An adjuvant that boosts immune cell proliferation in vivo

Abstract

The present invention provides an adjuvant that can promote the expansion of immune cells in vivo, and a drug combination comprising the adjuvant and immune cells. The booster adjuvant carries a booster antigen, which can activate a certain modified therapeutic immune cell in vivo and promote the expansion of the immune cell in vivo. With the help of the booster adjuvant, the number of therapeutic immune cells produced in vitro can be greatly reduced, thereby reducing the production cost and accelerating the preparation cycle.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. Specifically, the present invention provides a booster adjuvant that can help immune cells such as T cells to achieve artificial and relatively controllable number expansion in vivo, thereby improving the effect of immunotherapy. Background Art

[0002] There is a difficulty in immune cell therapy for solid tumors, that is, there are very few cells in the patient's own natural T cells that can recognize tumors. On the one hand, it is very difficult to separate tumor-recognizing T cells from autologous natural T cells. On the other hand, even if they are separated, it is a very difficult process to expand them in vitro to the number required to fight advanced tumors and produce significant therapeutic effects. Third, even if they are expanded to this number in vitro, the long cycle and high cost bring challenges to industrialization, and many tumor-recognizing T cells are already in a state of exhaustion and will die in large numbers after in vitro expansion. Summary of the invention

[0003] In view of the above background, there is an urgent need for methods, systems and products to improve the function of immune cells, and the present invention meets this need. The present invention provides an adjuvant for boosting immune cell therapy and a modified immune cell that matches the adjuvant. When the two are used together, the adjuvant can boost the number of the immune cells in the body to increase significantly, thereby enhancing the effect of immunotherapy.

[0004] Unless otherwise indicated, the practice of some 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); Series Molecular Biology (FM Ausubel, et al. eds.); Series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ Machersrs, BD 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 (RI Breshney, ed. (2010)).

[0005] The term "about" or "approximately" means within an acceptable error range for a 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, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean 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 can mean an order of magnitude of a value, preferably within 5 times, more preferably within 2 times. Where specific values ​​are described in the application and claims, the term "about" should be assumed to mean within an acceptable error range for the specific value unless otherwise stated.

[0006] As used herein, "cell" generally refers to a biological cell. A cell can be the basic structure, function and / or biological unit of an organism. A cell can 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, protozoan cells, algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C.Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primary cells, human cells, etc.), etc. Sometimes a cell is not derived from a natural organism (eg, a cell may be synthetic, sometimes called an artificial cell).

[0007] As used herein, the term "antigen" refers to a molecule or fragment thereof that can be bound by a selective binding agent. For example, an antigen can be a ligand that can be bound by a selective binding agent such as a receptor. As another example, an antigen can be an antigen molecule that can be bound by a selective binding agent such as an immune protein (e.g., an antibody). Antigen can also refer to a molecule or fragment thereof that can be used in an animal to produce an antibody that can bind to the antigen.

[0008] As used herein, the term "neoantigen" generally refers to a tumor-specific antigen caused by a gene mutation. The resulting mutant protein or fragment thereof can trigger an anti-tumor T cell response.

[0009] As used herein, the term "gene" refers to a nucleotide sequence of a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding coding RNA transcript. As used herein, the term for genomic DNA includes inserted non-coding regions and 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 include an "open reading frame" encoding a polypeptide. In some uses of the term, a "gene" only includes coding sequences (e.g., "open reading frames" or "coding regions") necessary for encoding polypeptides. In some cases, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. 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 natural gene in its natural position in the genome of an organism. A gene may refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not normally found in the host organism but are introduced into the host organism by gene transfer. Non-natural genes can also refer to genes that are not in the natural position in the genome of an organism. Non-natural genes can also refer to naturally occurring nucleic acids or polypeptide sequences that contain mutations, insertions and / or deletions (e.g., non-natural sequences).

