CN111676195A

CN111676195A - UCAR immune cell for treating T cell tumor

Info

Publication number: CN111676195A
Application number: CN201910178766.1A
Authority: CN
Inventors: 谷为岳
Original assignee: Beijing Cartesian Medical Technology Co ltd
Current assignee: Beijing Cartesian Medical Technology Co ltd
Priority date: 2019-03-11
Filing date: 2019-03-11
Publication date: 2020-09-18

Abstract

A UCAR immune cell for treating T cell tumor is provided. Given that T cell tumors are difficult to treat with CAR-T cells of autologous origin in patients. Therefore, the invention provides a universal CAR-T or universal CAR-NK cell that targets a portion of the TCR structure of a T cell tumor, which is allogeneic in origin, without relying on cells from the patient's own source, and will become a rational choice for treatment of T cell tumors.

Description

UCAR immune cell for treating T cell tumor

Technical Field

The invention belongs to the technical field of biological medicines, and particularly belongs to the field of immune cell therapy.

Background

The CAR-T cells applied by the current CAR-T immune cell therapy technology are mostly of autologous sources of patients, and in the case of T cell tumors, tumor cells are inevitably doped in autologous T cells, so that the CAR-T technology of autologous sources is difficult to apply.

Disclosure of Invention

In view of the above background, there is an urgent need for methods, systems and products for immune cell function in the treatment of T cell tumors, and the present invention satisfies this need. The invention provides a universal CAR-T or universal CAR-NK cell targeting a partial structure of the TCR of a T cell tumor, the T cell or NK cell being allogeneic in origin, without relying on cells of autologous origin of the patient, which would be a rational choice for treatment of T cell tumors.

Unless otherwise indicated, practice of some of the methods disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: a Laboratory Manual, 4th edition (2012); current protocols for molecular biology in series (f.m. ausubel, et al. eds.); the series of methods were performed in enzymology (Academic Press, Inc.), PCR 2: a Practical Approach (M.J.Machersrs, B.D.Hames and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A laboratory Manual, and Culture of Animal Cells: a Manual of Basic Technique and specialized Applications, 6th Edition (R.I. Breshney, ed. (2010)).

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or a standard deviation of greater than 1, according to practice in the art. Alternatively, "about" may represent a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may denote an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of the value. Where particular values are described in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within an acceptable error range for the particular value.

As used herein, "cell" may generally refer to a biological cell. A cell may be the basic structure, function and/or biological unit of an organism. The cells may be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, unicellular eukaryotic cells, protozoal cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, cereals, soybeans, corn, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmoss, hornworts, moss), algal cells (e.g., Botryococcus braunini, chlamydomonorenhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, rgassium patens c. agrahara, etc.), algae (e.g., kelp), fungal cells (e.g., yeast cells, a) from mushrooms), animal cells, cells from invertebrates (e.g., fruit, spiny, nematode, vertebrate cells (e.g., nematode), fish, amphibian, reptile, avian, mammalian) cells from mammals (e.g., primary cells of pigs, cows, goats, sheep, rodents, rats, mice, non-humans) te, humans, etc.). Sometimes the cells are not derived from a natural organism (e.g., the cells may be synthetic, sometimes referred to as artificial cells).

The term "antigen" as used herein refers to a molecule or fragment thereof capable of being bound by a selective binding agent. For example, the antigen may be a ligand that can be bound by a selective binding agent, such as a receptor. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent, such as an immunoprotein (e.g., an antibody). An antigen may also refer to a molecule or fragment thereof that can be used in an animal to produce antibodies that are capable of binding the antigen.

As used herein, the term "neoantigen" generally refers to a tumor-specific antigen caused by a gene mutation. The resulting muteins or fragments thereof can elicit an anti-tumor T cell response.

The term "gene" as used herein refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence encoding an RNA transcript. As used herein, the term with respect to genomic DNA includes intervening non-coding regions as well as regulatory regions, and may include 5 'and 3' ends. In some uses, the term includes transcribed sequences, including 5 'and 3' untranslated regions (5 '-UTR and 3' -UTR), exons and introns. In some genes, the transcribed region will comprise an "open reading frame" encoding the polypeptide. In some uses of this term, a "gene" comprises only coding sequences (e.g., "open reading frames" or "coding regions") necessary to encode a polypeptide. In some cases, the gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (trna) genes. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an "endogenous gene" or a native gene in its natural location in the genome of an organism. A gene may refer to a "foreign gene" or a non-native gene. A non-native gene may refer to a gene that is not normally found in a host organism but is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not in a native location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises a mutation, insertion, and/or deletion (e.g., a non-native sequence).

The term "antibody" as used herein refers to a protein binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal), as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). A derivative, variant or fragment thereof may refer to a functional derivative or fragment that retains the binding specificity (e.g., in whole and/or in part) of the corresponding antibody. Antigen binding fragments include Fab, Fab ', F (ab') 2, variable fragments (Fy), single chain variable fragments (scFv), minibodies, diabodies and single domain antibodies ("sdAb" or "nanobody" or "camelid"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (e.g., antibodies optimized in fragment crystallizable regions) and multispecific antibodies (e.g., bispecific antibodies).

The term "antibody" as used herein refers to a protein binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal), as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). A derivative, variant or fragment thereof may refer to a functional derivative or fragment that retains the binding specificity (e.g., in whole and/or in part) of the corresponding antibody. Antigen binding fragments include Fab, Fab ', F (ab') 2, variable fragments (Fv), single chain variable fragments (scFv), minibodies, diabodies and single domain antibodies ("sdAb" or "nanobody" or "camelid"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (e.g., antibodies optimized in fragment crystallizable regions) and multispecific antibodies (e.g., bispecific antibodies).

The term "nucleotide" as used herein generally refers to an alkali-sugar-phosphate combination. The nucleotides may comprise synthetic nucleotides. The nucleotides may comprise synthetic nucleotide analogs. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphate Adenosine Triphosphate (ATP), Uridine Triphosphate (UTP), Cytosine Triphosphate (CTP), Guanosine Triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof. . These derivatives may include, for example, [ α S ] dATP, 7-deaza-dGTP and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphate (ddNTP) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling can also be performed with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof, of any length, and can be in single-, double-, or multistrand form. The polynucleotide may be exogenous or endogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or a fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The polynucleotide may comprise one or more analogs (e.g., altered backbone, sugar or nucleobases).

The term "expression" refers to one or more of the processes of transcription of a polynucleotide from a DNA template (e.g., into mRNA or other RNA transcript) and/or the subsequent translation of the transcribed mRNA into a peptide. A polypeptide or a protein. The transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may comprise splicing of the mRNA in a eukaryotic cell. By reference to expression, "up-regulation" generally refers to an increase in the level of expression of a polynucleotide (e.g., RNA, e.g., mRNA) and/or polypeptide sequence, but "down-regulation," relative to its level of expression in the wild-type state. "generally refers to a decrease in the level of expression of a polynucleotide (e.g., RNA, e.g., mRNA) and/or polypeptide sequence relative to its expression in the wild-type state.

As used herein, the term "modulate" with respect to expression or activity refers to altering the level of expression or activity. Modulation may occur at the transcriptional level and/or the translational level.

The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bonds. The term does not imply a polymer of a particular length, nor does it imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins (e.g., domains) with or without secondary and/or tertiary structure. The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation, such as conjugation to a labeling component. The terms "amino acid" and "amino acid" as used herein generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids that have been chemically modified to include groups or chemical moieties that do not naturally occur on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes D-amino acids and L-amino acids.

The terms "derivative," "variant," and "fragment" when used herein with respect to a polypeptide refer to the polypeptide relative to the wild-type polypeptide, e.g., by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity), and/or function. Derivatives, variants, and fragments of the polypeptides may comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.

As used herein, "fusion" may refer to a protein and/or nucleic acid comprising one or more non-native sequences (e.g., portions). The fusion may comprise one or more of the same non-native sequences. The fusion may comprise one or more different non-native sequences. The fusion may be a chimera. The fusion may comprise a nucleic acid affinity tag. The fusion may include a barcode. The fusion may comprise a peptide affinity tag. Fusions can provide subcellular localization of site-directed polypeptides (e.g., Nuclear Localization Signals (NLS) for targeting the nucleus, mitochondrial localization signals for targeting mitochondria, chloroplast localization signals for targeting chloroplasts, Endoplasmic Reticulum (ER) retention signals, etc. fusions can provide non-native sequences (e.g., affinity tags) that can be used for tracking or purification.

The phrase "artificial TCR", as used herein, may be understood as "exogenous T Cell Receptor (TCR) complex", referring to a TCR complex in which one or more chains of a TCR are introduced into the genome of an immune cell. The TCR may or may not be expressed endogenously. In some cases, an exogenous TCR complex can refer to a TCR complex in which one or more chains of an endogenous TCR complex have one or more mutated sequences, e.g., at the nucleic acid or amino acid level. Expression of an exogenous TCR on an immune cell can confer binding specificity to an epitope or antigen (e.g., an epitope or antigen that preferentially resides on the surface of a cancer cell or other pathogenic cell or particle). The exogenous TCR complex can comprise a TCR- α, TCR- β chain, CD3- γ chain, CD 3-chain, CD 3-zeta chain, or any combination thereof introduced into the genome. In some cases, the strand introduced into the genome may replace the endogenous strand.

The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also included are tissues, cells and progeny of the biological entities obtained in vivo or cultured in vitro.

As used herein, the terms "treatment" and "treating" refer to a method for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and/or a prophylactic benefit. For example, treatment can include administration of a system or population of cells disclosed herein. Therapeutic benefit refers to any therapeutically relevant improvement or effect of one or more diseases, disorders or symptoms in treatment. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, disorder or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, disorder or symptom is. May not yet be present.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition, e.g., a composition comprising an immune cell of the present disclosure, such as a lymphocyte (e.g., a T lymphocyte and/or an NK cell), sufficient to produce a desired activity when administered to a subject in need thereof. In the context of the present disclosure, the term "therapeutically effective" means that the amount of the composition is sufficient to delay performance, arrest progression, alleviate or alleviate at least one symptom of the condition being treated by the method of the invention. Disclosed is a method for producing a semiconductor device.

As used herein, the term "genetic profile" refers to information about a particular gene, including variations and gene expression in an individual or in a certain type of tissue. Genetic profiles can be used for neoantigen selection. As used herein, the term "somatic mutation profile" refers to information about a particular gene associated with a somatic mutation, including, but not limited to, the particular gene resulting from the somatic mutation. Somatic mutation profiles can be used for neoantigen selection.

In one aspect, the present disclosure provides a modified T cell that specifically binds an antigen, the modified T cell comprising a Chimeric Antigen Receptor (CAR). The CAR can comprise an extracellular domain (ECD) of the protein. The ECD may be fused to the intracellular domain (ICD) of a costimulatory molecule that mediates signals for activation of immune cells. Binding of the CAR to the antigen can generate an immune cell activation signal in the modified T cell, rather than an immune cell inactivation signal.

Binding of a CAR-modified immune cell (e.g., a modified CAR-T cell or a modified CAR-NK as provided herein) to an antigen or a target cell expressing an antigen can activate the immune cell. The cellular modified CAR can be used to provide further control over immune cell activity, such as but not limited to immune cell activation and expansion. Binding of the CAR to the antigen or target cell expressing the antigen can elicit an immune cell activation signal in the modified immune cell rather than an immune cell inactivation signal. Priming immune cell activation signals in modified immune cells can minimize immunosuppression in immune cells. Minimizing immunosuppression in immune cells may increase the effectiveness of immune cells in an immune response, for example, by increasing immune cell cytotoxicity against target cells (e.g., tumor cells).

