CN112251452A

CN112251452A - TIL/TCR-T cell therapy platform

Info

Publication number: CN112251452A
Application number: CN202011138628.XA
Authority: CN
Inventors: 蒲程飞; 陈冬祺; 沈晓钢
Original assignee: Shanghai Sidansai Biotechnology Co ltd
Current assignee: Shanghai Xuxu Technology Co.,Ltd.
Priority date: 2019-10-22
Filing date: 2020-10-22
Publication date: 2021-01-22

Abstract

The invention relates to a TIL/TCR-T cell therapy platform. The present disclosure relates to modified cells engineered to comprise a modified TCR-CD3 complex, wherein the CD3 γ, CD3 ζ, CD3 ε, and/or CD3 δ chains of the modified TCR-CD3 complex are linked to one or more costimulatory signaling domains. For example, the modified TCR-CD3 complex can comprise SEQ ID NO: 18 or 19.

Description

TIL/TCR-T cell therapy platform

Technical Field

The present disclosure relates to compositions and methods for treating diseases, including cancer, using T cell therapy.

Background

Cellular immunotherapy of tumors has achieved great success, but cellular therapy is still difficult to treat certain malignancies and causes side effects in certain treatments. During CAR-T cell therapy, the physician will draw blood from the patient and harvest its cytotoxic T cells. Cells are engineered in the laboratory to attack specific cancers. Recent advances in genome editing technology have enabled scientists to disrupt gene expression in T cells to enhance effector function or bypass tumor immunosuppression and metabolically unfavorable tumor microenvironments. Therefore, there is a need for cellular immunotherapy that modulates T/NK cells to enhance tumors.

Disclosure of Invention

The common TCR only activates CD3, and the designed TCR adds a costimulatory domain and has stronger signal. At the same time, the new TCR we designed became a CAR, which could be a universal element. And CD3 is activated, and co-stimulation domain signals are generated, so that the killing effect of TCR-T can be enhanced. Embodiments relate to modified cells engineered to comprise a modified TCR-CD3 complex, wherein the CD3 γ, CD3 ζ, CD3 ε, and/or CD3 δ chains of the modified TCR-CD3 complex are linked to one or more costimulatory signaling domains. For example, the modified TCR-CD3 complex can comprise SEQ ID NO: 18 or 19.

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

FIGS. 1-4 show schematic diagrams of examples of the modifying components of the TCR-CD3 complex;

FIG. 5 shows a schematic of a polynucleotide of a modified TCR-CD3 complex and a modified cell;

FIG. 6 shows the results of flow cytometry of various vector expressions in T cells;

FIG. 7 shows the expression of HLA-A2 and NY-ESO-1 in substrate cells and the expression of CD3 ζ in these cells;

figure 8 shows western blot results demonstrating the structure of the modified TCR;

FIG. 9 shows a schematic of a polynucleotide and a modified cell;

FIGS. 10-13 are schematic diagrams showing metabolic processes.

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 more (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 amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length differs by up to 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

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

The term "antibody" is used in the broadest sense and refers to 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. The antibodies of the present disclosure may exist in a variety of forms, including, for example, polyclonal antibodies; a monoclonal antibody; fv, Fab 'and F (ab')2 fragments; and single chain and humanized antibodies (Harlow et al, 1999, antibodies for use: A laboratory Manual, Cold spring harbor laboratory Press, New York; Harlow et al, 1989, antibodies: a laboratory Manual, Spprink harbor, Cold New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-.

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 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 entire antigen recognition and binding site. The fragment consists of a dimer of one heavy and one light chain variable region domain in tight and non-covalent association. From the folding of these two domains, six hypervariable loops (3 loops each from the H and L chains) are evolved, which contribute amino acid residues to amino acid 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) specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site (dimer).

As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in a naturally occurring configuration in all antibody molecules. As used herein, "antibody light chain" refers to the smaller of two types of polypeptide chains present in a naturally occurring configuration in all antibody molecules. The kappa and lambda light chains refer to the 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 a bacteriophage. The term also includes antibodies produced by synthesizing a DNA molecule encoding the antibody and expressing the DNA molecule to obtain the antibody or to obtain the amino acids encoding the antibody. Synthetic DNA is obtained using techniques 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 immunologically active cells, or both. Antigens include any macromolecule, including all proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, DNA as used herein includes a nucleotide sequence or partial nucleotide sequence that encodes a protein or peptide that elicits an immune response, thus encoding an "antigen". An antigen need not be encoded by only the full-length nucleotide sequence of a 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 a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, a reduction in tumor cell proliferation, a reduction in tumor cell survival, an increase in the life expectancy of a subject having tumor cells, or an improvement in various physiological symptoms associated with a cancer condition. First, the ability of peptides, polynucleotides, cells and antibodies to prevent tumorigenesis may also exhibit an "anti-tumor effect".

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

The term "autologous" is used to describe material derived from a subject, which is subsequently reintroduced into the same subject.

The term "allogeneic" is used to describe grafts derived from different subjects of the same species. For example, the donor subject may be related or unrelated to the recipient subject, but the donor subject has similar immune system markers as the recipient subject.

The term "xenogeneic" is used to describe grafts derived from subjects of different species. For example, the donor subject and recipient subject are from different species, 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 rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally, but also to other parts of the body through the blood and lymphatic system. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprising" and "comprises" 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 an element or a set of steps or elements.

The phrase "consisting of … …" is intended to include and be limited to anything following the phrase "consisting of … …". Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that no other elements may be present.

The phrase "consisting essentially of … …" is meant to include any elements listed after the phrase, and may include other elements that do not interfere with or affect the activities or actions specified in the present disclosure for the listed elements. Thus, the phrase "consisting essentially of … …" means that the listed elements are required or mandatory, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements. For example, an element is not required if it does not affect the expansion, function, or phenotype of the cell, and is considered optional.

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 "partial," in which only some of the nucleic acids' bases 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 an important 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; (b) a peptide or polypeptide having an amino acid sequence substantially identical to an amino acid sequence in a reference peptide or protein.

The term "co-stimulatory ligand" refers to a molecule on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including proliferation, activation, differentiation and at least one other cellular response, in addition to the primary signal provided by, for example, the binding of the TCR/CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands can include B7-1(CD80), B7-2(CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, ligands for CD7, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also 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-related antigen 1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD 83.

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

The term "co-stimulatory signal" refers to a signal, such as TCR/CD3, that binds to a primary signal, resulting in the up-or down-regulation of T cell proliferation and/or key molecules.

The terms "disease" and "condition" are used interchangeably and may also be different, as a particular disease or condition may not have a known pathogen (and therefore the cause has not been resolved) and is therefore not recognized. It is a disease, but only an adverse condition or syndrome, in which more or less specific symptoms have been identified by the clinician. The term "disease" is a health state of a subject, wherein the subject is unable to maintain homeostasis, and wherein the health of the subject continues to deteriorate if the disease is not improved. In contrast, a "disease" in a subject is a healthy state in which the animal is able to maintain homeostasis, but in which the animal's healthy state is less favorable than the healthy state in the absence of the disease. If not treated in time, the disease does not necessarily lead to a further reduction in the health status of the animal.

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

The term "encoding" refers to the inherent property of a nucleotide of a particular sequence in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having defined structures. Nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or determined amino acid sequences and biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. The coding strand (except for "U" instead of "T") whose nucleotide sequence is identical to the mRNA sequence is typically provided in the sequence listing, and the "protein" whose non-coding strand is used as a template for transcription of a gene or cDNA may be referred to as the protein or other product encoding the gene or cDNA.

The term "exogenous" refers to a molecule that does not naturally occur in a wild-type cell or organism but is typically introduced into a cell by molecular biological techniques. Examples of exogenous polynucleotides include vectors, plasmids and/or artificial nucleic acid constructs encoding the desired proteins. With respect to polynucleotides and proteins, the term "endogenous" or "native" refers to a naturally occurring polynucleotide or amino acid sequence that may be found in a given wild-type cell or organism. Likewise, a particular polynucleotide sequence 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 relative to the second organism. In particular embodiments, a polynucleotide sequence may be "introduced" into a microorganism already containing such polynucleotide sequence by molecular biological techniques, e.g., to produce one or more additional copies of the originally naturally occurring polynucleotide sequence, thereby facilitating overexpression thereof. An encoded polypeptide.

The term "expression or overexpression" refers to the transcription and/or translation of a particular nucleotide sequence, for example, driven by its promoter, into a precursor or mature protein. By "over-expression" is meant that the production of the gene product in the transgenic organism or cell exceeds that in a normal or untransformed organism or cell. The term "expression" as defined herein 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 expression elements may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses), which incorporate the recombinant polynucleotide.

Viruses are useful for delivering 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, sonoporation, magnetic transfection, and the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles.

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

The term "immunoglobulin" or "Ig" refers to a class of proteins that act as antibodies. Five members contained in such proteins are IgA, IgG, IgM, IgD and IgE. IgA is a primary antibody present in human secretions (such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts). IgG is the most common circulating antibody. In most subjects, IgM is the primary immunoglobulin produced in the primary immune response. It is the most potent immunoglobulin in agglutination, complement fixation and other antibody reactions, and is important for defense 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 by causing mast cells and basophils to release mediators upon exposure to allergens.

The term "isolated" refers to a material that is substantially or essentially free of components that normally accompany it in its natural state. The material may be a cell or a macromolecule, such as a protein or nucleic acid. For example, as used herein, "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences flanking it with naturally occurring flanks, e.g., a DNA fragment that has been removed from the 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 native cellular environment and from its association with other components of a cell.