[0010] 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 antibodies), 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.). Its derivatives, variants or fragments may refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), miniantibodies, double antibodies and single-domain antibodies ("sdAb" or "nanoantibodies" or "camel"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of optimized antibodies include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (eg, antibodies optimized in the fragment crystallizable region) and multispecific antibodies (eg, bispecific antibodies).

[0011] As used herein, the term "gene" refers to a nucleotide sequence of a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding coding RNA transcript. As used herein, the term for genomic DNA includes inserted non-coding regions and 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 include an "open reading frame" encoding a polypeptide. In some uses of the term, a "gene" only includes coding sequences (e.g., "open reading frames" or "coding regions") necessary for encoding polypeptides. In some cases, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. 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 natural gene in its natural position in the genome of an organism. A gene may refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not normally found in the host organism but are introduced into the host organism by gene transfer. Non-natural genes can also refer to genes that are not in the natural position in the genome of an organism. Non-natural genes can also refer to naturally occurring nucleic acids or polypeptide sequences that contain mutations, insertions and / or deletions (e.g., non-natural sequences).

[0012] 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 antibodies), 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.). Its derivatives, variants or fragments may refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), miniantibodies, double antibodies and single-domain antibodies ("sdAb" or "nanoantibodies" or "camel"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of optimized antibodies include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (eg, antibodies optimized in the fragment crystallizable region) and multispecific antibodies (eg, bispecific antibodies).

[0013] The term "nucleotide" as used herein generally refers to an alkali-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates such as 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 used herein may refer to dideoxyribonucleoside triphosphates (ddNTP) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates may 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 using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

[0014] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof, which can be in single-, double-, or multi-stranded form. The polynucleotide can be exogenous or endogenous to the cell. The polynucleotide can be present in a cell-free environment. The polynucleotide can be a gene or fragment thereof. The polynucleotide can be DNA. The polynucleotide can be RNA. The polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The polynucleotide can contain one or more analogs (e.g., altered backbones, sugars or nucleobases).

[0015] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells. With reference to expression, "upregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state, while "downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state.

[0016] As used herein, the term "modulate" with respect to expression or activity refers to changing the level of expression or activity. Modulation can occur at the transcriptional level and / or the translational level.

[0017] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to a polymer of at least two amino acid residues connected by a peptide bond. The term does not imply a polymer of a specific length, nor does it imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally present. 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 a non-amino acid. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structures (e.g., domains). 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 operation, such as conjugation with a labeling component. The terms "amino acid" and "amino acid" used herein generally refer to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and non-natural amino acids that have been chemically modified to include groups or chemical moieties that are not naturally present on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes D-amino acids and L-amino acids.

[0018] When used herein with respect to polypeptides, the terms "derivatives", "variants" and "fragments" refer to polypeptides that are related to wild-type polypeptides, for example by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Derivatives, variants and fragments of polypeptides may comprise one or more amino acid variations (e.g., mutations, insertions and deletions), truncations, modifications or combinations thereof compared to wild-type polypeptides.

[0019] As used herein, "fusion" can refer to a protein and / or nucleic acid comprising one or more non-native sequences (e.g., portions). A fusion can comprise one or more identical non-native sequences. A fusion can comprise one or more different non-native sequences. A fusion can be a chimera. A fusion can comprise a nucleic acid affinity tag. A fusion can comprise a barcode. A fusion can comprise a peptide affinity tag. A fusion can provide subcellular localization of a site-directed polypeptide (e.g., a nuclear localization signal (NLS) for targeting the nucleus, a mitochondrial localization signal for targeting mitochondria, a chloroplast localization signal for targeting chloroplasts, an endoplasmic reticulum (ER) retention signal, etc. A fusion can provide a non-native sequence (e.g., an affinity tag) that can be used for tracking or purification. A fusion can be a small molecule, such as biotin or a dye, such as an Alexa fluor dye, a Cyanine3 dye, a Cyanine5 dye.