The ECD and ICD of the CAR may be connected by a transmembrane domain, for example by a transmembrane segment. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide may have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of a transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions, deletions, and polypeptides. The insertion is compared to the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-native polypeptide sequence, such as a sequence of a polypeptide linker. The polypeptide linker may be flexible or rigid. The polypeptide linker may be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from the ECD to the ICD, e.g., a signal indicative of ligand binding. In some embodiments, the ECD comprises a transmembrane domain. In some embodiments, the ICD comprises a transmembrane domain.

In some embodiments, ICD may mediate the production of immune cell activation signals (or immune cell activation signals) in immune cells. In some embodiments, the immune cell activation signal is mediated by an activating factor. The activator may be an immunomodulatory molecule. The activating factor may bind to, activate or stimulate T cells or other immune cells to modulate their activity. In some embodiments, the activator can be secreted from the immune cell. The activator can be, for example, a soluble cytokine, a soluble chemokine, or a growth factor molecule. Non-limiting examples of activating factors that can mediate immune cell activation include soluble cytokines such as IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, Tumor Necrosis Factor (TNF), Transforming Growth Factor (TGF), Interferon (IFN), or any functional fragment or variant thereof.

The immune cell activation signal may include or result in clonal expansion of a modified immune cell (e.g., a modified TIL or a modified T cell); release of cytokines by modified immune cells (e.g., modified TILs or modified T cells); cytotoxicity of modified immune cells (e.g., modified TILs or modified T cells); proliferation of modified immune cells (e.g., modified TILs or modified T cells); differentiation, dedifferentiation or transdifferentiation of modified immune cells (e.g., modified TILs or modified T cells); movement and/or transport of modified immune cells (e.g., modified TILs or modified T cells); depletion and/or reactivation of modified immune cells (e.g., modified TILs or modified T cells); other intercellular molecules, metabolites, chemical compounds, or combinations thereof are released by the modified immune cells (e.g., modified TILs or modified T cells).

In some embodiments, the immune cell activation signal comprises or results in clonal expansion of an immune cell. Clonal expansion may include the generation of daughter cells produced by immune cells. The daughter cells resulting from clonal expansion may comprise a CAR. The clonal expansion of the modified immune cell can be greater than the clonal expansion of a comparable immune cell lacking the CAR. Clonal expansion of the modified immune cell can be about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 200-fold. The ratio may be about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, or about 600-fold to about 700-fold greater than a ratio lacking the CAR. In some embodiments, determining clonal expansion can include quantifying a number of immune cells, e.g., with and without a CAR and after binding of an antigen or a target cell expressing an antigen to a CAR. Quantification of many immune cells can be achieved by a variety of techniques, non-limiting examples of which include flow cytometry, trypan blue exclusion, and blood cell counting.

In some embodiments, the immune cell activation signal comprises or causes an immune cell to release a cytokine. In some embodiments, the immune cell activity comprises or results in the release of an intercellular molecule, metabolite, chemical compound, or combination thereof. Cytokines released by the modified immune cells may include release of IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-21, IL-22, IFN γ, TNF α, CSF, TGF β, granzyme, and the like. In some embodiments, cytokine release may be quantified using enzyme-linked immunosorbent assay (ELISA), flow cytometry, western blot, and the like. The cytokine release of the modified immune cell can be greater than the cytokine release of a comparable immune cell lacking the CAR. The modified immune cells provided herein can be produced about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold. 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or more than 300-fold cytokine release, as compared to a comparable immune cell lacking a CAR. When the CAR binds to the antigen or a target cell expressing the antigen, the modified immune cell can exhibit increased cytokine secretion compared to a comparable immune cell lacking the CAR (e.g., unmodified). In some embodiments, the secreted cytokine is IFN gamma or IL-2. In some embodiments, cytokine release may be quantified in vitro or in vivo.

In some embodiments, the immune cell activation signal comprises or causes cytotoxicity of the immune cell. In some cases, the cytotoxicity of the modified immune cells provided herein can be used to kill target cells. The immune cell or population of immune cells expressing the CAR can induce death of the target cell. Killing the target cells can be used for a variety of applications, including but not limited to treating diseases or conditions where elimination of a cell population is desired or where inhibition of proliferation is desired. Cytotoxicity may also refer to the release of cytotoxic cytokines, such as IFN γ or granzyme, by immune cells. In some cases, the modified immune cells provided herein can alter (i) the release of cytotoxins, such as perforin, granzyme, and granulysin and/or (ii) induce apoptosis through Fas-Fas ligand interactions between T cells and target cells. In some embodiments, cytotoxicity can be quantified by a cytotoxicity assay, including a co-culture assay, an ELISPOT, a chromium release cytotoxicity assay, and the like. The modified immune cells provided herein can have cytotoxicity greater than a comparable immune cell lacking the CAR. The modified immune cell can exhibit increased cytotoxicity against a target cell when the CAR binds to the antigen or target cell expressing the antigen compared to a comparable immune cell lacking the CAR (e.g., unmodified). A cell. The modified immune cells of the invention may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. % 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175% or 200% more cytotoxic to target cells compared to a comparable immune cell lacking the CAR. The modified immune cells of the invention can induce target cell death by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200% of the molecules over comparable immune cells without a switch. In some embodiments, the immune cells provided herein can induce apoptosis in a target cell that displays a target epitope (e.g., a neoantigen) on its surface. In some embodiments, cytotoxicity can be determined in vitro or in vivo. In some embodiments, determining cytotoxicity can comprise determining a level of disease after administration of the modified immune cells provided herein as compared to the level of disease prior to administration. In some embodiments, determining cytotoxicity can comprise determining a level of disease after administration of a modified immune cell provided herein and a level of disease after administration of a comparable immune cell lacking the CAR.

In some embodiments, the immune cell activation signal comprises or results in the proliferation of an immune cell. The proliferation of immune cells may refer to the expansion of immune cells. Proliferation of an immune cell may refer to a phenotypic change in the immune cell. The proliferation of the modified immune cells of the present disclosure can be greater than the proliferation of comparable immune cells lacking the CAR. The modified immune cells provided herein can proliferate about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, or about 600-fold to about 700-fold greater than a comparable proliferative immune cell deficiency CAR. In some embodiments, proliferation can be determined by quantifying a number of immune cells. Quantifying many immune cells may include flow cytometry, trypan blue exclusion, and/or cytometry. Proliferation can also be determined by phenotypic analysis of immune cells.

In some embodiments, the immune cell activation signal may include or result in differentiation, dedifferentiation, or transdifferentiation of the immune cell. Differentiation, dedifferentiation or transdifferentiation of immune cells can be determined by flow cytometry evaluating the phenotypic expression of markers of differentiation, dedifferentiation or transdifferentiation on the cell surface. In some embodiments, the modified immune cells provided herein have an increased capacity to differentiate compared to a comparable immune cell lacking the CAR. In some embodiments, the modified immune cells provided herein have increased capacity to dedifferentiate compared to a comparable immune cell lacking the CAR. In some embodiments, the modified immune cells provided herein have increased capacity to dedifferentiate compared to a comparable immune cell lacking the CAR. In some embodiments, the modified immune cells provided herein have greater transdifferentiation capacity compared to a comparable immune cell lacking the CAR.

In some embodiments, the immune cell activation signal may include or result in movement and/or trafficking of immune cells. In some embodiments, movement can be determined by quantifying the localization of immune cells to a target site. For example, the modified immune cells provided herein can be quantified at a target site after administration, e.g., at a site that is not the target site. Quantification can be performed by isolating the lesion and quantifying many immune cells (e.g., tumor infiltrating lymphocytes) that contain the CAR. Movement and/or trafficking of an immune cell comprising the CAR can be greater than movement and/or trafficking of a comparable immune cell lacking the CAR. In some embodiments, the number of immune cells comprising the CAR at the target site (e.g., a tumor lesion) can be about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, or 40-fold greater than the comparable immune number. The cell lacks CAR. Trafficking can also be determined in vitro using the transwell migration assay. In some embodiments, the number of immune cells comprising the CAR at the target site, e.g., in a transwell migration assay, can be about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, or 40-fold the number. Comparable immune cells lack CAR.

In some embodiments, the immune cell activation signal may include or result in depletion and/or activation of immune cells. Depletion and/or activation of immune cells can be determined by phenotypic analysis by flow cytometry or microscopy analysis. For example, the expression levels of exhaustion markers, such as programmed cell death protein 1(PD1), lymphocyte activation gene 3 protein (LAG3), 2B4, CD160, Tim3, and T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), are determined quantitatively and/or qualitatively. In some cases, immune cells (e.g., T cells) lose effector function in a hierarchical manner and become exhausted. Due to fatigue, functions such as IL-2 production and cytokine expression, as well as high proliferation potency, may be lost. Exhaustion can also be a defect in the production and degranulation of IFN γ, TNF and chemokines. Depletion or activation of a modified immune cell provided herein can be greater than depletion or activation of a comparable immune cell lacking the CAR. In some embodiments, an immune cell provided herein can undergo at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold. 13-fold, 14-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, or more than 300-fold compared to a comparable immune cell lacking the CAR, in fatigue or activation. In some embodiments, the contained immune cells provided herein can undergo at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold. Fold, 13 fold, 14 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 250 fold, or more than 300 fold, has reduced failure or activation compared to a comparable immune cell lacking the CAR.

In some embodiments, binding of the target cell to the CAR can generate an immune cell activation signal at the CAR-modified immune cell.

In one aspect, the present disclosure provides a modified immune cell comprising a Chimeric Antigen Receptor (CAR) and a T Cell Receptor (TCR) complex that exhibits specific binding to a novel antigen. The CAR may comprise an antigen-interacting domain capable of binding to a T cell surface protein, a transmembrane domain, and an intracellular signaling domain.

The antigen binding domain may comprise any protein or molecule capable of binding an antigen, such as a T cell surface protein. Non-limiting examples of antigen binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies or functional derivatives, variants or fragments thereof. Including but not limited to Fab, Fab ', F (ab') 2, Fv, single chain Fv (scfv), minibodies, diabodies and single domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains (VHH) of camelid-derived nanobodies. In some embodiments, the first antigen binding domain comprises at least one of a Fab, Fab ', F (ab') 2, Fv, and scFv. In some embodiments, the antigen binding domain comprises an antibody mimetic. Antibody mimetics refer to molecules capable of binding a target molecule with an affinity comparable to an antibody, including single chain binding molecules, cytochrome b562 based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectins), lipocalin scaffolds, calixarene scaffolds, a domains, and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor or any derivative, variant or fragment thereof. For example, antigen binding domains

In some embodiments, the antigen binding domain may comprise a scFV. The scFv may be derived from an antibody of known variable region sequence. In some embodiments, the scFv may be derived from antibody sequences obtained from available mouse hybridomas. scFv can be obtained from sequencing of all exons from tumor cells or primary cells. In some embodiments, the scFv can be altered. For example, the scFv may be modified in various ways. In some cases, the scFv may be mutated such that the scFv may have a higher affinity for its target. In some cases, the affinity of an scFv for its target may be optimized for targets that are expressed at low levels on normal tissues. This optimization can be done to minimize potential toxicity, such as hypercytokinemia. In other cases, cloning of an scFv with higher affinity for the target membrane-bound form may be preferred over its soluble form counterpart. This modification can be made if certain targets can also be detected in soluble form at different levels, and their targeting can cause unintended toxicity, such as hypercytokinemia.

The antigen binding domain of the CAR of the present system may be linked to an intracellular signaling domain by a transmembrane domain. The transmembrane domain may be a transmembrane segment. The transmembrane domain of the subject CAR can anchor the CAR to the plasma membrane of a cell, e.g., an immune cell. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide linking the antigen binding domain and the intracellular signaling domain of the CAR can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of a transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions, deletions, and polypeptides. The insertion is compared to the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-native polypeptide sequence, such as a sequence of a polypeptide linker. The polypeptide linker may be flexible or rigid. The polypeptide linker may be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from an extracellular region of a cell to an intracellular region through an antigen binding domain. The natural transmembrane portion of CD28 is useful for CARs. In other cases, the native transmembrane portion of CD8 a may also be used for CARs.