The term "substantially purified" refers to a material that is substantially free of components with which it is normally associated in its native state. For example, a substantially purified cell refers to a cell that has been isolated from other cell types to which it is normally associated in its naturally occurring or native state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term simply refers to a cell that is separated from the cell with which it is naturally associated in its natural state. 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 common nucleic acid bases 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, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate versions of each other and that 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, in some forms, contain one or more introns.

The term "lentivirus" refers to a genus of the family retroviridae. Lentiviruses are unique among retroviruses and are capable of infecting non-dividing cells. They can transmit a large amount of genetic information into the DNA of host cells, and thus they are one of the most effective methods in gene delivery vectors. In addition, the use of lentiviruses enables integration of genetic information into the host chromosome, thereby stably transducing the genetic information. HIV, SIV and FIV are examples of lentiviruses. Vectors derived from lentiviruses 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 response in a subject as compared to the level of response in a subject in the absence of a treatment or compound, and/or as compared to the level of response in a subject. Response in otherwise identical but untreated subjects. The term encompasses perturbation and/or influence of 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 presequence or secretory leader is expressed as a preprotein that participates 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 sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

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

The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal expression level of the tumor antigen in cells from a disease region, such as a solid tumor, within a particular tissue or organ of a patient. To achieve expression levels of the tissue or organ in normal cells. Patients with solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be identified by standard assays known in the art.

Solid tumors are abnormal tissue masses that generally do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors vary with the cell type in which they are formed (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors (e.g., sarcomas and carcinomas) include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, bile duct carcinoma, choriocarcinoma, wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and central nervous system tumors (e.g., gliomas (e.g., brain stem glioma and mixed gliomas), glioblastomas (also referred to as glioblastoma multiforme)), (e), Astrocytoma, central nervous system lymphoma, germ cell tumor, medulloblastoma, schwann's sarcoma throat tumor, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, neuroblastoma, retinoblastoma, and brain metastasis).

A 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 human or animal suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human or an animal. In embodiments, the term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans and animals, such as dogs, cats, mice, rats, and transgenic species thereof.

Subjects in need of treatment or in need thereof include subjects having 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 a polymeric form of nucleotides of at least 10 bases in length, i.e., ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide. 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 a polynucleotide that exhibits substantial sequence identity to a reference polynucleotide sequence or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that are distinguished from a 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 by 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 such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide or has increased activity. Relationship to a reference polynucleotide (i.e., optimized). Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages between, for example, 90%, 95%, or 98%) sequence identity to a reference polynucleotide sequence. As described herein. The terms "polynucleotide variant" and "variant" 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 and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, e.g., a chemical analog of a corresponding naturally occurring amino acid, as well as to 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 embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In 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 other amino acids with substantially similar properties without changing the nature of the polypeptide activity. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted or substituted with a different amino acid residue.

The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery that is necessary to initiate specific transcription of a polynucleotide sequence. The term "expression control (regulatory) sequence" refers to a DNA sequence necessary for the 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 terms "bind", "bind" or "interact with … …" refer to a molecule that recognizes and attaches to a second molecule in a sample or organism, but does not substantially recognize or attach to other structurally unrelated molecules in the sample. As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen of one species may also bind to an antigen of one or more species. However, such inter-species reactivity does not change the specificity of the antibody itself. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, this cross-reactivity does not change the specificity of the antibody itself. In some cases, the term "specific binding" or "specific binding" may be used to refer to the interaction of an antibody, protein or peptide with a second chemical species to indicate that the interaction is dependent on presence. Specific structures (e.g., antigenic determinants or epitopes) on a chemical species; for example, antibodies recognize and bind to a specific protein structure, rather than to any protein. If the antibody is specific for epitope "A", then in a reaction comprising label "A" and the antibody, the presence of a molecule comprising epitope A (or free, unlabeled A) will reduce the amount of label A bound to the antibody.

By "statistically meaningful" is meant that the results are unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Commonly used significance measures include p-value, which is the frequency or probability of an event occurrence observed when an invalid hypothesis is true. If the obtained p-value is less than the significance level, the original hypothesis is rejected. In simple cases, the significance level is defined as a p-value equal to or less than 0.05. An amount that is "reduced" or "less" is typically a "statistically significant" or physiologically significant amount, and can include a reduction of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.61.7, 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 times) (including all integers and decimal points between 1 and greater than 1 (e.g., 1.5, 1.6, 1.7, 1.8, etc.)).

The term "stimulation" refers to the primary response induced by the binding of a stimulatory molecule (e.g., the TCR/CD3 complex) to its cognate ligand, thereby inducing a signaling event, e.g., signaling via the TCR/CD3 complex. Stimulation may mediate changes in the expression of certain molecules, such as 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, a functional signaling domain derived from a stimulatory molecule is the zeta chain associated with the T cell receptor complex. The stimulatory molecule includes a domain 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 the cell (e.g., 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, superagonist anti-CD 28 antibodies, and superagonist anti-CD 2 antibodies.

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

The term "therapeutically effective amount" means that 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 alleviate to some extent, one or more signs or symptoms of the condition or disease being treated. The therapeutically effective amount will depend on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

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. The cell includes a primary subject cell and its progeny.

The term "vector" refers to a polynucleotide that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to 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 an autonomously replicating plasmid or virus. 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 the viral vector include an adenovirus vector, an adeno-associated virus vector, a retrovirus vector and the like. For example, lentiviruses are complex retroviruses comprising, in addition to the common retroviral genes gag, pol and env, other genes with regulatory or structural functions. Lentiviral vectors are well known in the art. Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2 and simian immunodeficiency virus: and (6) SIV. Lentiviral vectors are created by multiple attenuation of HIV virulence genes, e.g., env, vif, vpr, vpu, and nef genes deleted, thus rendering 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 in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have all the possible subranges specifically disclosed as well as individual numerical values within that range. For example, a description of a range 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 breadth 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 or 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 expand and/or maintain the modified cells binds to an antigen on the surface of the WBC, such as a cell surface molecule or marker. In embodiments, the WBCs are GMP (granulocyte macrophage precursor), MDP (monocyte macrophage/dendritic cell precursor), cMyP (common monocyte precursor), basophils, eosinophils, neutrophils, SatM (atypical monocytes with heptalobal nuclei), macrophages, monocytes, CDP (conventional dendritic cell precursor), cDC (conventional DC), pDC (plasmacytoid DC), CLP (conventional lymphocyte precursor), B cells, ILC (innate lymphocytes), at least one of Natural Killer (NK) cells, megakaryocytes, myoblasts, myeloblasts, myeloid stromal cells, zonal cells, lymphoblasts, prolymphocytes, monocytes, megakaryocytes, prokaryotic cells, megakaryocytes, platelets, or MSDCs (myeloid-derived suppressor cells). In embodiments, the WBCs are granulocytes, monocytes and/or lymphocytes. In embodiments, WBCs are lymphocytes, such as B cells. In embodiments, the WBCs are B cells. In embodiments, the cell surface molecule of a B cell comprises CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11B, CD18, CD169, CD1c, CD33, CD38, CD138, or CD 13. In embodiments, the cell surface molecule of a B cell is CD19, CD20, CD22, or BCMA. In embodiments, the cell surface molecule of the B cell is CD 19.

Cells described herein, including modified cells, such as CAR cells and modified T cells, can be derived from stem cells. The stem cell may be an adult stem cell, an embryonic stem cell, more particularly a non-human stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell or a hematopoietic stem cell. The modified cell may also be a dendritic cell, NK cell, B cell or T cell selected from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte or a helper T lymphocyte. In embodiments, the modified cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. Prior to cell expansion and genetic modification, the cell source can be obtained from the 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 embodiments, 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 cell is 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 the population may be different such that the population is a mixed population or a heterogeneous population of cells. For example, a 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 types of cells. For example, a stem cell may give rise to two daughter stem cells (e.g., embryonic stem cells cultured in vitro) or one stem cell and cells undergoing differentiation (e.g., in the case of blood cell production as occurs with hematopoietic stem cells). Different classes of stem cells can be distinguished based on their source 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 are present in the inner cell mass of blastocysts and have an innate ability to differentiate. For example, pluripotent embryonic stem cells have the potential to form any type of cell in vivo. ES cells maintain pluripotency when grown in vitro for extended periods of time, as progeny 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 thought to have a lower differentiation capacity than pluripotent ES cells-fetal stem cells have a greater capacity than adult stem cells. Somatic stem cells apparently can only differentiate into a limited number of cell types and have been described as pluripotent stem cells. "tissue-specific" stem cells typically 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., red blood cells, platelets, white blood cells, etc.).

An induced pluripotent stem cell (i.e., iPS cell or iPSC) may include one that is artificially derived from a non-pluripotent cell (e.g., adult somatic cell) by inducing expression of a specific gene. Induced pluripotent stem cells resemble natural pluripotent stem cells, such as Embryonic Stem (ES) cells, in many ways, such as expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling times, embryoid body formation, teratoma formation, feasible chimera formation, and potency and differentiability. Induced pluripotent cells can be obtained from adult stomach, liver, skin and blood cells.