[0020] The phrase "artificial TCR" used herein may be understood as an "exogenous T cell receptor (TCR) complex", which refers to a TCR complex in which one or more chains of the 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 may refer to a TCR complex in which one or more chains of an endogenous TCR complex have one or more mutant sequences, such as at the nucleic acid or amino acid level. The expression of exogenous TCR on immune cells can confer binding specificity to an epitope or antigen (e.g., an epitope or antigen that is preferentially present on the surface of a cancer cell or other pathogenic cell or particle). The exogenous TCR complex may include TCR-α, TCR-β chain, CD3-γ chain, CD3-δ chain, CD3-ζ chain, or any combination thereof introduced into the genome. In some cases, the chain introduced into the genome can replace the endogenous chain.

[0021] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, such as humans. Mammals include, but are not limited to, rodents, monkeys, humans, farm animals, sports animals and pets. It also includes tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro.

[0022] As used herein, the terms "treat" and "treatment" refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. For example, treatment may include administration of a system or cell population disclosed herein. A therapeutic benefit refers to any treatment-related improvement or effect in one or more diseases, disorders, or symptoms being treated. For prophylactic benefit, a composition may be administered to a subject at risk for developing a particular disease, disorder, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, disorder, or symptom may not yet be manifested.

[0023] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition, e.g., a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells) of the disclosure, sufficient to produce the desired activity when administered to a subject in need thereof. In the context of the disclosure, the term "therapeutically effective" refers to an amount of a composition sufficient to delay manifestation, prevent progression, alleviate or relieve at least one symptom of a disorder treated by the methods of the disclosure.

[0024] As used herein, the term "genetic profile" refers to information about specific genes, including variation and gene expression in an individual or a type of tissue. Genetic profiles can be used for new antigen selection. As used herein, the term "somatic mutation spectrum" refers to information about specific genes associated with somatic mutations, including but not limited to specific genes generated by somatic mutations. Somatic mutation spectrum can be used for new antigen selection.

[0025] The present invention provides an adjuvant for assisting immune cell therapy (or "boosting adjuvant"), characterized in that the adjuvant carries a booster antigen (BA), which is a specific antigen that can be specifically recognized by a certain therapeutic immune cell in the body, and the adjuvant can activate and boost the number of the immune cell in the body with the help of the booster antigen;

[0026] In some cases, a specific therapeutic immune cell recognizes a booster antigen by expressing an artificially modified chimeric antigen receptor (CAR) on the therapeutic immune cell, and specifically recognizes the booster antigen by means of the CAR. The CAR is a chimeric antigen receptor targeting anBooster Antigen (hereinafter referred to as "Anti-Booster Antigen-CAR" or "aBA-CAR"), and its structure includes a variable region of an antibody that can recognize a booster antigen.

[0027] In some embodiments, the booster antigen can be a mutant of any membrane protein widely expressed by human cells, or a mutant of a membrane protein expressed by tumor cells, including class I RTKs (e.g., the epidermal growth factor (EGF) receptor family including EGFR; the ErbB family including ErbB-2, ErbB-3 and ErbB-4), class II RTKs (e.g., the insulin receptor family including INSR, IGF-1R and IRR), class III RTKs (e.g., the platelet-derived growth factor (PDGF) receptor family including PDGFR-α, PDGFR-β, CSF-1R, KIT / SCFR and FLK2 / FLT3), class IV RTKs (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, FGFR-3 and FGFR-4), class V RTKs (e.g., the vascular endothelial growth factor (VEGF) receptor family including VEGFR1, 3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6), Class IX RTKs (e.g., AXL receptor family such as AXL, MER, and TRYO3), Class X RTKs (e.g., LTK receptor family such as LTK and ALK), Class XI RTKs (e.g., LTK receptor family such as ALK and LTK), and Class IX RTKs. TIE receptor family such as TIE and TEK), Class XII RTK (e.g., ROR receptor family ROR1 and ROR2), Class XIII RTK (e.g., discoidin domain receptor (DDR) family such as DDR1 and DDR2), Class XIV RTK (e.g., RET receptor family such as RET), Class XV RTK (e.g., KLG receptor family, including PTK7), Class XVI RTK (e.g., RYK receptor family including Ryk), Class XVII RTK (e.g., MuSK receptor family such as MuSK), CD47, CD70, NKG2D, or any derivative, variant or fragment thereof.