The CAR of the present disclosure may comprise a signaling domain or any derivative, variant, or fragment thereof that is involved in immune cell signaling. The intracellular signaling domain of the CAR can induce the activity of an immune cell comprising the CAR. Intracellular signaling domains can transduce effector function signals and direct cells to perform specialized functions. The signaling domain may comprise a signaling domain of another molecule. In some cases, a truncated portion of the signal domain is used for CAR.

In some embodiments, the intracellular signaling domain comprises a plurality of signaling domains involved in immune cell signaling, or any derivative, variant, or fragment thereof. For example, an intracellular signaling domain may comprise at least 2 immune cell signaling domains, e.g., at least 2, 3, 4, 5, 7, 8, 9, or 10 immune cell signaling domains. The immune cell signaling domain may be involved in modulating primary activation of the TCR complex in either a stimulatory or inhibitory manner. The intracellular signaling domain may be a signaling domain of a T Cell Receptor (TCR) complex. The intracellular signaling domain of the subject CARs can comprise Fc γ receptor (fcyr), Fc receptor (FcR), Fc α receptor (fcar), neonatal Fc receptor (FcRn), CD3, CD3 ζ, CD3 γ signaling domain, CD3, CD3, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79B, CD80, CD86, CD278 (also known as ICOS), CD247 ζ, CD247 η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF- κ B, PLC- γ, iC3B, C3dg, C3d, and Zap 70. In some embodiments, the signaling domain comprises an immunoreceptor tyrosine-based activation motif or ITAM. The signaling domain comprising ITAM may comprise two repeated amino acid sequences YxxL/I, which are 6-8 amino acids apart, wherein each x is independently any amino acid, resulting in the conserved motif YxxL/Ix (6-8) YxxL/I. The signaling domain comprising ITAMs can be modified, e.g., by phosphorylation, when the antigen binding domain binds to an epitope. Phosphorylated ITAMs can serve as docking sites for other proteins, such as proteins involved in various signaling pathways. In some embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated, truncated, and/or optimized ITAM domain, having altered (e.g., increased or decreased) activity compared to a native ITAM domain.

In some embodiments, the intracellular signaling domain of the subject CAR comprises an fcyr signaling domain (e.g., ITAM). The Fc γ R signaling domain may be selected from Fc γ RI (CD64), Fc γ RIIA (CD32), Fc γ RIIB (CD32), Fc γ RIIIA (CD16a) and Fc γ RIIIB (CD16 b). In some embodiments, the intracellular signaling domain comprises an FcR signaling domain (e.g., ITAM). The FcR signaling domain may be selected from FcRI and FcRII (CD 23). In some embodiments, the intracellular signaling domain comprises an Fc α R signaling domain (e.g., ITAM). The Fc α R signaling domain may be selected from Fc α RI (CD89) and Fc α/μ R. In some embodiments, the intracellular signaling domain comprises a CD3 zeta signaling domain. In some embodiments, the primary signaling domain comprises ITAM of CD3 ζ.

In some embodiments, the intracellular signaling domain of the subject CAR comprises an immunoreceptor tyrosine-based inhibitory motif or ITIM. The ITIM-containing signaling domain may comprise a conserved amino acid sequence (S/I/V/LxYxxI I/V/L) that is present in the cytoplasmic tail of some inhibitory receptors of the immune system. The major signaling domain comprising ITIM may be modified, e.g., phosphorylated, by enzymes, e.g., Src kinase family members (e.g., Lck). After phosphorylation, other proteins, including enzymes, can be recruited to ITIMs. These other proteins include, but are not limited to, enzymes such as phosphotyrosine phosphatases SHP-1 and SHP-2, the phytases known as SHIPs, and proteins with one or more SH2 domains (e.g., ZAP 70). Intracellular signaling domains may comprise BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, fcyriib (CD32), Fc receptor-like protein 2(FCRL2), the signaling domain of an Fc receptor (e.g., ITIM). Analogous protein 3(FCRL3), Fc receptor-like protein 4(FCRL4), Fc receptor-like protein 5(FCRL5), Fc receptor-like protein 6(FCRL6), protein G6B (G6B), interleukin 4 receptor (IL4R), immunoglobulin superfamily receptor translocation related 1(IRTA1), immunoglobulin superfamily receptor translocation related 2(IRTA2), killer cell immunoglobulin-like receptor 2DL1(KIR2DL1), killer cell immunoglobulin-like receptor 2DL2(KIR2DL2), killer cell immunoglobulin-like receptor 2DL3(KIR2DL3), killer cell immunoglobulin-like receptor 2DL4(KIR2DL4), killer cell immunoglobulin-like receptor 2DL5(KIR2DL5), killer cell immunoglobulin-like receptor DL1(KIR3DL1), killer cell immunoglobulin-like receptor DL 3DL2(KIR3DL2), killer cell immunoglobulin-like receptor B subfamily 2), immunoglobulin B receptor subfamily (IRL 1), leukocyte immunoglobulin-like receptor subfamily B member 3(LIR3), leukocyte immunoglobulin-like receptor subfamily B member 5(LIR5), leukocyte immunoglobulin-like receptor subfamily B member 8(LIR8), leukocyte-associated immunoglobulin-like receptor 1(LAIR-1), mast cell function-associated antigen (MAFA), NKG2A, natural cytotoxicity trigger receptor 2 (lp 44), NTB-A, programmed cell death protein 1(PD-1), PILR, SIGLECL1, sialic acid-binding Ig such as lectin 2(SIGLEC2 or CD22), sialic acid-binding Ig such as lectin 3(SIGLEC3 or CD33), sialic acid-binding Ig such as lectin 5(SIGLEC 5or CD170), sialic acid-binding Ig such as lectin 6(SIGLEC6), sialic acid-binding Ig such as lectin 7(SIGLEC7), sialic acid-binding such as lectin 10(SIGLEC10), sialic acid-binding Ig such as lectin 11), sialic acid binding igs such as lectin 4(SIGLEC4), sialic acid binding igs such as lectin 8(SIGLEC8), sialic acid binding igs such as lectin 9 (SIGLEC EC9), platelet and endothelial cell adhesion molecule 1(PECAM-1), signal regulatory protein (SIRP2) and signal threshold regulating transmembrane adaptor 1 (SIT). In some embodiments, the intracellular signaling domain comprises a modified ITIM domain, e.g., a mutated, truncated, and/or optimized ITIM domain, having altered (e.g., increased or decreased) activity compared to a native ITIM domain.

In some embodiments, the intracellular signaling domain comprises at least 2 ITAM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains). In some embodiments, the intracellular signaling domain comprises at least 2 ITIM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITIM domains) (e.g., at least 2 major signaling domains). In some embodiments, the intracellular signaling domain comprises an ITAM and ITIM domain.

In some cases, the intracellular signaling domain of the subject CAR can include a costimulatory domain. In some embodiments, the co-stimulatory domain, e.g., from a co-stimulatory molecule, may provide a co-stimulatory signal for immune cell signaling, e.g., signaling from the ITAM and/or ITIM domains, e.g., for activation and/or inactivation. And (4) immune cell activity. In some embodiments, the co-stimulatory domain may be used to modulate proliferation and/or survival signaling in immune cells. In some embodiments, the co-stimulatory signaling domain comprises an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activated signaling domain. NK cell receptor, BTLA or Toll ligand receptor. In some embodiments, the co-stimulatory domain comprises a signaling domain of a molecule selected from the group consisting of: 2B4/CD244/SLAMF4, 4-1BB/TNFSF9/CD137, B7-1/CD80, B7-2/CD86 and B7. -H/PD-L, B-H, B-H, B-H, B-H, B-H, BAFF R/TNFRSF13, BAFF/BLyS/TNFRSF 13, BLAME/SLAMF, BTLA/CD272, CD100 (SEA 4), CD103, CD11, CD150, CD160 (BY), CD, CD, CD200, CD229/SLAMF, CD ligand/TNFRSF, CD, CD, CD 2-10/SLAMF, CD ligand/TNFRSF, CD 300/LMIR, CD, CD ligand/TNFRSF, CD/SLAMF, CD49, CD49, CD49, CD, CD, CD/A-3, CD, CD, CD, CD alpha, CD beta, CD/Kai-1, CD/SLAMF, CD1, CD/SLAMF, CD, CACA, CTLA/CAMC, CD1, TAC, CAMC-7, CAMC-1, CAMC-CTLA-7, CAMC-CAMF, DNAM (CD226), DPPIV/CD, DR/TNFRSF, EPHB, GADS, Gi/VISTA/B-H, GITR ligand/TNFRSF, GITR/TNFRSF, HLA class I, HLA-DR, HVEM/TNFRSF, IA, ICAM-1, ICOS/CD278, Ikaros gene, IL2 β, IL2 γ, IL7 α, integrin α 4/CD49, integrin α 4 β 1, integron α 4 β 7/LPAM-1, IPO-3, ITGA, ITGA, ITGAD, ITGAE, ITGAL, ITGAGAI, ITGB, ITGB, ITGB, KIRDS, LAG-3, LAT, LIGHT/TNFRSF, LTBR, Ly108, KL (LY 229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin- α/β, NKG2, NKG2, NKP 2, NKSFP/NKSFP, TNFRSF, TNFR-P/NKSF, TNFR-P, NKSF, TNFR-1, TNFR-P/NKSF, PAG/Cbp, PD-1, PDCD6, PD-L2/B7-DC, PSGL1, RELT/TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM/CD150, SLAMF4(CD244), SLAMF6(NTB-A)), SLAMF7, SLP-76, TACI/TNFRSF13B, TCL1A, TCL1B, TIM-1/KIM-1/HAVCR, TIM-4, TL1A/TNFSF15, TNF RII/TNFRSF1B, TNF-alpha, TRANCE/RANKL, TSLP, TSLP R, VLA1 and VLA-6. In some embodiments, the intracellular signaling domain comprises a plurality of co-stimulatory domains, e.g., at least two, e.g., at least 3, 4, or 5 co-stimulatory domains. The costimulatory signaling region can provide a signal in concert with the primary effector activation signal, and can fulfill the requirements for activating T cells. In some embodiments, the addition of a co-stimulatory domain to a CAR can enhance the efficacy and persistence of an immune cell provided herein.

Binding of the CAR to the target cell surface protein can enhance immune cell proliferation compared to an immune cell lacking the CAR. The proliferation of immune cells may refer to the expansion of immune cells. Proliferation of an immune cell may refer to a phenotypic change in the immune cell. The proliferation of an immune cell comprising a CAR provided herein can be greater than the proliferation of an immune cell lacking the CAR that exhibits binding to a target cell surface protein. The proliferation of an immune cell comprising the CAR can be about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, compared to the proliferation of an equivalent immunity of about 600-fold to about 700-fold. The cell lacks CAR. The proliferation of an immune cell comprising the CAR can be about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 200-fold, about 200-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 500-fold to about 600-fold, compared to the proliferation of an equivalent immunity of about 600-fold to about 700-fold. A cell that lacks the CAR, and wherein proliferation is determined at least about 12, 24, 36, 48, 60, 72, 84, or 96 hours after contacting the target cell with the target cell surface protein. Enhanced proliferation can be determined in vitro or in vivo. In some embodiments, proliferating may include quantifying the number of immune cells. Quantifying many immune cells may include flow cytometry, trypan blue exclusion, and/or cytometry. Proliferation can also be determined by phenotypic analysis of immune cells.