In embodiments, the antigen binding domain used to kill the tumor binds to an antigen on the surface of the tumor, such as a tumor antigen or a tumor marker. Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. Tumor antigens are well known in the art and include, for example, tumor-associated MUC1(tMUC1), glioma-associated antigen, carcinoembryonic antigen (CEA), β -human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2(AS), enterocarboxyesterase, mut hsp70-2, M-CSF, protease, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, survivin, telomerase, prostate cancer tumor antigen 1(PCTA-1), MAGE, ELF2M, neutrophilic 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 (19CAR, CD19CAR, or CD19-CAR), which is a CAR molecule that includes 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 linking the light and/or heavy chain variable regions using short linking 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, which bridges about 3.5nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences 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 amino acid residues or less. Single-stranded variants can be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer may be used. For recombinant production of the scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, 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 costimulatory 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 are derived from a stimulatory molecule and/or a co-stimulatory molecule, and a signaling domain is derived from a primary signaling domain, such as the CD3 zeta domain. In embodiments, the signaling domain further comprises one or more functional signaling domains derived from a co-stimulatory molecule. In embodiments, the co-stimulatory molecule is a cell surface molecule (other than an antigen receptor or ligand thereof) required to activate a cellular response to an antigen.

In embodiments, the co-stimulatory domain comprises CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, the intracellular domain of CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, or any combination thereof. In embodiments, the signaling domain comprises a CD3 zeta domain derived from a T cell receptor.

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

Between the extracellular and transmembrane domains of the CAR, a spacer domain may be incorporated. As used herein, the term "spacer" generally refers to any oligo-or polypeptide that has the function of linking a transmembrane domain to an extracellular domain and/or a 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.

Embodiments relate to polynucleotides encoding modified components of the TCR-CD3 complex. Embodiments relate to vectors comprising polynucleotides. Embodiments relate to modified cells comprising polynucleotides. Embodiments relate to modified cells engineered to express a modified component of the TCR-CD3 complex, wherein the modified cells comprise an antigen binding molecule. Embodiments relate to methods or uses of polynucleotides, the methods comprising providing a viral particle (e.g., AAV, lentivirus, or variants thereof) comprising a vector genome, the vector genome comprising a polynucleotide. Administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject. In embodiments, an AAV formulation can include AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids. Embodiments relate to pharmaceutical compositions comprising a population of cells. Embodiments relate to methods of eliciting or eliciting a T cell response and/or treating a tumor in a subject in need thereof comprising administering an effective amount of a composition. In embodiments, the polynucleotide comprises at least one of the sequences listed in table 2. For example, antigen-specific T Cell Receptors (TCRs) consist of disulfide-linked heterodimers comprising two clonally distributed, intact membrane glycoprotein chains, α and β, or γ and δ, non-covalently associated with a low molecular weight invariant protein complex, commonly referred to as CD3 (i.e., the TCR-CD3 complex). The TCR α and β chains determine the specificity of the antigen, while the CD3 structure is believed to represent accessory molecules that may be transduction elements of the activation signal that is triggered upon binding of TCR α β to its ligand. The TCR complex interacts with small peptide antigens presented in Major Histocompatibility Complex (MHC) proteins. MHC proteins represent another group of highly polymorphic molecules randomly distributed throughout a species.

The modified component of the TCR-CD3 complex can be expressed entirely in TIL or TCR T and can replace the peptide chain in CD3, thus when CD3 is activated there will be a signal from the costimulatory domain and enhance TIL/TCR-T. The common TCR only activates CD3, and the TCR-CD3 complex designed here has added a costimulatory domain, which is more strongly signaled. The designed TCR-CD3 complex can be associated with the use of CARs, and can be a universal component. When CD3 is activated, a costimulatory domain signal will be present, which enhances TCR-T killing. In embodiments, the TCR-CD3 complex comprises TCR α, TCR β, CD3 γ, CD3 ζ, CD3 ε, and CD3 δ chains. In embodiments, the TCR-CD3 complex comprises TCR γ, TCR δ, CD3 γ, CD3 ζ, CD3 ε, and CD3 δ chains. In embodiments, the modified component of the TCR-CD3 complex comprises a component of the TCR-CD3 complex linked to one or more costimulatory signaling domains.

In embodiments, the one or more co-stimulatory signaling domains comprise one or more functional signaling domains of one or more proteins, wherein the proteins comprise CD27, CD28, 4-1BB (CD137), 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, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT), SLAMF7, NKp80(KLRF1), CD160, CD19, CD4, CD8 α, CD8 β, IL 28 γ, IL 78 α, ITGA 8, VLA 8, CD8, VLITGA 8, CD8, GAITGB 72, GAITGB 8, GAITGA 8, GAITGB 8, GAITCD 8, GAITGB 8, GAITCD 8, GAITGB 36, 2B4) CD84, CD96 (tactle), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELLPG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, vFLIP K13, K13-opt, NEMO mutants, NEMO fusion proteins, IKKI-S176E-S180, IKK 2-S82177, IKK 181E, RIP α, KB β, Tcl-1, MyD88-L, My-S176, IKK-S E-S180, IKK 695 2-S82177, RIP 265, RIP, K β, a-NF-B-B-activating protein, or any gene-activating inhibitor capable of the NF-B-pathway selective gene editing system.

In embodiments, the one or more co-stimulatory signaling domains comprise one or more functional signaling domains of one or more proteins, wherein the proteins comprise CD27, CD28, 4-1BB (CD137), 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, CDS, ICAM-1, GITR, BAFFR, HV63EM (LIGHT TR), SLAMF7, NKp80(KLRF1), CD160, CD19, CD4 α, CD4 β, IL 24 γ, IL 74 α, ITGA 72, VLA 4, CD4, ITGA4, CD4, GAITGA 72, CD4, GAITGB 72, GAITGB 11-4, GAITGB 11, GAITGA 4, GAITGB 4, GAITGA 4, GAITGB 4, GAITX 4, GAITGB 4, GAITGA 4, CD4, GAITGB 11, CD4, GAITGB 4, CD4, GAITGB 4, GAITCD 4, CD4, CD84, CD96 (tactle), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELLPG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46 and NKG 2D.

In embodiments, the modified component of the TCR-CD3 complex comprises a CD3 γ, CD3 ζ, CD3 ε, and/or CD3 δ chain. In embodiments, the modified component of the TCR-CD3 complex comprises CD3 γ linked to one or more costimulatory signaling domains. In embodiments, the modified component of the TCR-CD3 complex comprises CD3 ζ linked to one or more costimulatory signaling domains. In embodiments, the modified component of the TCR-CD3 complex comprises CD3 epsilon linked to one or more costimulatory signaling domains. In embodiments, the modified component of the TCR-CD3 complex comprises CD3 δ linked to one or more costimulatory signaling domains. In embodiments, the CD3 γ, CD3 ζ, CD3 ∈, and/or CD3 δ chain and the one or more co-stimulatory signaling domains are linked by a linker (e.g., a GS linker). In embodiments, the one or more co-stimulatory signaling domains comprise at least two co-stimulatory signaling domains. In embodiments, the at least two co-stimulatory signaling domains are linked by a linker (e.g., a GS linker). In embodiments, the modified component of the TCR-CD3 complex is a modified CD3 domain. In embodiments, the modified TCR-CD3 complex is overexpressed by cells modified and/or the modified CD3 domain is overexpressed by cells modified.

In embodiments, the expression of the modified component of the TCR-CD3 complex can be modulated by an inducible expression system. Inducible expression systems allow for the temporally and spatially controlled activation and/or expression of genes. For example, tetracycline-controlled transcriptional activation is a method of inducible gene expression in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or a derivative thereof (e.g., doxycycline). For example, inducible suicide gene expression systems allow for temporally and spatially controlled activation and/or expression of suicide genes, which results in cells dying themselves by apoptosis.

In embodiments, the modified cell comprises a nucleic acid sequence encoding an inverse tetracycline transactivator (rtTA). In embodiments, the expression of one or more molecules is modulated by rtTA such that the modified component of the TCR-CD3 complex is expressed in the presence of tetracycline. In embodiments, the concentration of tetracycline in the cell culture medium is not less than about 2 μ g/ml. In embodiments, the tetracycline is selected from tetracycline, demeclocycline, meclocycline, doxycycline, leimecycline, mecycline, minocycline, oxytetracycline, tetracycline, and chlortetracycline. In embodiments, the tetracycline is doxycycline.

In embodiments, the inducible suicide system is the HSV-TK system or the inducible caspase 9 system. In embodiments, the modified cell comprises a nucleic acid sequence encoding a suicide gene such that when the nucleoside analog is present in the modified cell in a manner that allows expression of the suicide gene, the nucleoside analog is rendered cytotoxic to the modified cell. In embodiments, the suicide gene is selected from the group consisting of thymidine kinase of herpes simplex virus, thymidine kinase of varicella zoster virus and bacterial cytosine deaminase. In embodiments, the suicide gene is thymidine kinase of herpes simplex virus. In an embodiment, the nucleoside analog is selected from ganciclovir, acyclovir, ganciclovir, penciclovir, valacyclovir, trifluorothymidine, 1- [ 2-deoxy, 2-fluoro, β -D-arabinofuranosyl ] -5-iodouracil, ara-A, araT1- β -D-arabinofuranoxy thymine, 5-ethyl-2 ' -deoxyuridine, 5-iodo-5 ' -amino-2, 5' -dideoxyuridine, isoxyuridine, AZT, AIU, dideoxycytidine, and AraC. In embodiments, the nucleoside analog is ganciclovir.

In embodiments, expression of the modified component of the TCR-CD3 complex is regulated by one or more promoters. In embodiments, the polynucleotide comprises a promoter comprising a binding site for a transcriptional regulator that regulates expression and/or secretion of a modified component of the TCR-CD3 complex in a cell. For example, the transcriptional modulator is or includes Hif1a, NFAT, FOXP3, and/or NFkB. For example, a modified component of the TCR-CD3 complex comprises at least one costimulatory signaling domain associated with an oxygen-sensitive polypeptide domain, and the oxygen-sensitive polypeptide domain comprises a HIF VHL binding domain.