[0028] In some embodiments, the booster antigen may comprise at least one extracellular region (e.g., a ligand binding domain) of a catalytic receptor (e.g., a receptor tyrosine kinase (RTK)) or any derivative, variant or fragment thereof, or a mutant form of such fragments. In some embodiments, the enhancing receptor comprises a class I RTK (e.g., the epidermal growth factor (EGF) receptor family including EGFR; the ErbB family including ErbB-2, ErbB-3, and ErbB-4), a class II RTK (e.g., the insulin receptor family including INSR, IGF-1R, and IRR), a class III RTK (e.g., the platelet-derived growth factor (PDGF) receptor family including PDGFR-α, PDGFR-β, CSF-1R, KIT / SCFR, and FLK2 / FLT3), a class IV RTK (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), a class V RTK (e.g., the vascular endothelial growth factor (VEGF) receptor family including VEGFR1, VEGFR2, and VEGFR3), a class VI RTK (e.g., the hepatocyte growth factor (HGF) receptor family including hepatocyte growth factor receptor (HGFR / MET) and RON), a Class VII RTK (e.g., the tropomyosin receptor kinase (Trk) receptor family, including TRKA, TRKB and TRKC), a Class VIII RTK (e.g., the ephrin (Eph) receptor family, including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5 and EPHB6), a Class IX RTK (e.g., the AXL receptor family such as AXL, MER and TRYO3), a Class X RTK (e.g., the LTK receptor family such as LTK and ALK), a Class XI RTK (e.g., the TIE receptor family such as TIE and TEK), a Class XII RTK (e.g., the ROR receptor family ROR1 and ROR2), a Class XIII RTK (e.g., the discoidin domain receptor (DDR) families such as DDR1 and DDR2), Class XIV RTK (e.g., RET receptor family such as RET), Class XV RTK (e.g., KLG receptor family, including PTK7), Class XVI RTK (e.g., RYK receptor family including Ryk), Class XVII RTK (e.g., MuSK receptor family such as MuSK), CD47, CD70, NKG2D, or any derivative, variant or fragment thereof.

[0029] The booster antigen may comprise a mutant of an RTK or any derivative, variant or fragment thereof. Non-limiting examples of RTK ligands include growth factors, cytokines and hormones. Growth factors include, for example, members of the epidermal growth factor family (e.g., epidermal growth factor or EGF, heparin-binding EGF-like growth factor or HB-EGF, transforming growth factor-α or TGF-α, amphiregulin or AR, epiregulin or EPR, epigen, betacellulin or BTC, neuregulin-1 or NRG1, neuregulin-2 or NRG2, neuregulin-3 or NRG3, neuregulin-4 or NRG4), fibroblast growth factor family (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, FGF18, FGF20, FGF21 and FGF23), the vascular endothelial growth factor family (such as VEGF)-A, VEGF-B, VEGF-C, VEGF-D and PIGF) and the platelet-derived growth factor family (e.g., PDGFA, PDGFB, PDGFC and PDGFD). Hormones include, for example, members of the insulin / IGF / relaxin family (e.g., insulin, insulin-like growth factor, relaxin family peptides, including relaxin 1, relaxin 2, relaxin 3, Leydig cell-specific insulin-like peptide (gene INSL3), early placental insulin-like peptide (ELIP) (gene INSL4), insulin-like peptide 5 (gene INSL5) and insulin-like peptide 6).