Cytokines refer to proteins released by cells (e.g., chemokines, interferons, lymphokines, interleukins, and tumor necrosis factors) that can affect cell behavior. Cytokines are produced by a variety of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts and various stromal cells. A given cytokine may be produced by more than one cell. Cytokines may be involved in producing systemic or local immunomodulation.

Certain cytokines may act as pro-inflammatory cytokines. Proinflammatory cytokines refer to cytokines that are involved in inducing or amplifying an inflammatory response. Proinflammatory cytokines can produce an immune response with various cells of the immune system (e.g., neutrophils and leukocytes). Certain cytokines may act as anti-inflammatory cytokines. Anti-inflammatory cytokines refer to cytokines that are involved in reducing inflammatory responses. In some cases, anti-inflammatory cytokines may modulate pro-inflammatory cytokine responses. Some cytokines may act as both pro-inflammatory and anti-inflammatory cytokines. Certain cytokines, such as chemokines, may play a role in chemotaxis. Chemokines can induce directional chemotaxis in nearby responding cells.

In some embodiments, the expression of cytokines with pro-inflammatory and/or chemotactic functions can be up-regulated in immune cells. Upregulation of the expression of cytokines with pro-inflammatory and/or chemotactic functions can be used, for example, to stimulate an immune response against target cells in immunotherapy.

Examples of cytokines that may be overexpressed by the immune cells provided herein include, but are not limited to, lymphokines, monokines, and traditional polypeptide hormones. The cell factor includes growth hormone, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; loosening; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha; a Muller inhibitor; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; integrating; thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet growth factor; transforming Growth Factors (TGFs), such as TGF- α, TGF- β, TGF- β 1, TGF- β 2, and TGF- β 3; insulin-like growth factors-I and-II; erythropoietin (EPO); FLT-3L; stem Cell Factor (SCF); (ii) an osteoinductive factor; interferons (IFNs), such as IFN-alpha, IFN-beta, IFN-gamma; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF); macrophage stimulating factor (MSP); interleukins (ILs), such as IL-1, IL-1a, IL-1b, IL-1RA, IL-18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20; tumor necrosis factors such as CD154, LT- β, TNF- α, TNF- β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE; and other polypeptide factors including LIF, oncostatin M (OSM) and Kit Ligand (KL). Cytokine receptors refer to receptor proteins that bind cytokines. Cytokine receptors may be membrane bound and soluble.

In some embodiments, the over-expressed cytokine is an interleukin (IL-1) family member (e.g., ligand), an IL-1 receptor family member, an interleukin-6 (IL-6) family member (e.g., ligand). IL-6 receptor, an interleukin-10 (IL-10) family member (e.g., ligand), an IL-10 receptor, an interleukin-12 (IL-12) family member (e.g., ligand), an IL-12 receptor, an interleukin-17 (IL-17) family member (e.g., ligand), or an IL-17 receptor.

In some embodiments, the overexpressed cytokine is an interleukin-1 (IL-1) family member or related protein; tumor Necrosis Factor (TNF) family members or related proteins; an Interferon (IFN) family member or related protein; an interleukin-6 (IL-6) family member or related protein; or a chemokine or related protein. In some embodiments, the cytokine is selected from IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3/IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, BAFF/BLyS/TNFSF138, 4-1BBL, CD153/CD 30L. TNFSF8, CD40LG, CD70, Fas ligand/FASLG/CD 95L/CD178, EDA-A1, TNFSF 1/LIGHT/CD 258, TNFA, LTA/TNFB/TNFSF1, LTB/TNFC, CD 1/CD 27 1/TNFSF 1, TNFSF 1/TRAIL/APO-2L (CD253), RANKL/OPGL/TNFSF 1(CD254), TNFSF1, TNF- α/TNFA, TNFSF1, TL1 1/TNFSF 1, OX-40L/TNFSF 1/CD 252, CD40 1/CD 154/TNFSF 1, IFNA1, IFNA1, IFNA1, IFNA1, IFNA1, IFNA1, IFNA1, CCL/CCL 1, CCL1, CCL1, IFNA1, IFNA1, IFNA1, IFNA1, IFNA1, IFNA1, CCL/CCL 1, CCL1, IFNA1, IFNA1, IFNA1, IFNA1, IFNA1, CCL/CCL 1, IFN 1, IFNA1, IFNA1, IFNA1, IFNA1, IFNA1, IFNA1, CCL1, CCL, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL 10/MIP-2, CXCL 10/Ppbp, CXCL10, IL 10/CX 10, XCL10, FAM19 a10 and FAM19a 10.

Cytokine expression can be assessed using a variety of methods. Cytokine expression can be assessed by measuring cell culture medium in which the modified immune cells are grown (e.g., in vitro production) or serum obtained from one or more sera obtained from subjects having modified immune cells (e.g., in vivo production). Cytokine levels can be quantified in various suitable units, including concentrations, using any suitable assay. In some embodiments, cytokine proteins are detected. In some embodiments, mRNA transcripts of the cytokine are detected. Examples of cytokine assays include enzyme-linked immunosorbent assays (ELISA), immunoblots, immunofluorescent assays, radioimmunoassays, antibody arrays allowing parallel detection of various cytokines in a sample, bead-based arrays, quantitative PCR, microarrays, and the like. Other suitable methods may include proteomics methods (2-D gel, MS analysis, etc.).

In some embodiments, the cytokine overexpressed by the modified immune cells provided herein is a chemokine. Chemokines can be, for example, CC chemokines, CXC chemokines, C chemokines and CX3C chemokines. In some embodiments, the chemokine overexpressed by the modified immune cell is a CC chemokine selected from the group consisting of CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL 28. The chemokine is a CXC chemokine selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL 17. In some embodiments, the chemokine overexpressed by the modified immune cell is a C chemokine selected from the group consisting of XCL1 and XCL 2. In some embodiments, the chemokine over-expressed by the immune cells is a CX3C chemokine, and the CX3C chemokine is CX3CL 1.

In one aspect, the present disclosure provides a Universal CAR (UCAR) immune cell characterized in that,

a. the UCAR immune cells express a Chimeric Antigen Receptor (CAR) on the surface;

b. the UCAR immune cells are used for treating a patient with a T cell tumor;

c. the CAR of the UCAR immune cell comprises an antibody that targets a T cell surface protein or a variable region of an antibody;

d. the UCAR immune cells are not autologous to the patient;

in some embodiments, the UCAR immune cells are UCAR-T cells.

In some embodiments, the UCAR immune cells are UCAR-NK cells. In some embodiments, the NK cells are immortalized cells that are destroyed by irradiation prior to reinfusion.

In some embodiments, the UCAR-T cells are derived from a semi-allogeneic source, i.e., from the patient's immediate relative.

In some embodiments, the UCAR immune cells are from allogenic sources, but not from HLA full or semi-allogenic sources, nor from the patient's immediate relatives.

In some embodiments, UCAR-T cells have knocked out all or part of the TCR gene (e.g., TRAC), and all or part of the HLA gene (e.g., B2M). By knocking out the two genes, UCAR-T cells can be prevented from attacking autologous cells of a patient on one hand, and UCAR-T cells can be prevented from attacking autologous T cells of the patient on the other hand.

In some embodiments, the gene knockout employs a zinc finger ribonuclease (ZFN) technology system; in some embodiments, gene knock-out employs a transcription activator-like effector nuclease (TALEN) technology system; in some embodiments, the gene knockout employs the CRISPER system; in some embodiments, the gene knockout employs the CRISPER-Cas9 system.

In some embodiments, UCAR-T cells knock out the FAS (CD95) gene; in some embodiments, UCAR-T cells are knocked out for TRAC, B2M, FAS (CD95) genes simultaneously.

In some embodiments, the CAR of the UCAR-T cell comprises an antibody or variable region of an antibody that targets the T cell receptor TRBC1 or TRBC 2. In embodiments involving clinical treatment against T cell tumors, specifically targeting TRBC1 versus TRBC2 is determined by whether the TCR of the T cell tumor cell expresses TRBC1 versus TRBC 2. CAR of UCAR-T cells targets TRBC1 if the TCR of the T cell tumor cell is TRBC1 expressing; CAR of UCAR-T cells targets TRBC2 if the TCR of the T cell tumor cell is TRBC2 expressing;

in one aspect, the present disclosure provides a method of treating a T cell tumor in a subject, comprising: returning UCAR-T targeting TRBC1 or TRBC2 expressed by the tumor cells to the subject, depending on whether the tumor cells in the subject's T cells express TRBC1 or TRBC 2.

In various embodiments of the aspects herein, promoters that can be used with the compositions of the present disclosure include promoters that are active in eukaryotic, mammalian, non-human mammalian or human cells. The promoter may be an inducible or a constitutively active promoter. Alternatively or additionally, the promoter may be tissue or cell specific.

Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that function in eukaryotic cells) may include those from early Cytomegalovirus (CMV), Herpes Simplex Virus (HSV) thymidine kinase, early and late SV40, Long Terminal Repeats (LTR). From a retrovirus, the human elongation factor-1 promoter (EF1), a hybrid construct comprising the Cytomegalovirus (CMV) enhancer fused to the chicken beta-active promoter (CAG), the murine stem cell virus promoter (MSCV), the phosphoglycerate kinase-1 locus Promoter (PGK) and the mouse metallothionein-I. The promoter may be a fungal promoter. The promoter may be a plant promoter. A database of plant promoters can be found (e.g., plantarperm). The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression.

In various embodiments of aspects herein, the modified immune cell can specifically bind to a neoantigen and/or a neoepitope. Neoantigens and neoepitopes are commonly referred to as tumor-specific mutations, which in some cases elicit an anti-tumor T cell response. For example, these endogenous mutations can be identified using a whole-body exon sequencing method. Tran E et al, "cancer immunotherapy based on mutation-specific CD4+ T cells in patients with epithelial cancer", Science 344: 641-644(2014). Modified immune cells (e.g., modified TILs or modified T cells) comprising the CARs can exhibit specific binding to a tumor-specific neoantigen. The neoantigen bound by the immune cell may be expressed on the target cell and, for example, encoded by a mutation in an endogenous gene. In some cases, the neoantigen or neoepitope specifically bound by the immune cell may be encoded by a mutant gene. The gene may be selected from: ABL, ACO 11997, ACVR2, AFP, AKT, ALK, ALPPL, ANAPC, APC, ARID1, AR-v, ASCL, β 2M, BRAF, BTK, C15ORF, CDH, CLDN, CNOT, CT45A, CTAG1 (encoding NY-ESO-1), DCT, DKK, EEF1B, EEF1DP, EGFR, EIF2B, env, EPHB, ERBB, ESR, ESRP, FAM111, FGFR, FRG1, GAGE 10, GATA, GBP, HER, IDH, JAK, KIT, KRAS, LMAN, mab16, MAGEA, MAGEB 17, MAGEB, MAGEC, MEK, MLANA, MLL, MMP, MSH, myndc, nrnyc, nrnas, typ-p, SLC, tfap, tpel, SMAP, tpel, tfap, tpo, tfsp. In some embodiments, the neoantigen is selected based on a genetic profile of a tumor sample from the individual. In some embodiments, neoantigens may be selected based on the somatic mutation profile of a tumor sample from an individual.

In various embodiments of aspects herein, the modified immune cell further comprises an inactivation switch (or suicide switch). In cases of severe toxicity, such as hypercytokinemia, a kill switch can be activated to eliminate immune cells. This may occur when the immune system has such a strong response that many inflammatory cytokines are released, causing mild to severe symptoms including fever, headache, rash, accelerated heartbeat, hypotension and dyspnea. The kill switch may be a drug induced kill switch. The kill switch may comprise inducible caspase 9.