In embodiments, the polynucleotide may be integrated into the genome of the modified cell, and progeny of the modified cell will also express the polynucleotide, thereby producing a stably transfected modified cell. In embodiments, the modified cell may express a polynucleotide encoding a CAR, but the polynucleotide is not integrated into the genome of the modified cell, such that the modified cell expresses the transiently transfected polynucleotide for a limited period of time (e.g., several days), after which the polynucleotide is lost due to cell division or other factors. For example, the polynucleotide is present in the modified cell as a recombinant DNA construct, mRNA or viral vector, and/or the polynucleotide is an mRNA that is not integrated into the genome of the modified cell.

Embodiments related to a method or use of a polynucleotide, the method comprising providing a viral particle (e.g., an AAV, a lentivirus, or variant thereof) comprising a vector genome comprising a polynucleotide encoding one or more molecules and a polynucleotide encoding a binding molecule, said polynucleotide operably linked to an expression control element that confers transcription of said polynucleotide; administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject. In embodiments, an AAV formulation can include AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids.

Embodiments relate to methods or uses of polynucleotides. The method or use comprises: providing a viral particle (e.g., an AAV, lentivirus, or variant thereof) comprising a vector genome comprising the polynucleotide, wherein the polynucleotide is operably linked to an expression control element that confers transcription of the polynucleotide, and administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject. In embodiments, an AAV formulation can include AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids. More information on the administration and preparation of viral particles can be found in U.S. patent nos.: 9840719 and Milani et al, Sci.Natl.Acad.Sci. In translation 11, eaav7325(2019) May 222019, which is incorporated herein by reference.

In embodiments, the bioreactor may be seeded at a cell density of about 0.5 x 106 cells/mL with a viability of greater than 95%. When the cell density reaches about 1.0X 106 cells/ml, a PEI/DNA ratio of 2: 1 (PEI/DNA complex) transfected cells. At the time of harvest, AAV can be released from the cell culture in the bioreactor using the Triton X-100 method. All solutions can be added directly to the bioreactor and the lysate centrifuged at 4000 × g for 20 minutes. The supernatant may be stored at-80 ℃ for further processing. AAV may be further purified. For example, purification can be achieved by overlaying a series of step gradients with AAV samples (12.3mL) at concentrations of 1, 5, 7, and 5mL, respectively, using iodixanol concentrations of 15, 25, 40, and 54%, respectively. The concentration of 15% iodixanol also contained 1M NaCl to avoid aggregation of AAV with other cellular proteins and negatively charged nuclear components. After centrifugation is complete, 5mL of the solution can be drawn 2 mm below the interface labeled 40/54 and then ultracentrifuged at 385,000 Xg for 1 hour 45 minutes in a Sorval T-865 rotor in a Sorval ultracentrifuge. The viral vector can then be quantified. For example, in all cases, the infectivity of the vector AAV can be determined by Gene Transfer Assay (GTA) using GFP as a reporter gene. AAV infectivity assay, samples can be diluted prior to addition to cells to bring GFP positive cells in the range of 2% to 20% to ensure that only one virus enters the cell to express GFP. GFP positive cells can be quantified by FACS using suspended HEK293 cells. AAV may then be administered to the subject. For example, AAV may be diluted in a 0.9% sterile NaCl salt solution (supplemented with 0.25% human serum albumin [ HSA ]) to be infused into a patient, the final infusion amount will be calculated to be 3mL/kg based on the patient's body weight.

In an embodiment, the modified cell comprises an antigen binding molecule that 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 binds to a tumor antigen including TSHR, CD123, CD171, CS-1, CLL-1, CD, EGFRvIII, GD, BCMA, TnAg, PSMA, ROR, FLT, FAP, TAG, CD44v, CEA, EPCAM, B7H, KIT, IL-13Ra, Mesothelin, IL-11Ra, PSCA, PRSS, VEGFR, LewisY, CD, PDGFR-beta, SSEA-4, CD, folate receptor alpha, ERBB (Her/neu), MUC, EGFR, NCAM, prostatase, PAP, ELF2, Ephrin B, IGF-I receptor, CAIX, LMP, gp100, bcr-abl, EphA, fucosyl GM, sLe, GM, TGS, HMAA, O-acetyl-TEM, WMAA, O-TEM, CD-I receptor, CAIX, LMP, gp100, bcr-abl, CD179, CD-5, CD-LR, GMA, CD-D, GMA, CD-D, GMA, GM, UPK2, HAVCR1, ADRB3, PANX3, GPR 3, LY 63, OR51E 3, TARP, WT 3, NY-ESO-1, LAGE-1a, MAGE-A3, legumain, HPV E3, MAGE A3, ETV 3-AML, sperm protein 17, XAGE 3, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p3 mutant, protein, survivin and telomerase, PCTA-1/Galectin 8, Melana/MART 3, Ras mutant, hTERT, sar-translocation, ML-myiap, ERG (TMPRSS2ETS fusion gene), 36NA 72, PAX3, androgen receptor 3, cyclin B3, CN, BOC-3, BO-IRE 2-RACR 72, RACK 3, RACK-3, RACK-3, RACK-3, RACK-MAG, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL 1. In embodiments, the intracellular signaling domain comprises a costimulatory signaling domain, or a major signaling domain and a costimulatory signaling domain, wherein the costimulatory signaling domain comprises a functional signaling domain of a protein, wherein the protein comprises CD27, 4-1BB (CD137), OX 27, CD27, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD27, LIGHT, NKG2 27, B27-H27, a ligand that specifically binds to CD27, CDs, ICAM-1, GITR, BAFFR, HVEM (LIGHT), SLAMF 27, NKp 27 (rf 27), CD160, CD27 α, CD27 β, IL2 27, gaily 7, vlitga 72, vlitga 27, CD27, gaiitgi ITGA 27, CD 36, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BL AME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46 and NKG 2D.

In embodiments, the modified cell comprises an antigen binding molecule that 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 TCR comprises TCR γ and TCR δ chains or TCR α and TCR β chains, or a combination thereof.

In embodiments, the first antigen binding domain is on a CAR and the second antigen binding domain is on a T Cell Receptor (TCR). In embodiments, the TCR 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, T cell clones expressing TCRs with high affinity for the target antigen can be isolated. Tumor Infiltrating Lymphocytes (TILs) or Peripheral Blood Mononuclear Cells (PBMCs) can be cultured in the presence of Antigen Presenting Cells (APCs) pulsed with peptides representing epitopes known to elicit a dominant T cell response when presented in defined HLA alleles. High affinity clones can then be selected based on the ability of the MHC-peptide tetramer to stain and/or recognize and lyse target cells pulsed with low titer concentrations of the cognate peptide antigen. After selection of clones, the TCR α and TCR β chains or TCR γ and TCR δ chains were identified and isolated by molecular cloning. For example, for TCR α and TCR β chains, TCR α and TCR β gene sequences are then used to generate expression constructs that ideally promote stable, high level expression of both TCR chains in human TC cells. Transduction vectors, such as gamma retroviruses or lentiviruses, can then be generated and tested for functionality (antigen specificity and functional affinity) and used to produce large quantities of vectors for clinical use. Aliquots of the final product may then be used to transduce a population of target T cells (usually purified from patient PBMCs) which are then expanded prior to infusion into the patient.

Various methods can be implemented to obtain a gene encoding a tumor-reactive TCR. More information is provided in the Clin Transl Immunology of Kershaw et al. 5 months in 2014; 3(5): e16. in embodiments, the specific TCR may be derived from a tumor-specific T cell that occurs spontaneously in the patient. Included within this class are 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. TCRs specific for virus-associated malignancies can also be isolated as long as the viral proteins are expressed by the transformed cells. This category of malignancies includes liver cancer and cervical cancer associated with hepatitis and papillomaviruses, and malignancies associated with epstein-barr virus. In embodiments, target antigens for the TCR can include CEA (e.g., for colorectal cancer), gp100, MART-1, p53 (e.g., for melanoma), MAGE-A3 (e.g., melanoma, esophageal and synovial sarcomas), NY-ESO-1 (e.g., melanoma and sarcomas, and multiple myeloma).

In embodiments, the preparation and infusion of Tumor Infiltrating Lymphocytes (TILs) may be accomplished in the following manner. For example, tumor tissue is from surgical or biopsy specimens, can be obtained under sterile conditions, and transported to a cell culture room in a refrigerator. Necrotic tissue and adipose tissue may be removed. Tumor tissue can be cut into pieces of about 1-3 cubic millimeters. Collagenase, hyaluronidase and DNase may be added and digested overnight at 4 ℃. The cells were separated and collected by filtration through a 0.2um filter for 5 minutes by means of a lymphocyte separator (1500 rpm). Cells were expanded with medium containing PHA, 2-mercaptoethanol and CD3 monoclonal antibody, and a small dose of IL-2(10-20IU/ml) was added to induce activation and proliferation. According to the growth situation, the cell density can be carefully detected and maintained at 0.5X 10 under the conditions of 37 ℃ and 5% CO2 for 7-14 days⁶-2×10⁶In the range of/ml. TIL positive cells have the ability to kill syngeneic cancer cells and can be screened by co-culture. Positive cells can be expanded in serum-free medium with high doses of IL2(5000-¹¹The TIL of (1). To administer TIL, it can be first collected in saline using continuous flow centrifugation, then filtered through platelet administration to a volume of 200-300ml of a solution containing 5% albumin and 450000 IU IL-2. The TIL may be injected into the patient via a central venous catheter within 30-60 minutes. In embodiments, the TIL may often be injected into two to four separate bags; for example in a bag.Infusion may take several hours apart. If applicable, more than about 1.5X 10¹¹The TIL of (3) may be administered usually for 2 consecutive days.