[0030] In some embodiments, the enhancing receptor comprises at least the extracellular region (e.g., ligand binding domain) of a catalytic receptor, such as a receptor threonine / serine kinase (RTSK), or any derivative, variant or fragment thereof, or a fragment comprising an antibody variable region with these fragments as antigens. The enhancing receptor may comprise a type I RTSK, a type II RTSK, or any derivative, variant or fragment thereof. The enhancing receptor may comprise a type I receptor, or any derivative, variant or fragment thereof, selected from the group consisting of: ALK1 (ACVRL1), ALK2 (ACVR1A), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGF βR1), ALK6 (BMPR1B) and ALK7 (ACVR1C). The enhancing receptor may comprise a type II receptor, or any derivative, variant or fragment thereof selected from the group consisting of: TGFβR2, BMPR2, ACVR2A, ACVR2B and AMHR2 (AMHR). In some embodiments, the booster antigen comprises TGF-β.

[0031] The booster antigen comprises RTSK or any derivative or mutant thereof.

[0032] In some cases, the booster antigen is a B cell surface protein or a fragment thereof. The B cell surface protein can be any protein that can be found on the surface of a B cell. Non-limiting examples include CD1d, CD5, CD10, CD11a, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD29, CD34, CD37, CD38, CD40, CD44, CD45, CD49b, CD69, CD72. CD74, CD80, CD83, CD84, CD86, CD93, CD95, CD117, CD127, CD138, CD147, CD148, CD185, CD270, CD284 and CD360. In some embodiments, the antigen interaction domain of the CAR is capable of binding to a surface protein on a non-B cell, as long as the binding to the surface protein does not significantly impair the general health or immune system of the host. . In some embodiments, the surface protein is a surfing protein on an immune cell. In some embodiments, the surface protein is a surface protein on a cell other than an immune cell. In some embodiments, the surface protein can be selected from, provided that binding to the surface protein does not significantly impair the general health or immune system of the host CD31, CD32, A, B, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, a, b, c, d, CD43, CD44, CD45, CD46, CD47, CD48, CD49 (a, b, c, d, e, f), CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD61, CD62 (E, L, P), CD63, CD64 (A, B, C), CD66 (a, b, c), d, e, f), CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD78, CD79(a, b), CD80, CD81, CD82, CD83, CD84, CD85(a, d, e, h, j, k), CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD1(ac), 1A, 1D, 1E, CD2, CD3(γ, δ, ε), CD4, CD5, CD6, CD7, CD8,a,CD9,CD10,CD11(a,b,c,d),CD13,CD14,CD15,CD16,A,B,CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,CD29,CD30, CD101,CD102,CD103,CD104,CD105,CD106,CD107(a,b),CD108,CD109,CD110, CD111,CD112,CD113,CD114,CD115,CD116,CD117,CD118,CD119,CD120(a, b),CD121(a,b),CD122,CD123,CD124,CD125,CD126,CD127,CD129,CD130,CD131,CD132,CD133,CD134,CD135,CD136,CD137,CD138,CD140b,CD141, CD142,CD143,CD144,CD146,CD147,CD148,CD150,CD191,CD192,CD193,CD194, CD195,CD196,CD197,CDw198,CDw199,CD200,CD201,CD202b,CD204,CD205, CD206,CD207,CD208,CD209,CDw210(a,b),CD212,CD213a(1,2),CD217,CD218, (a,b),CD220,CD221,CD222,CD223,CD224,CD225,CD226,CD227,CD228, CD229,CD230,CD233,CD234,CD235(a,b),CD236,CD238,CD239,CD240CE, CD240D,CD241,CD243,CD244,CD246,CD247,CD248,CD249,CD252,CD253,CD254,CD256,CD257,CD258,CD261,CD262,CD263,CD264,CD265,CD266,CD267, CD268,CD269,CD271,CD272,CD273,CD274,CD275,CD276,CD278,CD279,CD280, CD281,CD282,CD283,CD284,CD286,CD288,CD289,CD290,CD292,CDw293, CD294,CD295,CD297,CD298,CD299,CD300A,CD301,CD302,CD303,CD304, CD305,CD306,CD307,CD309,CD312,CD314,CD315,CD316,CD317,CD318, CD320,CD321,CD322,CD324,CD325,CD326,CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD151, CD152, CD153, CD154, CD155, CD156(a,b,c),CD157,CD158,(a,d,e,i,k),CD159(a,c),CD160,CD161,CD162,CD163,CD164,CD166,CD167(a,b),CD168, CD169, CD170, CD171, CD172(a, b, g), CD174, CD177, CD178, CD179(a, b), CD180, CD181, CD182, CD183, CD184, CD185 and CD186.

