or CCR7 and/or the enhanced memory T cell phenotype comprises a decrease in the gene expression level of CD137 and/or KLRG.

7. The method of claim 1, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

8. The method of claim 7, wherein the antigen binding domain that binds to a tumor antigen is selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1,CD33,EGFRvIII, GD2, GD3, BCMA, tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra,PSCA,PRSS21,VEGFR2,LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her 2/neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephA2, fucosyl GM1, sLe, GM3, TGS5, HMWA, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, 7R, CLDN6, RC5D, CX 61, CD97, shock absorber, saliva, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, W1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17,XAGE1,Tie 2,MAD-CT-1, MAD-CT-2, fos associated antigen 1, p53, p53 mutants, proline, survivin and telomerase, PCTA-1/Galectin 8, melanA/MART1, ra mutants, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, enterocarboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1; the intracellular signaling domain is selected from the group consisting of CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte-function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT), SLAMF7, NKp80 (KLRF 1), CD160, CD19, CD4, CD8 alphase:Sub>A, CD8 betase:Sub>A, IL2R betase:Sub>A, IL2R gammase:Sub>A, IL7R alphase:Sub>A, ITGA4, VLA1, CD49 ase:Sub>A, ITGA4, IA4, CD49D, ITGA6, the functional signaling domain of the ligand protein to which VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11 ase:Sub>A, LFA-1,ITGAM,CD11b,ITGAX,CD11c,ITGB1,CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (tactile), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLAMME (SLAMF 8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46 and NKG2D bind specifically.

9. A pharmaceutical composition comprising a population of cells produced by the method of claim 1.