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 an NK cell. In embodiments, the immune effector cell is a T cell. In embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof. In embodiments, the cell is a human cell.

In embodiments, enhanced expression and/or function of the modified component of the TCR-CD3 complex is achieved by introducing a nucleic acid sequence encoding the modified component of the TCR-CD3 complex and/or the binding molecule, which is present in the modified cell in the form of a recombinant DNA construct, mRNA, or viral vector.

In embodiments, the nucleic acid sequence is an mRNA that is not integrated into the genome of the modified cell. In embodiments, the nucleic acid sequence is associated with an oxygen-sensitive polypeptide domain. In embodiments, an oxygen-sensitive polypeptide domain comprises a HIF VHL binding domain. In embodiments, the nucleic acid sequence is regulated by a promoter that comprises a binding site for a transcriptional regulator that regulates expression and/or secretion of the therapeutic agent in the cell. In embodiments, the transcriptional modulator is or comprises Hif1a, NFAT, FOXP3, and/or NFkB.

Embodiments relate to compositions and methods for enhancing the metabolism of modified cells (e.g., immune cells) in a tumor microenvironment. For example, lactate metabolism can be increased (e.g., transporters MCT1 and MCT 4). Anaerobic and aerobic respiration and mitochondrial function and/or amino acid metabolism can be enhanced. The modified cells enhance metabolism and/or amino acid metabolism (e.g., the molecules listed in table 9) by enhancing the pathway of metabolizing lactate (e.g., using the transporters MCT1, MCT4 to enhance lactate metabolism) (e.g., the molecules listed in table 7), anaerobic and aerobic respiratory mitochondrial function (e.g., the molecules listed).

Conditions of the tumor microenvironment (e.g., hypoxia, hyperacidity, etc.) inhibit the viability of T cells, and enhancing the function of T cell monocarboxylate transporters can effectively help T cells survive in the tumor microenvironment. MCT1 is normally expressed on T cells, regulates bidirectional transport of lactate both inward and outward, and is also expressed in tumor cells. MCT4 is highly expressed in some tumor cells, regulates the outward transport of lactate, and is not expressed on normal T cells. MCT2 is expressed on normal T cells and on some other cells in the body, regulating the transport of lactate into the cell. CD147 is an accessory protein of MCT family protein and can regulate the correct positioning of the MCT family protein on cell membrane. LDHB converts lactic acid to pyruvic acid. Pyruvate can enter the mitochondria through pyruvate transporters (MPCs) on the mitochondrial membrane and eventually undergo oxidation and breakdown by the mitochondria. Overexpression of MCT3 prevented lactate from entering T cells by knock-out/knock-out MCT 1/2. By overexpressing MCT1/2, knocking down/knocking out MCT3, and overexpressing LDHB and MPC, it can enhance the entry of lactate into T cells and enhance lactate metabolism, helping immune cells to enhance resistance to solids. A tumor. Mitochondrial oxidative function can be achieved by over-expressing mitochondrial proteins such as Frataxin, HBA, HBB, HBD, HBE, and/or HBG, etc., which promote the synthesis of heme and hemoglobin and enhance the oxygen storage capacity of mitochondria; the assembly of mitochondria is promoted by over-expressing TOMM20, TOMM22, TOMM40 and/or TOM70, the functions of the mitochondria are finally enhanced, immune cells adapt to the microenvironment of tumors, and the treatment capability of the tumors is improved. To make T cells with more amino acids, one can use: overexpression of the amino acid transporters CD98, SNAT1, SNAT2, and/or ASCT2, etc., transports amino acids from outside the cell into the cell, or can convert them into glutamine salts into the tricarboxylic acid cycle via GLS glutamine. It is then converted from POA to PEP by PCK enzyme.

Embodiments relate to cells modified to express one or more molecules at a level that is higher or lower than one or more expression levels expressed by cells that are not modified to express the one or more molecules, wherein the one or more molecules are associated with modifying the metabolism of the cells. Embodiments relate to modified cells engineered to express antigen binding molecules, wherein the expression and/or function of one or more molecules in the modified cell has been enhanced or reduced (including eliminated), wherein the one or more molecules associate with the metabolically modified cell. In some embodiments, the modified cell comprises a disruption of an endogenous gene or addition of an exogenous gene associated with a biosynthetic or transport pathway of one or more molecules. Embodiments relate to pharmaceutical compositions comprising a population of cells. Embodiments relate to a method of eliciting or eliciting a T cell response and/or treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of the composition of claim 6. Embodiments relate to isolated nucleic acid sequences encoding one or more molecules associated with the metabolism of a modified cell.

Embodiments relate to a method or use of a polynucleotide, the method comprising providing a viral particle (e.g., an AAV, a lentivirus, or variant thereof) comprising a vector genome comprising a polynucleotide encoding one or more molecules and a polynucleotide encoding a binding molecule, operably linking the polynucleotide to an expression control element that confers transcription of the polynucleotide; administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject, wherein the one or more molecules are overexpressed in cancer cells, associated with recruitment of immune cells, and/or associated with autoimmunity. In embodiments, an AAV formulation can include AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids.

In embodiments, the one or more molecules comprise at least one of MCT1, MCT2, MCT3, LDHB, and MPC, a functional variant of one or more molecules, or a functional fragment of one or more molecules; and/or metabolism includes metabolism of lactic acid.

In embodiments, metabolism comprises altered transport of lactate by the modified cell.

In embodiments, the modified cell transports less or more lactate into the modified cell as compared to a corresponding wild-type cell.

In embodiments, the modified cell overexpresses MCT3 and expresses fewer MCT1 and MCT2, and transports less lactate into the modified cell, as compared to a corresponding wild-type cell.

In embodiments, the modified cell overexpresses MCT1, MCT2, LDHB, and MPC, expresses fewer MCT3, and transports more lactate into the modified cell as compared to a corresponding wild-type cell.

In some embodiments, the one or more molecules comprise at least one of: frataxin, HBA, HBB, HBD, HBE, HBG, TOMM20, and TOMM22, a functional variant of one or more molecules, or a functional fragment of one or more molecules; and/or metabolism includes metabolism of lactic acid.

In embodiments, metabolism comprises enhanced oxidative function of the mitochondria of the modified cell.

In embodiments, the modified cell overexpresses Frataxin, HBA, HBB, HBD, HBE, HBG to enhance the mitochondrial oxygen storage capacity of the modified cell.

In embodiments, the modified cell overexpresses TOMM20 and TOMM22 to enhance mitochondrial function of the modified cell.

In embodiments, the one or more molecules comprise at least one of CD98, SNAT1, SNAT2, ASCT2, a functional variant of the one or more molecules, or a functional fragment of the one or more molecules; and/or metabolism includes metabolism of lactic acid.

In embodiments, the metabolism comprises enhanced amino acid metabolism of the modified cell.

In embodiments, the modified cell overexpresses CD98, SNAT1, SNAT2, ASCT2 to enhance the transport ability of the modified cell to transport amino acids into the modified cell.

Embodiments relate to a method of modifying a target genomic locus in a T cell to down-regulate a gene of interest, the method comprising: introducing a nuclease that produces a single-or double-strand break within a target genomic locus into a T cell; introducing the nucleic acid insert into a cell to knock out or knock out a gene of interest; selecting a cell comprising the nucleic acid insert integrated into the target genomic locus. In some embodiments, the nucleic acid insert is flanked by a 5 'homology arm and a 3' homology arm, and the 3 'homology arm of the nucleic acid insert and the 5' homology arm of the nucleic acid insert are fragments within a corresponding genomic target genomic locus of genomic homology. In some embodiments, the nuclease agent is a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a meganuclease. In certain embodiments, the nuclease agent comprises a cluster-like regularly interspaced short palindromic repeats (CRISPR) -associated (Cas) protein and a guide rna (grna). For example, the Cas protein is Cas 9.

In embodiments, expression of the polynucleotide is regulated or modulated by a synthetic Notch receptor comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising an antibody (e.g., single chain fv (scfv)) or nanobody) specifically binds an antigen; b) the Notch Regulatory Region (NRR) and c) comprises the intracellular domain of a transcriptional activator comprising a DNA binding domain. In embodiments, the Notch regulatory region comprises a Lin 12-Notch repeat, a heterodimerization domain comprising a S2 proteolytic cleavage site and a transmembrane domain comprising a S3 proteolytic cleavage site. The intracellular domain is heterologous to the Notch regulatory region. In some embodiments, the transcriptional activator replaces a naturally occurring intracellular notch domain, and binding of the antibody to the antigen induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain. The release of the intracellular domain results in the transcriptional activator inducing expression of a polynucleotide encoding one or more target proteins in the modified cell. In embodiments, the modified cell comprises a polynucleotide encoding a synthetic Notch receptor and a polynucleotide encoding a transcriptional control element that is responsive to a transcriptional activator and is operably linked to a polynucleotide encoding one or more target proteins (e.g., overexpression of a metabolic-related molecule)

Exemplary embodiments

The following are exemplary embodiments:

1. a polynucleotide encoding a modified component of the TCR-CD3 complex.

2. A vector comprising the polynucleotide of embodiment 1.

2. A modified cell comprising the polynucleotide of embodiment 1.

3. A modified cell engineered to express a modified component of the TCR-CD3 complex, wherein the modified cell comprises an antigen binding molecule.