Various embodiments of aspects herein include cells, such as modified immune cells. Cells, such as immune cells (e.g., lymphocytes including T cells and NK cells), can be obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. Examples of samples from subjects from which cells can be obtained include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, stomach and digestive fluids, tears, stool, semen, vaginal fluids, interstitial fluid from tumor tissue, ocular fluids, sweat, mucus, cerumen, oil, glandular secretions, spinal fluid, hair, nails, plasma, nasal swabs or nasopharyngeal washes, spinal fluid, cerebrospinal fluid, tissue, throat swabs, biopsies, placental fluid, amniotic fluid, umbilical cord blood, strength fluid, luminal fluid, sputum, pus, microflora, meconium, milk and/or other excretions or body tissue.

In some cases, the cell can be a population of T cells, NK cells, B cells, and the like obtained from a subject. T cells can be obtained from a number of sources, including PBMCs, bone marrow, lymph node tissue, cord blood, thymus tissue and tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In some embodiments, T cells can be obtained from a blood unit collected from a subject using any number of techniques, such as ficoll (tm) separation. In one embodiment, the cells from the circulating blood of the individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing steps.

Any of a variety of immune cells may be used in aspects herein. In some embodiments, the immune cells include granulocytes, such as ascophils, eosinophils, and neutrophils; mast cells; monocytes may develop into macrophages; antigen presenting cells, such as dendritic cells; and lymphocytes such as natural killer cells (NK cells), B cells and T cells. In some embodiments, the immune cell is an immune effector cell. Immune effector cells are immune cells that are capable of performing a specific function in response to a stimulus. In some embodiments, the immune cell is an immune effector cell that can induce cell death. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is an NK cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is an activated T cell. T cells include naive and memory cells (e.g., central memory or TCM, effector memory or TEM and effector memory RA or TEMRA), effector cells (e.g., cytotoxic T cells or CTLs or Tc cells), helper cells (e.g., Th1, Th2, Th3), Th9, Th7, TFH), regulatory cells (e.g., Treg and Tr1 cells), natural killer T cells (NKT cells), Tumor Infiltrating Lymphocytes (TIL), lymphocyte activated killer cells (LAK), α β T cells, γ T cells, and similarly distinct classes of T cell lineages. T cells can be divided into two broad categories: CD8+ T cells and CD4+ T cells, based on the presence of proteins on the cell surface. T cells expressing the subject system can perform a variety of functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are called cytotoxic T cells or Cytotoxic T Lymphocytes (CTLs). CTLs expressing the subject system may be involved in the recognition and removal of virus-infected cells and cancer cells. CTLs have specialized compartments or particles that contain cytotoxins that cause apoptosis, such as programmed cell death. CD4+ T cells can be subdivided into four subgroups-Th 1, Th2, Th17 and tregs, "Th" refers to "T helper cells", although other subgroups may exist. Th1 cells can coordinate the immune response against intracellular microorganisms, particularly bacteria. They can produce and secrete molecules that alert and activate other immune cells, such as bacteria that take up macrophages. Th2 cells are involved in coordinating immune responses against extracellular pathogens, such as helminths (parasites), by alerting B cells, granulocytes and mast cells to participate. Th17 cells produce interleukin 17(IL-17), a signaling molecule that activates immune and non-immune cells. Th17 cells are important for the recruitment of neutrophils.

In some embodiments, the immune cell populations provided herein can be heterogeneous. In some embodiments, the cells used may consist of a heterogeneous mixture of CD4 and CD8T cells. CD4 and CD8 cells may have the phenotypic characteristics of circulating effector T cells. The CD4 and CD8 cells may also have phenotypic characteristics of effector memory cells. In some embodiments, the cell may be a central memory cell.

In some embodiments, the cells include Peripheral Blood Mononuclear Cells (PBMCs), Peripheral Blood Lymphocytes (PBLs) and other subpopulations of blood cells such as, but not limited to, T cells, natural killer cells, monocytes, natural cells. Killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells. In some cases, the cell may be any immune cell, including any T cell, such as a tumor infiltrating cell (TIL), e.g., a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, or any other type of T-cell. A cell. T cells may also include memory T cells, memory stem T cells or effector T cells. T cells may also be selected from a large population, for example, T cells selected from whole blood. T cells can also be expanded from a large population. T cells may also be biased towards a particular population and phenotype. For example, T cells may be tilted to a phenotype including CD45RO (-), CCR7(+), CD45RA (+), CD62L (+), CD27(+), CD28(+) and/or IL-7R α (+). Suitable cells may be selected which comprise one or more markers selected from the following list: CD45RO (-), CCR7(+), CD45RA (+), CD62L (+), CD27(+), CD28(+) and/or IL. -7R α (+). Cells also include stem cells, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells. The cells may comprise any number of primary cells, such as human cells, non-human cells, and/or mouse cells. The cells may be progenitor cells. The cells can be from a subject (e.g., a patient) to be treated. The cells may be derived from a human donor. The host cell may be a stem memory TSCM cell consisting of CD45RO (-), CCR7(+), CD45RA (+), CD62L + (L-selectin), CD27+, CD28+, and IL-7R α +, which may also express CD95, IL-2R β, CXCR3, and LFA-1 and exhibit a number of different functional attributes than the stem memory cell. The host cell may be a central memory TCM cell containing L-selectin and CCR7, which may secrete, for example, IL-2, but not IFN γ or IL-4. The cells may also be effector memory TEM cells comprising L-selectin or CCR7 and producing, for example, effector cytokines such as IFN γ and IL-4.

In various embodiments of aspects herein, the immune cell comprises a lymphocyte. In some embodiments, the lymphocyte is a natural killer cell (NK cell). In some embodiments, the lymphocyte is a T cell. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord and tumors. In some embodiments, any number of available T cell lines may be used. Immune cells such as lymphocytes (e.g., cytotoxic lymphocytes) may preferably be autologous cells, but heterologous cells may also be used. T cells can be obtained from a blood unit collected from a subject using any number of techniques, such as Ficoll separation. Cells from the circulating blood of an individual may be obtained by apheresis or leukopheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium, such as Phosphate Buffered Saline (PBS), for subsequent processing steps. After washing, the cells can be resuspended in various biocompatible buffers, such as MgPBS without Ca. Alternatively, the sample of apheresis may be freed of unwanted components and the cells resuspended directly in culture medium. The sample may be provided directly by the subject, or indirectly through one or more intermediaries, such as a sample collection service provider or a medical provider (e.g., a doctor or nurse). In some embodiments, separating T cells from peripheral blood leukocytes can comprise lysing erythrocytes and separating peripheral blood leukocytes from monocytes by, for example, by PERCOL (TM) gradient centrifugation.

Specific subsets of T cells, such as CD4+ or CD8+ T cells, may be further isolated by positive or negative selection techniques. For example, negative selection of a population of T cells can be achieved with a combination of antibodies directed against surface markers specific to the negatively selected cells. One suitable technique involves cell sorting by negative magnetic immunoadhesion using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to isolate CD4+ cells, the monoclonal antibody cocktail may include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. Negative selection procedures can be used to generate a predominantly homogeneous population of desired T cells. In some embodiments, the composition comprises a mixture of two or more (e.g., 2, 3, 4, 5or more) different types of T cells.

In some embodiments, the immune cell is a member of an enriched cell population. One or more desired cell types may be enriched by any suitable method, non-limiting examples of which include treatment of a population of cells to trigger expansion and/or differentiation into a desired cell type, treatment to prevent growth of an undesired cell type, treatment to kill or lyse an undesired cell type, purification of a desired cell type (e.g., purification on an affinity column to retain a desired or undesired cell type based on one or more cell surface markers). In some embodiments, the enriched population of cells is a population of cells enriched for cytotoxic lymphocytes selected from the group consisting of cytotoxic T cells (also referred to as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, and killer T cells). ) Natural Killer (NK) cells and lymphokine-activated killer (LAK) cells.

To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., beads, etc.) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact of the cells and beads. For example, a concentration of 20 hundred million cells/mL may be used. In some embodiments, a concentration of 10 hundred million cells/mL is used. In some embodiments, greater than 1 hundred million cells/mL are used. Cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45 or 5000 ten thousand cells/mL may be used. In another embodiment, cell concentrations of 75, 80, 85, 90, 95, or 1 hundred million cells/mL may be used. In further embodiments, concentrations of 125 or 1.5 million cells/mL may be used. The use of high concentrations can lead to increased cell yield, cell activation and cell expansion.

A variety of target cells can be killed using the systems and methods of the present disclosure. Target cells to which the method can be applied include a variety of cell types. The target cell may be in vitro. The target cell may be in vivo. The target cell may be ex vivo. The target cell may be an isolated cell. The target cell may be a cell within an organism. The target cell may be an organism. The target cell may be a cell in cell culture. The target cell may be one of a collection of cells. The target cell may be a mammalian cell or derived from a mammalian cell. The target cell may be a rodent cell or derived from a rodent cell. The target cell may be a human cell or derived from a human cell. The target cell may be a prokaryotic cell or derived from a prokaryotic cell. The target cell may be a bacterial cell or may be derived from a bacterial cell. The target cell may be an archaeal cell or derived from an archaeal cell. The target cell may be or be derived from a eukaryotic cell. The target cell may be a pluripotent stem cell. The target cell may be a plant cell or derived from a plant cell. The target cell may be an animal cell or derived from an animal cell. The target cell may be an invertebrate cell or derived from an invertebrate cell. The target cell may be a vertebrate cell or derived from a vertebrate cell. The target cell may be a microbial cell or derived from a microbial cell. The target cell may be a fungal cell or derived from a fungal cell. The target cells may be from a particular organ or tissue.

The target cell may be a stem cell or a progenitor cell. Target cells can include stem cells (e.g., adult stem cells, embryonic stem cells, Induced Pluripotent Stem (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, and the like. The cloned cells may comprise progeny of the cells. The target cell can comprise a target nucleic acid. The target cell may be in a living organism. The target cell may be a genetically modified cell. The target cell may be a host cell.

The target cell may be a primary cell. For example, a culture of primary cells may be passaged 0, 1, 2, 4, 5, 10, 15or more times. The cell may be a unicellular organism. The cells may be grown in culture.

The target cell may be a diseased cell. Diseased cells may have altered metabolism, gene expression and/or morphological characteristics. The diseased cells may be cancer cells, diabetic cells and apoptotic cells. The diseased cell can be a cell from a diseased subject. Exemplary diseases may include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disorders, and the like.

If the target cells are primary cells, they may be harvested from the individual by any method. For example, leukocytes can be harvested by apheresis, leukopheresis, density gradient separation, and the like. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. Suitable solutions may be used to isolate or suspend the harvested cells. Such solutions may typically be balanced salt solutions (e.g., physiological saline, Phosphate Buffered Saline (PBS), Hank's balanced salt solution, etc.), conveniently supplemented with fetal bovine serum or other naturally occurring factors, and an acceptable low concentration of buffer. The buffer may include HEPES, phosphate buffer, lactate buffer, and the like. The cells may be used immediately, or they may be stored (e.g., by freezing). The frozen cells can be thawed and can be reused. Cells can be frozen in DMSO, serum, media buffer (e.g., 10% DMSO, 50% serum, 40% buffered media), and/or some other such common solution for preserving cells at freezing temperatures.