[0033] In some cases, the booster antigen is a whole or partial fragment of a surface protein of a NK cell, granulocyte, erythrocyte, or platelet.

[0034] In some cases, the booster antigen is a surface marker or transmembrane protein or a fragment thereof of a viral pathogen, including HPV virus, Epstein-Barr virus, Helicobacter pylori (HP), hepatitis B virus (HBV), and human immunodeficiency virus (HIV).

[0035] In some cases, the booster antigen is any human intracellular protein or a fragment thereof.

[0036] In some cases, the booster antigen is any human extracellular secretory protein or a fragment thereof.

[0037] In some cases, the booster adjuvant is a protein, compound, complex, or cell (hereinafter referred to as "boosting cell"), or artificial nanomaterial containing a booster antigen;

[0038] In some embodiments, the booster antigen can be coupled (e.g., by covalent and / or non-covalent bonds) to the surface of a particle (e.g., a nanoparticle). The particle can be any particulate material comprising organic and / or inorganic materials. The particle can have various shapes and sizes. The particle can be from about 1 nanometer (nm) to about 50 microns (μm) in at least one dimension. The particle can be at least about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 50 μm or more in at least one dimension. The particle can be at most about 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm or less in at least one dimension. The particle can be a nanoparticle, a microparticle, a nanosphere, a microsphere, a nanorod, a microrod, a nanofiber, a nanobelt, etc. Examples of particles include metal nanoparticles (e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic nano-semiconductor nanoparticles, core-shell nanoparticles, particles of inorganic cores with polymer shells, particles of organic cores with polymer shells, and mixtures thereof. Alternatively, the particles may be organic nanoparticles, such as cross-linked polymers, hydrogel polymers, biodegradable polymers, polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), copolymers, polysaccharides, starch, cellulose, chitosan, polyhydroxyalkanoate (PHA), PHB, PHV, lipids, peptides, peptide amphiphiles, polypeptides (e.g., proteins), or combinations thereof. Particles presenting B cell surface proteins on the surface may be introduced in vitro into immune cells comprising CARs binding to B cell surface proteins. Alternatively or in addition, particles presenting B cell surface proteins may be introduced in vivo with immune cells comprising CARs (e.g., local or systemic injection). These particles may be used to amplify immune cell populations comprising CARs in vitro or in vivo.

[0039] In some cases, the boosting adjuvant is a boosting cell, and the boosting cell is derived from the patient's autologous or allogeneic peripheral blood mononuclear cells (PBMC) or any type of cells such as B cells, T cells, NK cells, or a mixture of multiple types of cells, and the cell is a natural unmodified cell, or a modified cell, or an immortalized B cell or NK cell (such as NK92) or K562 cell;

[0040] In some cases, the booster adjuvant is a booster cell (such as a T cell), and the booster antigen is expressed transmembrane on the booster cell, and the booster antigen is a fusion protein consisting of an extracellular binding domain and a transmembrane region, wherein the extracellular binding domain is a structure recognizable by specific therapeutic immune cells (such as EGFRviii or CD19), and the transmembrane region is a partial or complete structure of a protein naturally expressed by the booster cell (such as CD3);

[0041] The antigen binding domain of aBA-CAR may include any protein or molecule capable of binding to boost antigens.Non-limiting examples of antigen binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, mouse antibodies, or their functional derivatives, variants or fragments, including but not limited to Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), microantibodies, double antibodies and single-domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains (VHH) of nanoantibodies derived from camelids.In some embodiments, the first antigen binding domain includes at least one of Fab, Fab', F(ab')2, Fv and scFv.In some embodiments, the antigen binding domain includes an antibody mimetic. Antibody mimetics refer to molecules that can bind to target molecules with an affinity comparable to that of antibodies, including single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectin), 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, the antigen binding domain may comprise at least a ligand binding domain of a transmembrane receptor.