4. A method or use of a polynucleotide, the method comprising

Providing a viral particle (e.g., AAV, lentivirus, or variant thereof) comprising a vector genome comprising the polynucleotide of embodiment 1; and

administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject.

5. The method of embodiment 4, wherein the AAV preparation can comprise AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids.

6. A pharmaceutical composition comprising a population of cells of any one of

embodiments

2 and 3.

7. A method of eliciting or eliciting a T cell response and/or treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of the composition of embodiment 6.

8. The polynucleotide of any ongoing embodiment 1-7, wherein said polynucleotide comprises at least one of the sequences listed in table 2.

9. The polynucleotide, vector, modified cell and method of any one of embodiments 1-8, wherein said TCR-CD3 complex comprises TCR α, TCR β, CD3 γ, CD3 ζ, CD3 ε, and CD3 δ chains.

10. The polynucleotide, vector, modified cell and method of any one of embodiments 1-8, wherein the TCR-CD3 complex comprises TCR γ, TCR δ, CD3 γ, CD3 ζ, CD3 ε, and CD3 δ chains.

11. The polynucleotide, vector, modified cell and method of any one of embodiments 1-10, wherein the modified component of the TCR-CD3 complex comprises a component of the TCR-CD3 complex linked to one or more costimulatory signaling domains.

12. The polynucleotides, vectors, modified cells and methods of embodiment 11, wherein the one or more co-stimulatory signaling domains comprise one or more functional signaling domains of one or more proteins, wherein the proteins comprise CD, 4-1BB, OX, CD, PD-1, ICOS, lymphocyte function-associated antigen 1(LFA-1), CD, LIGHT, NKG2, B-H, a ligand that specifically binds to CD, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF, NKp (KLRF), CD160, CD alpha, CD beta, IL2 gamma, IL7 alpha, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITLFGAL, CD11, GAMA-1, GAMMA, GAGB, ITGAX, ITGB, ITGAX-11, ITGB, ITGAX-1, ITGB, ITGAX, CD103, ITGAX-1, ITGB, CD, ITGB, CD-1, CD-1, CD-, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-6), SLAM (SLAMF 6, CD150, IPO-3), BLAME (SLAMF 6), LPG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp 6, NKG 26, vFLIKIK 6, K6-opt, NEMO mutant, NEMO fusion protein, 6-S6-6, NKS 177-6, DNAM-36177, MyK-6, RIKB-6, My-K-36265, NF-K-activating gene or any fragment of the gene capable of inhibiting the activation system of the activation of the gene.

13. The polynucleotides, vectors, modified cells and methods of embodiment 11, wherein the one or more co-stimulatory signaling domains comprise one or more functional signaling domains of one or more proteins, wherein the proteins comprise CD, 4-1BB, OX, CD, PD-1, ICOS, lymphocyte function-associated antigen 1(LFA-1), CD, LIGHT, NKG2, B-H, a ligand that specifically binds to CD, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF, NKp (KLRF), CD160, CD alpha, CD beta, IL2 gamma, IL7 alpha, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITLFGAL, CD11, GAMA-1, GAMMA, GAGB, ITGAX, ITGB, ITGAX-11, ITGB, ITGAX-1, ITGB, ITGAX, CD103, ITGAX-1, ITGB, CD, ITGB, CD-1, CD-1, CD-, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96 (tactle), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), LPG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG 2D.

14. The polynucleotide, vector, modified cell and method of any one of embodiments 1-13 wherein the modified component of the TCR-CD3 complex comprises a CD3 γ, CD3 ζ, CD3 ε, and/or CD3 δ chain.

15. The polynucleotide, vector, modified cell and method of any one of embodiments 1-13 wherein the modified component of the TCR-CD3 complex comprises CD3 γ linked to one or more costimulatory signaling domains and/or the modified component of the TCR-CD3 complex comprises CD3 ζ linked to one or more costimulatory signaling domains.

16. The polynucleotides, vectors, modified cells, and methods of any one of embodiments 1-13, wherein the modified component of the TCR-CD3 complex comprises CD3 epsilon linked to one or more costimulatory signaling domains.

17. The polynucleotides, vectors, modified cells, and methods of any one of embodiments 1-13, wherein the modified component of the TCR-CD3 complex comprises CD3 δ linked to one or more costimulatory signaling domains.

18. The polynucleotides, vectors, modified cells, and methods of any one of embodiments 14-17, wherein the CD3 γ, CD3 ζ, CD3 ε, and/or CD3 δ chains and one or more costimulatory signaling domains are linked by a linker (e.g., a GS linker).

19. The polynucleotide, vector, modified cell and method of any one of embodiments 14-17, wherein the one or more costimulatory signaling domains comprises at least two costimulatory signaling domains.

20. The polynucleotide, vector, modified cell and method of embodiment 19, wherein said at least two co-stimulatory signaling domains are linked by a linker (e.g., a GS linker).

21. The modified cell of any proceeding embodiment 1-20, wherein the modified component of the TCR-CD3 complex is a modified CD3 domain.

22. The modified cell of any preceding embodiment 1-21, wherein the modified cell overexpresses the modified TCR-CD3 complex and/or the modified cell overexpresses the modified CD3 domain.

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

24. The modified cell of embodiment 23, wherein said 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, TnAG, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra 3, mesothelin, IL-11Ra, PSCA, PRSS 3, VEGFR 3, LewisY, CD3, PDGFR-beta, SSEA-4, CD3, folate receptor alpha, ERBB 3 (Her 3/neu), MUC 3, EGFR, NCAM, protease, ELF 23, Ephrin B3, CX-1 receptor, CAIX, LMP 3, NYb 36100, bcr-3, tyrosine-GM, EPHA-72, PEG 3, MAGE-A1, legumain, HPV E1, MAGE A1, ETV 1-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p1 mutant, prostein, survivin and telomerase, PCTA-1/Galetin 8, Mela A/MART1, Ras mutant, hTERT, sarcoma translocation, ML-IAP, ERG (TMPRSS2ETS fusion gene), 36NA 72, PAX 1, RAG, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART 1, PAHsX 1, OY-1, LCK, AKAKAP-4, SSX 1, human RAKE-1, telomerase reverse transcriptase, LRRU-IRU 1, LRRU-7, CD1, LRRU 1, CD1, EMR 1, CD1, and MRRU 3679.

25. The modified cell of any one of embodiments 23 and 24, wherein the intracellular signaling domain comprises a costimulatory signaling domain, or a primary signaling domain and a costimulatory signaling domain, wherein the costimulatory signaling domain comprises a functional signaling domain of a protein, wherein the protein comprises CD27, CD28, 4-1BB (CD137), 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 CD3, CDs, ICAM-1, GITR, BAFFR, HVEM (LIGHT), SLAMF 3, NKp3 (KLRF 3), CD160, CD3 α, CD β, IL 23, vly IL7, vlitga 3, CD3 a3, CD3 a3, CD3 a3, CD3 a3, LFA-1, ITGAM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96 (tactle), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF 69 (NTB-69), SLAM (SLAMF 69, CD150, IPO-3), BLAME (SLAMF 69), SEL (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp 69, NKG2 and NKG 69.

26. The modified cell of any one of embodiments 1-22, wherein the modified cell comprises an antigen binding molecule that is a modified tcr (tcr) or tcr (til).

27. The modified cell of embodiment 26, wherein the TCR is derived from a spontaneously occurring tumor-specific T cell in a patient.

28. The modified cell of embodiment 27, wherein the TCR is bound to a tumor antigen.

29. The modified cell of embodiment 28, wherein said tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1.

30. The modified cell of embodiment 28, wherein the TCR comprises TCR γ and TCR δ chains or TCR α and TCR β chains, or a combination thereof.

31. 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).

32. The modified cell of embodiment 31, wherein said immune effector cell is a T cell or an NK cell.

33. The modified cell of embodiment 32, wherein said immune effector cell is a T cell.

34. The modified cell of embodiment 32, wherein said T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof.

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

36. The modified cell of any of the preceding embodiments, wherein enhanced expression and/or function of the modified component of the TCR-CD3 complex is achieved by introducing a polynucleotide encoding the modified component of the TCR-CD3 complex and/or the binding molecule, said polynucleotide being present in the modified cell in the form of a recombinant DNA construct, mRNA or viral vector.

37. The modified cell of embodiment 36, wherein said polynucleotide is an mRNA that is not integrated into the genome of said modified cell.

38. The modified cell of embodiment 36, wherein said polynucleotide is associated with an oxygen-sensitive polypeptide domain.

39. The modified cell of embodiment 38, wherein the oxygen-sensitive polypeptide domain comprises a HIF VHL binding domain.

40. The modified cell of embodiment 36, wherein said polynucleotide is regulated by a promoter comprising a binding site for a transcriptional regulator that regulates expression and/or secretion of a therapeutic agent in the cell.

41. The modified cell of embodiment 40, wherein said transcriptional modulator is or comprises Hif1a, NFAT, FOXP3, and/or NFkB.

42. A cell modified to express one or more molecules at a level that is higher or lower than the level of one or more molecules expressed by a cell that is not modified to express the one or more molecules, wherein the one or more molecules are associated with the metabolism of the modified cell.

43. A modified cell engineered to express an antigen binding molecule, wherein the expression and/or function of one or more molecules in the modified cell has been enhanced or reduced (including eliminated), wherein the one or more molecules are associated with the metabolism of the antigen. The modified cell.

44. The modified cell of any one of embodiments 42 and 43, wherein the modified cell comprises a disruption of an endogenous gene or addition of an exogenous gene associated with a biosynthetic or transport pathway for one or more molecules.