Non-limiting examples of cells that can be target cells include, but are not limited to, lymphocytes, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., US 20080241194); bone marrow cells, such as granulocytes (basophils, eosinophils, neutrophils/metaneutrophils), monocytes/macrophages, erythrocytes (reticulocytes), mast cells, platelets/megakaryocytes, dendritic cells; cells from the endocrine system, including thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, Oxyphil cells), adrenal (chromaffin cells), pineal (pineal cells); nervous system cells, including glial cells (astrocytes, microglia), giant cell neuroendocrine cells, astrocytes, Boettcher cells, and pituitary gland (gonadotropins, corticosteroids, thyroid hormone, growth hormone, lactate hormone); respiratory system cells including pneumocytes (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells; cells of the circulatory system, including cardiomyocytes, pericytes; cells of the digestive system, including stomach (gastric chief cell, parietal cell), goblet cell, panne cell, G cell, D cell, ECL cell, I cell, K cell, S cell; enteroendocrine cells including enterochromaffin cells, APUD cells, liver (hepatocytes, kupffer cells), cartilage/bone/muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, teeth (osteoblasts, ameloblasts); chondrocytes, including chondrocytes, chondrocytes; skin cells, including hair cells, keratinocytes, melanocytes (nevus cells); muscle cells, including muscle cells; urinary system cells, including podocytes, pericytes, mesangial/mesangial cells, proximal tubular brush border cells, macular cells; reproductive system cells including sperm, sertoli cells, Leydig cells, ova; and other cells, including adipocytes, fibroblasts, tenocytes, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), nail and toenail keratinocytes, nail basal cells (stem cells), medullary hair cells, cortical hair shaft cells, keratinocyte hair stem cells, keratinocyte root sheath cells, Huxley layer hair root sheath cells, Henle layer hair root sheath cells, outer hair sheath cells, hair stromal cells (stem cells), wet layer barrier epithelial cells, cornea, tongue, oral cavity, esophagus, anal canal, double-layered squamous surface epithelial cells of the distal urethra and vagina, corneal epithelium, tongue, oral cavity, esophagus, anal canal, basal cells (stem cells) of the distal urethra and vagina, urinary epithelial cells (urinary bladder and urinary duct), exocrine epithelial cells, salivary gland mucus (polysaccharide-rich secretions), salivary gland serous cell (glycoprotein-ri) ch secretion), Von Ebner in glandular cells of the tongue (washing taste buds), mammary cells (milk secretion), lacrimal cells (tear secretion), coccygeal cells in the ear (wax secretion), Eccrine sweat gland dark cells (glycoprotein secretion)), Eccrine sweat gland clear cells (small molecule secretion). Apocrine sweat gland cells (odor secretions, sex hormone sensitive), eyelid Moll cell glands (specialized sweat glands), sebaceous gland cells (lipid-rich sebaceous secretions), Bowman gland cells in the nose (cleansing olfactory epithelium), cells in the duodenum of the Brunner gland (enzymes and alkaline mucus), seminal vesicle cells (secretory seminal fluid components including fructose for swimming sperm), prostate cells (secretory seminal fluid components), bulboretral gland cells (mucus secretions), vestibular gland cell (vaginal lubricant) secretions), Littre cell glands (mucus secretions), endometrial cells (carbohydrate secretions), goblet cells isolated from the respiratory and digestive tracts (mucus secretions), gastric mucosal cells (mucus secretions), gastric gland zymogen cells (pepsinogen secretions), gastric gland secretory cells (hydrochloric acid secretions), pancreatic acinar cells (bicarbonate and digestive enzyme secretions), small intestinal Paneth cells (lysozyme secretion), type II pneumocytes (surfactant secretion), lung Clara cells, hormone secreting cells, anterior pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, intermediate pituitary cells, Magnocellular nerve secreting cells, intestinal and respiratory tract cells, thyroid gland epithelial cells, parafollicular cells, parathyroid chief cells, Oxyphil cells, adrenal gland cells, chromaffin cells, testis Ley digging cells, ovarian follicular intimal cells, ruptured ovarian follicle cells, granulocytic xanthophyll cells, Theca xanthophyll cells, Juxtaglomerular cells (renin secretion), renal Macula densa cells, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands and urogenital tract), kidney, type I pneumocytes (lung lining air space), pancreatic ductal cells (central cell), noncyclic duct cells (sweat gland, salivary gland, mammary gland, etc.), duct cells (seminal vesicle, prostate, etc.), epithelial cells lining the closed body lumen, fibrohair cells with propulsive function, extracellular matrix secreting cells, contracting cells; skeletal muscle cells, stem cells, cardiac muscle cells, blood and immune system cells, erythrocytes (erythrocytes), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (of various types), epidermal langerhans cells, osteoclasts (in bone), dendritic cells (lymphoid tissue), microglia (central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and committed progenitors for the blood and immune system (of various types), pluripotent stem cells, Totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory sensor cells, autonomic nerve cells, sensory organs and peripheral neuron support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oxonium/oxocyte, specmatid, specatocyte, specatogonium cells (stem cells of spermatocytes), sperm, nurse cells, ovarian follicular cells, supporting cells (testes), thymic epithelial cells, mesenchymal cells and mesenchymal kidney cells, or non-human cells engineered or irradiated such as K562, NK92, and the like. The target cell may be a natural cell or a modified cell.

Of particular interest are cancer cells. In some embodiments, the target cell is a cancer cell. Non-limiting examples of cancer cells include cancer cells, including acanthoma, Acinic cell carcinoma, acoustic neuroma, Acral lentiginous melanoma, Acrospira, acute eosinophilic leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute myeloid dendritic cell leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, adamantine disease, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenal cortex carcinoma, adult T cell leukemia, aggressive NK cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic tumor, anal carcinoma, anaplastic large cell lymphoma, thyroid undifferentiated carcinoma, angioimmunoblastic T cell lymphoma, vascular smooth muscle sarcoma, angiosarcoma, adnexal cancer, astrocytoma, atypical teratocarcinoma, basal cell carcinoma, basal-like carcinoma, B cell leukemia, B cell lymphoma, Bellini ductal carcinoma, Bilia rytract cancer, bladder cancer, Blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt's lymphoma, unknown primary cancer, carcinoid tumor, carcinoma in situ, penile cancer, unknown primary cancer, Carcinosarcomas, Castleman's disease, central nervous system embryonic tumor, cerebellar astrocytoma, brain astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroidal papillary tumor, chronic lymphocytic leukemia, chronic monocytic leukemia, myeloproliferative disease, chronic neutrophilic leukemia, clear cell tumors, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos ' disease, cutaneous fibrosarcoma, dermoid cysts, proliferative small round cell tumors, diffuse large B-cell lymphoma, dysplastic neuroepithelial tumors, embryonic carcinoma, endoblastoma, endometrial carcinoma, endometrioid tumors, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, Ethesieuroblastoma, Ewing's family tumors, Wing's sarcoma, Ewing's sarcoma, extracranial germ cell tumors, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetal fetus, fibroma fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, glioma, gangliocytoma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, germ cell tumors, choriocarcinoma of pregnancy, trophoblastic tumor of pregnancy, giant cell tumor of bone, glioblastoma multiforme, glioma, brain glioma, Glomus tumors, Glucagonoma, gonadublas toma, Granulosa cytoma, hairy cell leukemia, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiothecoma, angiosarcoma, cardiac cancer, hemangioblastoma, hemangiosarcoma, hematological sarcoma, hematological malignancy, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer, hodgkin lymphoma, hypopharynx cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell cancer, islet cell carcinoma, islet cell tumor, juvenile myelocytic leukemia, kaposi's sarcoma, kidney cancer, Klatz's tumor, Krukenberg tumor, laryngeal cancer, malignant melanoma, leukemia, lip cavity and oral cavity cancer, liposarcoma, lung cancer, luteal tumor, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdomyosarcoma, malignant tritium karyoma, MALT lymphoma, mantle cell lymphoma a, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma, occult primary, metastatic urothelial cancer, mixed muller's tumor, monocytic leukemia, oral cancer, myxoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelogenous leukemia, myeloma, myeloproliferative disease, myxoma, nasal cancer, nasopharyngeal cancer, tumor, neuroma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology, oligolymphomas, oligodendroglioma, eosinophilic tumors, optic nerve sheath meningiomas, oral cancer, or al cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, breast Paget's disease, Pancoast tumor, pancreatic cancer, papillary thyroid carcinoma, papillomatosis, paragangliomas, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, mesodifferentiated pineal parenchymal tumor, pineal cytoma, pituitary adenoma, pituitary tumor, plasmacytoma, pleuropneumoblastoma, polyembryonic tumor, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primary neuroectodermal tumor, prostate cancer, pseudoperitoneal myxoma, rectal cancer, renal cell carcinoma, respiratory tract cancer, NUT gene 15 involved in Chromoso, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, lirious transformation, sacrococcal tail teratoma, salivary gland cancer, sarcoma, schwannoma, sebaceous gland cancer, secondary tumor, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord stromal tumor, Sezary syndrome, signet ring cell cancer, skin cancer, small blue cell tumor, small cell cancer, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatin tumor, verruca, spinal cord tumor, spinal column tumor, marginal zone lymphoma, squamous cell cancer, gastric cancer, superficial diffuse melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial-stromal tumor, synovial sarcoma, T cell acute lymphocytic leukemia, T cell large granular lymphocytic leukemia, T cell lymphoma, T cell prolymphocytic leukemia, teratocarcinoma, advanced lymphocytic carcinoma, testicular cancer, Thecoma, laryngeal cancer, thymus cancer, thymoma, thyoid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, cancer of the umbilical duct, cancer of the urethra, genitourinary tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous cancer, glioma of the visual pathway, cancer of the vulva, Waldenstrom's macroglobulinemia, tumors of Warthin, tumors of Wilms, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation in a population of cancer cells, such as cancer stem cells. In some embodiments, the cancer is of hematopoietic lineage, e.g., lymphoma. The antigen may be a tumor associated antigen.

In some embodiments, the target cell forms a tumor. Tumors treated with the methods herein can result in stable tumor growth (e.g., one or more tumors do not increase in volume by more than 1%, 5%, 10%, 15% or 20%, and/or do not metastasize). In some embodiments, the tumor is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. In some embodiments, the tumor is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. In some embodiments, the tumor is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years. In some embodiments, the size of the tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the tumor is completely eliminated, or reduced below detection levels. In some embodiments, the subject remains tumor-free (e.g., remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years after treatment.

Death of the target cells can be determined by any suitable method, including but not limited to counting cells before and after treatment, or measuring the level of a marker associated with live or dead cells (e.g., live or dead target cells). The extent of cell death can be determined by any suitable method. In some embodiments, the extent of cell death is determined relative to the starting conditions. For example, an individual may have a known starting amount of target cells, such as a starting cell mass of known size or a known concentration of circulating target cells. In this case, the degree of cell death can be expressed as the ratio of viable cells to the starting cell population after treatment. In some embodiments, the extent of cell death may be determined by a suitable cell death assay. Various cell death assays can be used, and a variety of detection methods can be used. Examples of detection methods include, but are not limited to, cell staining, microscopy, flow cytometry, cell sorting, and the use of combinations of these.

When a tumor is surgically resected after the end of the treatment period, the efficacy of the treatment in reducing the size of the tumor can be determined by measuring the percentage of necrotic (i.e., dead) resected tissue. In some embodiments, the treatment is effective if the percentage of necrosis of the resected tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%). Or 100%). In some embodiments, the percentage of necrosis of the resected tissue is 100%, i.e., no viable tumor tissue is present or detectable.

Exposure of a target cell to an immune cell or population of immune cells disclosed herein can be performed in vitro or in vivo. Exposing a target cell to an immune cell or population of immune cells generally refers to contacting the target cell with an immune cell and/or being sufficiently close that an antigen (e.g., membrane-bound or non-membrane-bound)) of the target cell can bind to a CAR expressed in the immune cell. Exposing a target cell to an immune cell or a population of immune cells also generally refers to contacting the target cell with an immune cell and/or being in sufficient proximity to allow antigen (e.g., membrane bound or unbound) of the target cell. Membrane-bound) can bind to CARs expressed in immune cells. By co-culturing the target cell and the immune cell, the immune cell or immune cell population can be exposed to the target cell in vitro. The target cells and immune cells may be co-cultured, for example, as adherent cells or as a suspension. The target cells and immune cells can be co-cultured in various suitable types of cell culture media, e.g., co-cultured with supplements, growth factors, ions, and the like. Target cells can be exposed to an immune cell or population of immune cells in vivo, in some cases, by administering an immune cell to a subject (e.g., a human subject), and allowing the immune cell to localize to the target cell through the circulatory system. In some cases, the immune cells can be delivered to the direct region where the target cells are located, for example by direct injection.