[0042] In some embodiments, the antigen binding domain of aBA-CAR may include scFV. scFv can be derived from antibodies with known variable region sequences. In some embodiments, scFv can be derived from antibody sequences obtained from available mouse hybridomas. scFv can be obtained from all exon sequencing of tumor cells or primary cells. In some embodiments, scFv can be changed. For example, scFv can be modified in various ways. In some cases, scFv can be mutated so that scFv can have a higher affinity for its target. In some cases, the affinity of scFv to its target can be optimized for targets expressed at low levels on normal tissues. This optimization can be performed to minimize potential toxicity, such as hypercytokinemia. In other cases, cloning scFv with a higher affinity for target membrane-bound forms can be superior to its soluble counterpart. If certain targets can also be detected in soluble forms at different levels, and their targeting can cause unexpected toxicity, such as hypercytokinemia, this modification can be performed.

[0043] The antigen binding domain of the aBA-CAR of this system can be connected to the intracellular signaling domain through a transmembrane domain. The transmembrane domain can be a transmembrane segment. The transmembrane domain of the subject CAR can anchor CAR to the plasma membrane of the cell, such as an immune cell. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide connecting the antigen binding domain of CAR and the intracellular signaling domain may have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of the 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. Insertion is compared to the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as a sequence of a polypeptide linker. The polypeptide linker can be flexible or rigid. The polypeptide linker can be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits the signal from the extracellular region of the cell to the intracellular region through the antigen binding domain. The natural transmembrane portion of CD28 can be used for aBA-CAR. In other cases, the natural transmembrane portion of CD8α can also be used for aBA-CAR.

[0044] The CAR of the present disclosure may include a signaling domain or any derivative, variant or fragment thereof that participates in immune cell signaling. The intracellular signaling domain of CAR can induce the activity of immune cells containing CAR. The intracellular signaling domain can transduce effector function signals and instruct cells to perform specialized functions. The signaling domain may include the signaling domain of other molecules. In some cases, a truncated portion of the signaling domain is used for CAR.

[0045] 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, the intracellular signaling domain may comprise at least 2 immune cell signaling domains, such as at least 2, 3, 4, 5, 7, 8, 9 or 10 immune cell signaling domains. The immune cell signaling domain may be involved in regulating the primary activation of the TCR complex in 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 CAR may comprise a signaling domain of an Fcγ receptor (FcγR), an Fcε receptor (FcεR), an Fcα receptor (FcαR), a neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the signaling domain comprises an immunoreceptor tyrosine-based activation motif or ITAM. The ITAM-containing signaling domain may comprise two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids, wherein each x is independently any amino acid, resulting in a conserved motif YxxL / Ix(6-8)YxxL / I. When the antigen binding domain binds to the epitope, the ITAM-containing signaling domain may be modified, for example, by phosphorylation. The phosphorylated ITAM may serve as a docking site for other proteins, such as proteins involved in various signaling pathways. In some embodiments, the primary signaling domain comprises a modified ITAM domain, such as a mutated, truncated and / or optimized ITAM domain, which has an altered (e.g., increased or decreased) activity compared to a native ITAM domain.

[0046] In some embodiments, the intracellular signaling domain of a CAR (including aBA-CAR) comprises an FcγR signaling domain (e.g., ITAM). The FcγR signaling domain may be selected from FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In some embodiments, the intracellular signaling domain comprises an FcεR signaling domain (e.g., ITAM). The FcεR signaling domain may be selected from FcεRI and FcεRII (CD23). In some embodiments, the intracellular signaling domain comprises an FcαR signaling domain (e.g., ITAM). The FcαR signaling domain may be selected from FcαRI (CD89) and Fcα / μR. In some embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain. In some embodiments, the primary signaling domain comprises an ITAM of CD3ζ.