45. A method or use of a polynucleotide, the method comprising

Providing a viral particle (e.g., an AAV, lentivirus, or variant thereof) comprising a vector genome comprising a polynucleotide encoding one or more molecules and a polynucleotide encoding an antigen binding molecule operably linked to an expression control element polynucleotide that confers transcription; and

administering to the subject an amount of the viral particle such that the polynucleotide is expressed in the subject, wherein the one or more molecules are associated with the metabolism of the modified cell.

46. The method of embodiment 45, wherein the AAV preparation may comprise AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids.

47. A pharmaceutical composition comprising a population of cells according to any one of embodiments 1-3.

48. A method of eliciting or eliciting a T cell response and/or treating a tumor in a subject in need thereof comprising administering to the subject an effective amount of the composition of embodiment 47 or an isolated molecule or molecules encoding the same associated with the metabolism of the modified cells.

49. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 42-48, wherein the one or more molecules comprises at least one of MCT1, MCT2, MCT3, LDHB and MPC (functional variants thereof), a functional variant of one or more molecules or a functional fragment of one or more molecules; and/or metabolism includes metabolism of lactic acid.

50. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 42-48, wherein said metabolism comprises altered transport of lactate in the modified cell.

51. The modified cell of any preceding embodiment 42-50, wherein the modified cell transports less or more lactate into the modified cell as compared to a corresponding wild-type cell.

52. The modified cell of any preceding embodiment 42-50, wherein the modified cell overexpresses MCT3 and expresses less MCT1 and MCT2 and transports less lactate into the modified cell as compared to a corresponding wild-type cell.

53. The modified cell of any preceding embodiment 42-50, wherein the modified cell overexpresses MCT1, MCT2, LDHB, and MPC, expresses less MCT3, and transports more lactate into the modified cell as compared to a corresponding wild-type cell.

54. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 42-53, wherein the one or more molecules comprises a pelastatin, HBA, HBB, HBD, HBE, HBG, TOMM20, and TOMM22, a functional variant of one or more molecules, or a functional fragment of one or more molecules; and/or metabolism includes metabolism of lactic acid.

55. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 42-54, wherein said metabolism comprises an enhancement of the oxidative function of mitochondria of said modified cell.

56. The modified cell of any proceeding embodiment 42-55, wherein the modified cell overexpresses Frataxin, HBA, HBB, HBD, HBE, HBG, thereby enhancing the mitochondrial oxygen storage capacity of the modified cell.

57. The modified cell of any preceding embodiment 42-55, wherein the modified cell overexpresses TOMM20 and TOMM22, thereby enhancing mitochondrial function of the modified cell.

58. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 42-47, wherein the one or more molecules comprises at least one of CD98, SNAT1, SNAT2, ASCT2, one or more functional variants, or a functional fragment of one or more molecules; and/or metabolism includes metabolism of amino acids.

59. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of any one of embodiments 42-48, wherein said metabolism comprises enhanced metabolism of lactate and/or amino acids of the modified cell.

60. The modified cell of any preceding embodiment 42-58, wherein the modified cell overexpresses CD98, SNAT1, SNAT2, ASCT2 to enhance transport capacity of the modified cell to transport an amino acid to the modified cell.

61. The isolated nucleic acid sequence, modified cell, method or pharmaceutical composition of embodiments 42-60, wherein one or more of the sequences listed in tables 7, 8 and 9 are overexpressed or downregulated in the modified cell.

62. The modified cell of any one of the preceding embodiments 42-61, wherein the modified cell comprises an antigen binding molecule.

63. The modified cell of embodiment 62, wherein the antigen binding molecule is a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

64. The modified cell of embodiment 63, wherein said 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, TnAG, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra 3, mesothelin, IL-11Ra, PSCA, PRSS 3, VEGFR 3, LewisY, CD3, PDGFR-beta, SSEA-4, CD3, folate receptor alpha, ERBB 3 (Her 3/neu), MUC 3, EGFR, NCAM, protease, ELF 23, Ephrin B3, CX-1 receptor, CAIX, LMP 3, NYb 36100, bcr-3, tyrosine-GM, EPHA-72, PEG 3, MAGE-A1, legumain, HPV E1, MAGE A1, ETV 1-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p1 mutant, proline, survivin and telomerase, PCTA-1/Galetin 8, Mela/MART 1, mutants of hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA1, PAX 1, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART 1, PAX 1, OhsS-EMR 1, LCK, AK-4, SSX 1, RAGE-1, human reverse transcriptase, telomerase, LRRU 1, CD 3679, CD1, and CD 1.

65. The modified cell of any one of embodiments 63 and 64, wherein the intracellular signaling domain comprises a costimulatory signaling domain, or a major signaling domain and a costimulatory signaling domain, wherein the costimulatory signaling domain comprises a functional signaling domain of a protein, wherein the protein comprises CD27, CD28, 4-1BB (CD137), 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 CD3, CDs, ICAM-1, GITR, BAFFR, HVEM (LIGHT), SLAMF 3, NKp3 (KLRF 3), CD160, CD3 α, CD3 β, IL2 β, vly 2, vly 3, VLA 3, CD3 ga 3, CD3, ga 3, CD3 a3, CD3 a3, CD 36, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229, CD160(BY 9), PSGL 9, CD100(SEMA4 9), CD9, SLAMF 9 (NTB-9), SLAAM (SLAMF 9, CD150, IPO-3), BLE (SLAMF 9), LPG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKP 72, NKP 9, NKG2 and NKG 9.

66. The modified cell of embodiment 62, wherein the modified cell comprises an antigen binding molecule that is a modified TCR (TCR) or TCR (TIL).

67. The modified cell of embodiment 65, wherein the TCR is derived from a spontaneously occurring tumor-specific T cell in the patient.

68. The modified cell of embodiment 67, wherein the TCR binds a tumor antigen.

69. The modified cell of embodiment 68, wherein said tumor antigen comprises CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1.

70. The modified cell of embodiment 68, wherein the TCR comprises TCR γ and TCR δ chains or TCR α and TCR β chains, or a combination thereof.

71. The modified cell of embodiment 70, wherein the cell is an immune effector cell (e.g., a population of immune effector cells), and the immune effector cell is a DC, a macrophage, a T cell, or an NK cell.

72. The modified cell of embodiment 71, wherein said immune effector cell is a T cell.

73. The modified cell of embodiment 71, wherein said T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof.

74. The modified cell of any one of the preceding embodiments 42-73, wherein said cell is a human cell.

75. The modified cell of any of the preceding embodiments 42-74, wherein enhanced expression and/or function of the one or more molecules is achieved by introducing a nucleic acid sequence encoding the one or more molecules and/or binding molecules, which nucleic acid sequence is present in the modified cell in the form of a recombinant DNA construct, mRNA or viral vector.

76. The modified cell of embodiment 75, wherein said nucleic acid sequence is an mRNA that is not integrated into the genome of said modified cell.

77. The modified cell of embodiment 75, wherein said nucleic acid sequence is associated with an oxygen-sensitive polypeptide domain.

78. The modified cell of embodiment 77, wherein the oxygen-sensitive polypeptide domain comprises a HIF VHL binding domain.

79. The modified cell of embodiment 75, wherein said nucleic acid sequence is regulated by a promoter comprising a binding site for a transcriptional regulator that regulates expression and/or secretion of a therapeutic agent in the cell.

80. The modified cell of embodiment 79, wherein said transcriptional modulator is or comprises Hif1a, NFAT, FOXP3, and/or NFkB.

Examples of the present invention

Lentiviral vectors encoding a single CAR molecule were generated and transfected with T cells, as described in detail below. Techniques related to cell culture, the construction of cytotoxic T lymphocyte paper can be found in "control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains", PNAS, 3.3.2009, volume one, No. 106(9), 3360-.

On day 0, peripheral blood was extracted from healthy volunteers. CD3+ T cells were selected by the pan T Kit and ranked every 1X 10⁷T cells were supplemented with 100ul TransAct. On day 1, 1X 10⁶T cells were transfected with vector 8301. Will be 1 × 10⁶T cells were transfected with vector 8307 and 2X 10⁶The T cells are non-transduced T cells (NTs). Lentiviruses and TransAct were removed and cells were resuspended in fresh medium. On day 7, flow measurements of TCR ratios were performed. FIG. 6 shows flow cytometry results for expression of various vectors in T cells. Since both vectors encode V β 13.1 (a variant of the TCR β chain), anti-TCR V β 13.1 was used. The expression rate of V β 13.1 in the vector 8301 was 83.58%. The expression rate of V β 13.1 in the vector 8307 was 51.38%. The experiments were performed according to tables 3 and 4. The samples were co-cultured for 24h and subjected to FCM staining with V.beta.13.1 + multiple colorsColor and detection of amplification by FCM staining with cell micro-markers at 120 h. The sequences can be found in table 2 below. More information on the sequence, composition and related clinical trials can be found in WO2020106843 and WO2020146743, which are incorporated herein by reference in their entirety.

TABLE 2

TABLE 3

TABLE 4

FIGS. 7 and 8 show the expression of HLA-A2 and NY-ESO-1 in substrate cells and the expression of zeta chain at 8301 and 8307. TCRT from NY-ESO-1 recognizes the expression of HLA-A2, and the experiment serves as an experimental control material (8301), whereby the flow expression of HLA-A2 in substrate cells was detected, and K19 appeared to be HLA-A2 negative, 293T positive for weak HLA-A2, and 8505C and SAOS-2 positive for HLA-A2. RT-PCR detects the mRNA expression of NY-ESO-1 in 4 substrate cells, and the result shows that 8505C and SAOS-2 are positive to NY-ESO-1. The primer information for RT-PCR is shown in Table 6 below. Figure 8 shows western blot results of the structure of the modified TCR. According to the western blot results, 8307 cells were approximately 23.3kDa in size and were the zeta chain of CD137 after recombination.