The exposure can be for any suitable length of time, for example, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, or longer.

The various domains of the CARs provided herein can be linked by chemical bonds, such as amide or disulfide bonds; a small organic molecule (e.g., a hydrocarbon chain); an amino acid sequence, such as a peptide linker (e.g., an amino acid sequence of about 3-200 amino acids in length), or a combination of a small organic molecule and a peptide linker. Peptide linkers may provide the desired flexibility to allow for the desired expression, activity and/or conformational positioning of the chimeric polypeptide. The peptide linker may be of any suitable length to link at least two domains of interest, and is preferably designed to be sufficiently flexible to allow proper folding and/or function and/or activity of one or both domains to which it is linked. The peptide linker can have a length of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. In some embodiments, the peptide linker is about 0 to 200 amino acids, about 10 to 190 amino acids, about 20 to 180 amino acids, about 30 to 170 amino acids, about 40 to 160 amino acids, about 50 to 150 amino acids, about 60 to 140 amino acids, about 70 to 130 amino acids, about 80 to 120 amino acids, about 90 to 110 amino acids in length. In some embodiments, the linker sequence may comprise an endogenous protein sequence. In some embodiments, the linker sequence comprises glycine, alanine and/or serine amino acid residues. In some embodiments, the linker may contain a motif, e.g., a multiple or repeat motif, of GS, GGS, GGGGS, GGSG, or SGGG. The linker sequence may comprise any naturally occurring amino acid, non-naturally occurring amino acid, or a combination thereof.

The compositions and molecules of the present disclosure (e.g., polypeptides and/or nucleic acids encoding polypeptides) can be introduced into a host cell, such as an immune cell, using any suitable delivery method. The various components may be delivered simultaneously or separated in time. The choice of method may depend on the type of transformed cell and/or the environment in which the transformation occurs (e.g., in vitro, ex vivo, or in vivo).

The delivery method may comprise contacting the target polynucleotide or introducing one or more nucleic acids comprising a nucleotide sequence encoding a composition of the invention into a cell (or population of cells such as immune cells). Suitable nucleic acids comprising nucleotide sequences encoding compositions of the disclosure can include expression vectors, wherein the expression vector comprising a nucleotide sequence encoding one or more compositions of the disclosure is a recombinant expression vector.

Non-limiting examples of delivery methods or transformations include, for example, viral or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, Polyethyleneimine (PEI) mediated transfection, DEAE-dextran mediated transfection, liposome mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle mediated nucleic acid delivery.

In some aspects, the disclosure provides methods comprising delivering one or more polynucleotides, or one or more vectors as described herein, or one or more transcripts thereof, and/or one or more proteins transcribed therefrom, to a host cell. In some aspects, the disclosure also provides cells produced by these methods, and organisms (e.g., animals, plants, or fungi) comprising or produced by these cells.

Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding the compositions of the disclosure to cells or host organisms in culture. Non-viral vector delivery systems can include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vectors, e.g., liposomes. Viral vector delivery systems may include DNA and RNA viruses, which may have episomal or integrated genomes after delivery to cells.

Methods for non-viral delivery of nucleic acids may include lipofection, nuclear transfection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial viral particles and agents of DNA enhance uptake. Lipid-transfected cationic and neutral lipids can be recognized using efficient receptors suitable for polynucleotides. Delivery may be to a cell (e.g., in vitro or ex vivo administration) or to a target tissue (e.g., in vivo administration). Preparation of lipids can be used: nucleic acid complexes, including targeted liposomes, such as immunoliposome complexes.

RNA or DNA virus based systems can be used to target specific cells in the body and transport viral payloads to the nucleus. Viral vectors may be administered directly (in vivo), or they may be used to treat cells in vitro, and the modified cells may optionally be administered (ex vivo). Virus-based systems may include retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration into the host genome can be achieved using retroviral, lentiviral and adeno-associated viral gene transfer methods, which can result in long-term expression of the inserted transgene. High transduction efficiencies can be observed in many different cell types and target tissues.

Lentiviruses can integrate their genome into a host cell (e.g., 293 cell, or T cell). Lentiviruses can employ a three-plasmid system, or a four-plasmid system.

The tropism of retroviruses can be altered by the incorporation of foreign envelope proteins, thereby amplifying the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and produce high viral titers. The choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors may contain cis-acting long terminal repeats with a packaging capacity of up to 6-10kb of exogenous sequence. The minimal cis-acting LTRs may be sufficient for replication and packaging of the vector, which may be used to integrate a therapeutic gene into a target cell to provide permanent transgene expression. Retroviral vectors can include vectors based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV), and combinations thereof.

Adenovirus-based systems may be used. Adenovirus-based systems can result in transient expression of the transgene. Adenovirus-based vectors may have high transduction efficiency in cells and may not require cell division. High titers and expression levels can be achieved with adenovirus-based vectors. Adeno-associated virus ("AAV") vectors can be used to transduce cells with target nucleic acids, e.g., to produce nucleic acids and peptides in vitro, and for in vivo and ex vivo gene therapy procedures.

The packaging cells can be used to form viral particles capable of infecting host cells. Such cells may include 293 cells (e.g., for packaging lentiviruses or adenoviruses) and Psi2 cells or PA317 cells (e.g., for packaging retroviruses). Viral vectors can be produced by generating cell lines that package nucleic acid vectors into viral particles. The vector may contain the minimal viral sequences required for packaging and subsequent integration into the host. The vector may contain other viral sequences, which are replaced by an expression cassette for the polynucleotide to be expressed. The missing viral functions may be provided in trans by the packaging cell line. For example, an AAV vector may comprise ITR sequences from the AAV genome that are necessary for packaging and integration into the host genome. Viral DNA can be packaged in cell lines that can contain helper plasmids encoding other AAV genes, i.e., rep and cap, but lack ITR sequences. Cell lines can also be infected with adenovirus as a helper cell. Helper viruses can promote replication of AAV vectors and expression of AAV genes from helper plasmids. Contamination with adenovirus can be reduced by, for example, heat treatment in which adenovirus is more sensitive than AAV. Other methods for delivering nucleic acids to cells can be used, for example, as described in US20030087817, which is incorporated herein by reference.

Host cells may be transfected transiently or non-transiently with one or more of the vectors described herein. The cell may be transfected because it is naturally present in the subject. Cells may be taken from or derived from a subject and transfected. The cells may be derived from cells taken from the subject, e.g., cell lines. In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines comprising one or more vector-derived sequences. In some embodiments, cells transiently transfected with a composition of the disclosure (e.g., by transient transfection of one or more vectors, or transfection with RNA) are used to establish new cell lines comprising cells containing modifications but lacking any other exogenous sequences. .

Any suitable vector compatible with the host cell may be used with the methods of the present disclosure. Non-limiting examples of vectors for eukaryotic host cells include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (pharmacia).

Contacting the cells with the composition can occur in any medium and under any culture conditions that promote cell survival. For example, the cells may be suspended in any convenient suitable nutrient medium, such as Iscove's modified DMEM or RPMI 1640, supplemented with fetal bovine serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols, especially 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which the cells respond. A growth factor as defined herein is a molecule capable of promoting the survival, growth and/or differentiation of cells in culture or intact tissue by specific action on transmembrane receptors. Growth factors may include polypeptide and non-polypeptide factors.

In many embodiments, the delivery system selected targets a particular tissue or cell type. In some cases, tissue or cell targeting of the delivery system is achieved by combining the delivery system with tissue or cell specific markers (e.g., cell surface proteins). Viral and non-viral delivery systems can be tailored to target tissues or cell types of interest.

Pharmaceutical compositions containing the molecules (e.g., polypeptides and/or nucleic acids or proteins encoding polypeptides) or immune cells described herein can be administered for prophylactic and/or therapeutic treatment. In therapeutic applications, the composition may be administered to a subject already suffering from a disease or disorder in an amount sufficient to cure or at least partially arrest the symptoms of the disease or disorder, or cure, heal, ameliorate, or improve the condition. The amount effective for this use will vary depending on the severity and course of the disease or disorder, previous treatments, the subject's health, weight and response to the drug, and the judgment of the treating physician.

The multiple therapeutic agents may be administered in any order or simultaneously. If simultaneous, multiple therapeutic agents may be provided in a single, unified form or in multiple forms, e.g., as multiple individual pills or cell solutions. The molecule or cell solution may be packaged together or separately in a single package or in multiple packages. One or all of the therapeutic agents may be administered in multiple doses. The time between doses may vary from about 1 to 24 months if different.

The molecules or cells described herein can be administered before, during, or after the onset of a disease or condition, and the time at which the composition containing the compound is administered can vary. For example, the pharmaceutical composition may be used as a prophylactic and may be administered continuously to a subject having a condition or predisposition to a disease, in order to prevent the occurrence of the disease or condition. The molecules, cells and pharmaceutical compositions can be administered to a subject during the onset of symptoms or as soon as possible. Administration of the molecule can begin within the first 48 hours of onset of symptoms, within the first 24 hours of onset of symptoms, within the first 6 hours of onset of symptoms, or within 3 hours of onset of symptoms. Onset of symptoms. Initial administration may be by any practical route, e.g., by any route described herein using any of the formulations described herein. The molecule may be administered as soon as possible after the onset of the disease or disorder is detected or suspected, and for the length of time required to treat the disease, e.g., about 1 month to about 3 months, where feasible. The treatment time may vary from subject to subject.

The molecules may be packaged into biological compartments. The biological compartment comprising the molecule can be administered to a subject. Biological compartments may include, but are not limited to, viruses (lentiviruses, adenoviruses), nanospheres, liposomes, quantum dots, nanoparticles, microparticles, nanocapsules, vesicles, polyethylene glycol particles, hydrogels, and micelles.

For example, the biological compartment can comprise a liposome. Liposomes can be self-assembled structures comprising one or more lipid bilayers, each of which can comprise two monolayers containing oppositely oriented amphiphilic lipid molecules. Amphiphilic lipids may comprise a polar (hydrophilic) head group covalently linked to one or two or more non-polar (hydrophobic) acyl groups or alkyl chains. Energetically unfavorable contact between hydrophobic acyl chains and the surrounding aqueous medium induces self-alignment of the amphiphilic lipid molecules such that the polar head groups can be oriented towards the bilayer surface and the acyl chains are oriented towards the bilayer interior, effectively shielding the acyl chains from contact with the aqueous environment.

The phospholipid used in the liposome may include phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoyl lecithin (DMPC), dipalmitoyl lecithin, preferably of amphiphilic compounds (examples of DPPC), dioleoyl phosphatidylcholine, distearoyl phosphatidylcholine (DSPC), dilinoleoyl phosphatidylcholine and egg sphingomyelin, or any combination thereof.

The biological compartment may comprise nanoparticles. The nanoparticles may have a diameter of about 40 nanometers to about 1.5 microns, about 50 nanometers to about 1.2 microns, about 60 nanometers to about 1 micron, about 70 nanometers to about 800 nanometers, about 80 nanometers. From about nm to about 600 nm, from about 90 nm to about 400 nm, from about 100 nm to about 200 nm.

In some cases, the release rate may slow or prolong as the size of the nanoparticle increases, and the release rate may increase as the size of the nanoparticle decreases.

The amount of albumin in the nanoparticle may be about 5% to about 85% albumin (v/v), about 10% to about 80%, about 15% to about 80%, about 20% to about 20%. About 70% albumin (v/v), about 25% to about 60%, about 30% to about 50%, or about 35% to about 40%. The pharmaceutical composition may comprise up to 30, 40, 50, 60, 70 or 80% or more of the nanoparticles. In some cases, the nucleic acid molecules of the present disclosure can bind to the surface of a nanoparticle.

The biological compartment may contain a virus. The virus may be a delivery system for a pharmaceutical composition of the present disclosure. Exemplary viruses may include lentiviruses, retroviruses, adenoviruses, herpes simplex virus I or II, parvoviruses, reticuloendotheliosis virus and adeno-associated virus (AAV). The pharmaceutical compositions of the present disclosure can be delivered to cells using viruses. The virus may infect and transduce cells in vivo, ex vivo or in vitro. In ex vivo and in vitro delivery, the transduced cells can be administered to a subject in need of treatment.

The pharmaceutical composition may be packaged into a viral delivery system. For example, the composition can be packaged into a viral particle by an HSV-1 helper-free viral packaging system.

Viral delivery systems (e.g., viruses comprising the pharmaceutical compositions of the present disclosure) can be administered by direct injection, stereotactic injection, intracerebroventricular, by a micro-pump infusion system, by convection, catheter, intravenous, parenteral, intraperitoneal, and/or subcutaneous injection. Injected into a cell, tissue or organ of a subject in need thereof. In some cases, cells may be transduced in vitro or ex vivo with a viral delivery system. The transduced cells can be administered to a subject having a disease. For example, stem cells can be transduced with a viral delivery system comprising a pharmaceutical composition, and the stem cells can be implanted in a patient to treat a disease.

In some cases, the dose of cells administered to a subject may be less than 1 × 10⁴Individual cell/kg, or about 1 × 10⁴Individual cell/kg, about 2 × 10⁴Individual cell/kg, about 3 × 10⁴Cell/kg, about 4 × 10⁴Individual cell/kg, about 5 × 10⁴Individual cell/kg, about 6 × 10⁴Individual cell/kg, about 7 × 10⁴Individual cell/kg, about 8 × 10⁴Individual cell/kg, about 9 × 10⁴One cell, about 1 × 10⁵Individual cell/kg, about 2 × 10⁵Individual cell/kg, about 3 × 10⁵Cell/kg, about 4 × 10⁵Individual cell/kg, about 5 × 10⁵Individual cell/kg, about 6 × 10⁵Individual cell/kg, about 7 × 10⁵Individual cell/kg, about 8 × 10⁵Individual cell/kg, about 9 × 10⁵One cell, about 1 × 10⁶Individual cell/kg, about 2 × 10⁶Individual cell/kg, about 3 × 10⁶Cell/kg, about 4 × 10⁶Individual cell/kg, about 5 × 10⁶Individual cell/kg, about 6 × 10⁶Individual cell/kg, about 7 × 10⁶Individual cell/kg, about 8 × 10⁶Individual cell/kg, about 9 × 10⁶One cell, about 1 × 10⁷Individual cell/kg, about 5 × 10⁷Individual cell/kg, about 1 × 10⁸Individual cells/kg or more. The cell dose can be calculated here as the number of effectively modified cells successfully transfected, but also as the total number of cells.

The biological compartment can be introduced into the cell by viral or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, Polyethyleneimine (PEI) mediated transfection, DEAE-dextran mediated transfection, liposome mediated. Transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

In some embodiments, immune cells expressing the subject's system are administered. The immune cells expressing the subject's system can be administered before, during or after the onset of the disease or disorder, and the timing of administration of the immune cells can vary. For example, immune cells expressing the subject's system can be used as a prophylactic and can be continuously administered to a subject having a disorder or predisposition to a disease, in order to prevent the occurrence of the disease or disorder. The immune cells can be administered to the subject during the onset of symptoms or as soon as possible. Administration can begin within the first 48 hours of symptom onset, within the first 24 hours of symptom onset, within the first 6 hours of symptom onset, or within 3 hours of symptom onset. Symptoms are presented. Initial administration may be by any suitable route, e.g., by any route described herein using any of the formulations described herein. Following detection or suspicion of the onset of a disease or condition, immune cells can be administered as soon as possible, if feasible, and treatment of the disease requires a period of time, e.g., about 1 month to about 3 months. And (5) the product is taken for a month. The treatment time may vary from subject to subject.

The molecules (e.g., polypeptides and/or nucleic acids) described herein can be present in the composition in a range of about 1mg to about 2000 mg; about 5mg to about 1000mg, about 10mg to about 25mg to 500mg, about 50mg to about 250mg, about 100mg to about 200mg, about 1mg to about 50 mg. 50mg to about 100mg, about 100mg to about 150mg, about 150mg to about 200mg, about 200mg to about 250mg, about 250mg to about 300mg, about 300mg to about 350 mg. mg, about 350mg to about 400mg, about 400mg to about 450mg, about 450mg to about 500mg, about 500mg to about 550mg, about 550mg to about 600mg, about 600mg to about 650mg, about 650mg to about 700mg, about 700mg to about 750mg, about 750mg to about 800mg, about 800mg to about 850mg, about 850mg to about 900 mg. From about 900mg to about 950mg, or from about 950mg to about 1000 mg.

The molecules (e.g., polypeptides and/or nucleic acids) described herein can be present in the composition in an amount of about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 10 mg. About 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 125mg, about 150mg, about 175mg, about 200mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 550mg, about 600mg, about 650mg, about 700mg, about 750mg, about 800mg, about 850mg, about 900mg, about 950mg, about 1000mg, about 1050mg, about 1100mg, about 1150mg, about 1200mg, about 1250mg, about 1300mg, about 1350mg, about 1400mg, about 1450mg, about 1500mg, about 1550mg, about 1600mg, about 1650 mg, about 1750 mg, about 1900 mg, about 2000mg, about 1850 mg, about 2000mg, or about 1850 mg.

The molecules (e.g., polypeptides and/or nucleic acids) described herein can be present in a composition that provides at least 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 10 or more units of activity per mg of molecule. The activity may be modulation of gene expression. In some embodiments, the total number of active units of the molecule delivered to the subject is at least 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000 or more units. In some embodiments, the total number of active units of the molecule delivered to the subject is at most 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000, or 250,000 or more units.

Description of the figures:

FIG. 1. preparation of U-CART cells

FIG. 2 flow assay for knock-out efficiency of CD3/B2M/CD95

FIG. 3 identification of CD3 and CAR expression in UCART cells

FIG. 4 flow-assay of TRBC1 expression in different cells

FIG. 5 flow assay of expression of CD107a from UCART co-cultured with tumor cells

FIG. 6 ELISA detection of cytokine secretion by UCART

FIG. 7 tumor burden profile of Jurkat-CBG tumor bearing mice

FIG. 8 statistics of survival of Jurkat-CBG tumor-bearing mice

Detailed Description

For a more complete understanding and appreciation of the invention, the invention will be described in detail below with reference to examples and the accompanying drawings, which are intended to illustrate the invention and not to limit the scope thereof. The scope of the invention is specifically defined by the appended claims.

Example (b): in vitro and animal experiments of UCAR-T (U-CART-TRBC1, or UCART-TRBC1) cells targeting TRBC1

A,General preparation of UCART-TRBC1 cells:

as shown in FIG. 1, peripheral blood of a healthy human was collected intravenously, PBMCs were separated from the Ficoll lymphocyte separation medium, T cells were sorted and stimulated using magnetic beads of anti-CD3/CD28, and cultured overnight. Corresponding amount of lentivirus was added at MOI of 1 and cultured for 72 h. On day 4, the magnetic beads are removed, CART cells are collected, and genes to be knocked out, namely TRAC, B2M, gRNA of CD95 and caspase9 protein are jointly electroporated into the CART cells by using CRISPR/CAS9 technology. On day 8, knockout efficiency was measured by flow. On the 10 th day, CD3 negative sorting is carried out, T cells with CD 3-enriched are UCART cells, the culture is continued for 4 to 5 days, and final product cells are collected.

II,Detection of CRISPR/CAS9 knockout efficiency

As shown in FIG. 2, on day 8, the efficiency of knocking out the gene was examined by flow. In this example, we co-electroporated and knocked out T cells for TRAC, B2M and CD 95. The flow detection result shows that the knockout efficiency (CD3-) of the TCR is 80-90%, and the proportion of the triple negative cell CD3-/B2M-/CD 95-is 40-60%.

III,Identification of UCART-TRBC1 cells

On

day

14 or 15, UCART cells were collected and flow-assayed for CD3 and CAR. Flow-through results showed (fig. 3) that CD3+ T cells were only 0.9%, CD3 negative selection was successful, and the positivity of lentiviral transfected CARs was 38.5%.

Fourthly,Detection of in vitro function of UCART-TRBC1 cells

We sorted untransfected T by TRBC1 and TRBC2 as positive and negative target cells, respectively, and selected Jurkat cells from T-cell tumors as target cells, TRBC1 flow analysis showed (fig. 4) that substantially all of the Jurkat cells expressed TRBC1, whereas none of the sorted TRBC2-T cells expressed TRBC1, and 80% of the TRBC1-T cells expressed TRBC1.

The different target cells were co-cultured with UCART cells for 3h, and the expression of CD107a in the UCART cells was detected. As shown in FIG. 5, UCART cells were significantly killed against TRBC 1-positive Jurkat and TRBC1-T cells, and CD107a was significantly expressed.

Different target cells were co-cultured with UCART cells for 24h, and secretion of IL2 and IFN-. gamma.was detected by ELISA, as shown in FIG. 6, UCART cells were significantly killed by TRBC1 positive Jurkat and TRBC1-T cells, and secretion of IL2 and IFN-. gamma.was up-regulated.

V, V,Detection of in vivo function of UCART-TRBC1 cell

To further test the in vivo function of UCART, Jurkat cells stably expressed luciferase (CBG) and were tumor-loaded into NSG mice, approximately day 5-6, and divided into two groups after CBG imaging: treatment group (U-CART) and control group (U-T), and treatment was performed by administering corresponding T cells, respectively. Imaging for 1-2 times per week and counting. As shown in fig. 7, the tumor burden was significantly reduced in the treated group relative to the control group from day 7 of treatment. The survival of both groups of mice was counted (fig. 8), and it was also seen that the survival of the treated group was significantly longer than that of the control group (44 days vs 26.17 days).

Jurkat tumor-bearing mice experiments prove that UCART also has obvious tumor killing effect in vivo.

Claims

1. A Universal CAR (UCAR) immune cell,

a) the UCAR immune cells express a Chimeric Antigen Receptor (CAR) on the surface;

b) the UCAR immune cells are used for treating a patient with a T cell tumor;

c) the CAR of the UCAR immune cell comprises an antibody that targets a T cell surface protein or a variable region of an antibody;

d) the UCAR immune cells are not autologous to the patient.

2. The UCAR immune cell of claim 1, wherein said immune cell is a T cell.

3. The UCAR immune cell of claim 1, wherein said immune cell is an NK cell.

4. The UCAR-T cells according to any of claims 1 to 3, wherein said UCAR-T cells are derived from a semi-allogeneic source, i.e., from the patient's immediate relative.

5. The UCAR immune cells of any of claims 1 to 3, wherein said UCAR immune cells are allogeneic, but not HLA-homozygous or semi-allogeneic, or from the patient's immediate relative.

6. The UCAR-T cell according to any one of claims 1 to 5, wherein all or a portion of the TCR gene (e.g., TRAC) and all or a portion of the HLA gene (e.g., B2M) are knocked out by said UCAR-T cell.

7. The UCAR-T cells according to any one of claims 1 to 6, wherein said UCAR-T cells have the FAS (CD95) gene knocked out.

8. The UCAR-T cell according to any one of claims 1 to 7, wherein the CAR of said UCAR-T cell comprises an antibody or variable region of an antibody that targets the T cell receptor TRBC1 or TRBC 2.