TABLE 5

TABLE 6

Primer and method for producing the same	Sequences (5 'to 3')	SEQ ID NO
			NY-ESO-1-RTF1	CGGCAACATACTGACTATCCG	12
NY-ESO-1-RTR1	CTGGAGACAGGAGCTGATGGA	13
			NY-ESO-1-RTF2	TGCAGACCACCGCCAACT	14
NY-ESO-1-RTR2	TCCACATCAACAGGGAAAGCT					15
			β-actin-RTF	CGCCCAGCACGATGAAA	16
β-actin-RTR	CCGCCGATCCACACAGAG	17

TABLE 7 lactic acid metabolism-related molecules

TABLE 8 molecules associated with mitochondrial function

TABLE 9 molecules involved in amino acid metabolism

The disclosure is further described by reference to the following exemplary embodiments and examples. These exemplary embodiments and examples are provided for the purpose of illustration 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 which become apparent as a result of the teachings provided herein.

Sequence listing

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Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

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His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

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Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

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Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val Leu Leu Thr Cys Asp Ala

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Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp Gly Lys Met Ile Gly Phe

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Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp

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Pro Arg Gly Met Tyr Gln Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro

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Leu Gln Val Tyr Tyr Arg Met Cys Gln Asn Cys Ile Glu Leu Asn Ala

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Ala Thr Ile Ser Gly Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val

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Leu Ala Val Gly Val Tyr Phe Ile Ala Gly Gln Asp Gly Val Arg Gln

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Ser Arg Ala Ser Asp Lys Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr

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Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln Gly

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Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val

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Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala

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Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr

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Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg

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Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile

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Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg

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Pro Thr Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser

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Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

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Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

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Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

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

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Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

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Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

210 215 220

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225 230 235 240

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

245 250 255

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

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Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu

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Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His

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Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu

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Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro

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Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg

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Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser

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Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg

115 120 125

Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala

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Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr

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Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

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Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ser Leu

195 200 205

Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn

210 215 220

His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu

225 230 235 240

Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu

245 250 255

Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln

260 265 270

Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala

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Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val

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Lys Arg Lys Asp Ser Arg Gly

305 310

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Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

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Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

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Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

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Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

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

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Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

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Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

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Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

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Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

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Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 12

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cggcaacata ctgactatcc g 21

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<211> 21

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ctggagacag gagctgatgg a 21

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<211> 18

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tgcagaccac cgccaact 18

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<211> 21

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tccacatcaa cagggaaagc t 21

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<211> 17

<212> DNA

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cgcccagcac gatgaaa 17

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ccgccgatcc acacagag 18

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Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu

1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys

20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

85 90 95

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

100 105 110

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

115 120 125

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

130 135 140

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

145 150 155 160

Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

165 170 175

Asp Val Glu Glu Asn Pro Gly Pro Lys Arg Gly Arg Lys Lys Leu Leu

180 185 190

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

195 200 205

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

210 215 220

Glu Leu

225

<210> 19

<211> 833

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<213> Artificial Sequence (Artificial Sequence)

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Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp

1 5 10 15

Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val

20 25 30

Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala

35 40 45

Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr

50 55 60

Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg

65 70 75 80

Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile

85 90 95

Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg

100 105 110

Pro Thr Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser

115 120 125

Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

130 135 140

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

145 150 155 160

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

165 170 175

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

180 185 190

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

195 200 205

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

210 215 220

Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp

225 230 235 240

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

245 250 255

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

260 265 270

Ser Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly

275 280 285

Asp Val Glu Glu Asn Pro Gly Pro Met Ser Ile Gly Leu Leu Cys Cys

290 295 300

Ala Ala Leu Ser Leu Leu Trp Ala Gly Pro Val Asn Ala Gly Val Thr

305 310 315 320

Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln Ser Met Thr Leu

325 330 335

Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser Trp Tyr Arg Gln

340 345 350

Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser Val Gly Ala Gly

355 360 365

Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr Asn Val Ser Arg Ser

370 375 380

Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser Ala Ala Pro Ser Gln

385 390 395 400

Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Val Gly Asn Thr Gly Glu

405 410 415

Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys

420 425 430

Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu

435 440 445

Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe

450 455 460

Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val

465 470 475 480

His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala

485 490 495

Leu Asn Asp Ser Arg Tyr Ser Leu Ser Ser Arg Leu Arg Val Ser Ala

500 505 510

Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe

515 520 525

Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro

530 535 540

Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly

545 550 555 560

Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu

565 570 575

Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser

580 585 590

Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly Gly

595 600 605

Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu

610 615 620

Asn Pro Gly Pro Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu

625 630 635 640

Gln Ala Gln Leu Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp

645 650 655

Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val

660 665 670

Ile Leu Thr Ala Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

675 680 685

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

690 695 700

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

705 710 715 720

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

725 730 735

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

740 745 750

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

755 760 765

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

770 775 780

Met Gln Ala Leu Pro Pro Arg Lys Arg Gly Arg Lys Lys Leu Leu Tyr

785 790 795 800

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

805 810 815

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

820 825 830

Leu

Claims

1. A polynucleotide comprising a sequence encoding the zeta chain of a TCR-CD3 complex linked to one or more costimulatory signaling domains.

2. The polynucleotide of claim 1, wherein the polynucleotide further comprises a sequence encoding a modified TCR-CD3 complex comprising TCR α, TCR β, CD3 γ, CD3 ε, and CD3 δ.

3. The polynucleotide of claim 1, wherein the polynucleotide further comprises a sequence encoding a modified TCR-CD3 complex comprising TCR γ, TCR δ, CD3 γ, CD3 ε, and CD3 δ.

4. The polynucleotide of claim 1, wherein the one or more co-stimulatory signaling domains comprise one or more functional signaling domains of one or more proteins comprising CD, 4-1BB (CD137), OX, CD, PD-1, ICOS, lymphocyte function-associated antigen 1(LFA-1), CD, LIGHT, NKG2, B-H, a ligand that specifically binds to CD, CDs, ICAM-1, GITR, BAFFR, HVEM (LIGHT), SLAMF, KLRF, CD160, CD α, CD β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, itlfgal, CD11, VLA-1, gamma, ITGB, ITGA, ITGB, CD11, ITGB, CD11, ITGB, CD103, ITGB, CD1, ITGB, CD-1, ITGB, CD-1, ITGB, CD, TRANCE/RANKL, DNAM1(CD226), SLAMF4, CD244, 2B4, CD84, CD96, CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELLPG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D or a combination thereof.

5. The polynucleotide of claim 1, wherein the one or more co-stimulatory signaling domains comprises a functional signaling domain of 4-1BB or CD 28.

6. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence encoding the amino acid sequence of SEQ ID NO: 18 or 19.

7. A vector comprising the polynucleotide of any one of claims 1-6.

8. A modified cell comprising the polynucleotide of any one of claims 1-6.

9. The modified cell of claim 8, wherein the modified cell comprises a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen binding domain binds to a tumor antigen comprising TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, eppsma, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- β, SSEA-4, CD20, folate receptor alpha, ERBB2 (ERBB 56/herr), MUC 82695, EGFR 8653, fucokinase, IGF 1, EGFR-848672, fucokinase, IGF-B8414, IGF 368672, IGF 36865, EGFR-B36865, beha-7, beha-4, beha-9, bevacc 695, EGFR-B695, ephora-7, ephora receptor alpha, ephora-B-1, EGFR-H, EGFR-B-H8414, and a receptor therefor, sLe, GM, TGS, HMWMAA, o-acetyl GD, folate receptor beta, TEM/CD 248, TEM7, CLDN, GPRC5, CXORF, CD179, ALK, polysialic acid, PLAC, GloboH, NY-BR-1, UPK, HAVCR, ADRB, PANX, GPR, LY6, OR51E, TARP, WT, NY-ESO-1, LAGE-1a, MAGE-A, legumain, HPV E, MAGE A, ETV-AML, sperm protein 17, XAGE, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p mutant, prostein, survivin and telomerase, telomere PCTA-1/Galectin 8, MelanA/MART, Ras mutant, hTERT sarcoma, translocation breakpoint, PRIAP, ERG (gene), androgen receptor fusion, BOML, BOTMS, SART-1, CYP-MYRT-2, CYP-B, CYP-MYRT, MY-1, MYGE, MYRT-2, MART, RNA, and RNA, 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, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL 1; wherein the intracellular signaling domain comprises a costimulatory signaling domain, or a major signaling domain and a costimulatory signaling domain, and wherein the costimulatory signaling domain comprises one or more functional signaling domains of one or more proteins comprising CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1(LFA-1), CD2, CD7, LIGHT, NKG 27, B7-H7, a ligand that specifically binds to CD7, CDs, ICAM-1, GITR, BAFFR, HVEM (LIGHT), SLAMF7, NKp 7 (KLRF 7), CD160, CD7 a, CD7 β, IL 27 β, vly 2 IL 72, vly 7, VLA 7, CD7 ga 7, CD7 a 7, CD7 a 7, CD7, LFA-1, ITGAM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96 (tactle), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF 69 (NTB-69), SLAM (SLAMF 69, CD150, IPO-3), BLAME (SLAMF 69), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp 69, NK3672, NKG 69, NKG2, or a combination thereof.

10. The modified cell of claim 8, wherein the modified cell comprises a TCR derived from a spontaneously occurring tumor-specific T cell in a patient, the modified cell comprising a TCR that binds a tumor antigen comprising CEA, gp100, MART-1, p53, MAGE-A3, or NY-ESO-1.