[0047] In some embodiments, the intracellular signaling domain of CAR (including aBA-CAR) comprises an immunoreceptor tyrosine-based inhibitory motif or ITIM. The signaling domain comprising ITIM may comprise a conserved amino acid sequence (S / I / V / LxYxxI / V / L), which is present in the cytoplasmic tail of some inhibitory receptors of the immune system. The main signaling domain comprising ITIM can be modified, for example, by an enzyme (e.g., a member of the Src kinase family (e.g., Lck)), such as phosphorylation. After phosphorylation, other proteins, including enzymes, can be recruited to ITIM. These other proteins include, but are not limited to, enzymes such as phosphotyrosine phosphatases SHP-1 and SHP-2, inositol phosphatases called SHIP, and proteins having one or more SH2 domains (e.g., ZAP70). The intracellular signaling domain may comprise BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (CD32), Fc receptor-like protein 2 (FCRL2), a signaling domain of an Fc receptor (eg, ITIM).Fc receptor-like 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-associated 1 (IRTA1), Immunoglobulin superfamily receptor translocation-associated 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 3DL1 (KIR3DL1), Killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2), Leukocyte immunoglobulin-like receptor subfamily B member 1 (LIR1), Leukocyte immunoglobulin-like receptor subfamily B member 2 (LIR2), 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 triggering receptor 2 (NKp44), 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 (SIGLEC5 or CD170), sialic acid binding Ig such as lectin 6 (SIGLEC6), sialic acid binding Ig such as lectin 7 (SIGLEC7), sialic acid binding Ig such as lectin 10 (SIGLEC10), sialic acid binding Ig such as lectin 11 (SIGLEC11), sialic acid binding Ig such as lectin 4 (SIGLEC4), sialic acid binding Ig such as lectin 8 (SIGLEC8), sialic acid binding Ig such as lectin 9 (SIGL EC9), platelet and endothelial cell adhesion molecule 1 (PECAM-1), signal regulatory protein 2 (SIRP2) and signaling 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, that has an altered (e.g., increased or decreased) activity compared to a native ITIM domain.

[0048] In some embodiments, the intracellular signaling domain of a CAR (including aBA-CAR) 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 primary signaling domains). In some embodiments, the intracellular signaling domain includes ITAM and ITIM domains.

[0049] In some cases, the intracellular signaling domain of CAR (including aBA-CAR) may include a costimulatory domain. In some embodiments, the costimulatory domain, for example, from a costimulatory molecule, can provide a costimulatory signal for immune cell signaling, such as signaling from ITAM and / or ITIM domains, for example, for activation and / or inactivation. Immune cell activity. In some embodiments, the costimulatory domain can be used to regulate proliferation and / or survival signals in immune cells. In some embodiments, the costimulatory 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 costimulatory 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, B7. -H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100(SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160(BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49A, CD49D, CD49f, CD5, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EPHB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR,HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, LY9(CD229), lymphocyte function-associated antigen-1(LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, 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-α, TRANCE / RANKL, TSLP, TSLP R, VLA1 and VLA-6. In some embodiments, the intracellular signaling domain comprises multiple costimulatory domains, such as at least two, such as at least 3, 4 or 5 costimulatory domains. The costimulatory signaling region can provide a signal that cooperates with the primary effector activation signal and can complete the requirements for activating T cells. In some embodiments, adding a costimulatory domain to a CAR can enhance the efficacy and persistence of the immune cells provided herein. ,

[0050] Compared to immune cells lacking CAR (including aBA-CAR), the combination of CAR (including aBA-CAR) and booster antigen can enhance immune cell proliferation. The proliferation of immune cells can refer to the expansion of immune cells. The proliferation of immune cells can refer to the phenotypic change of immune cells. The proliferation of immune cells comprising CAR provided herein can be greater than the proliferation of immune cells lacking CAR, and the immune cells show binding to B cell surface proteins. The proliferation of immune cells comprising 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, about 600-fold to about 700-fold greater than the proliferation of comparable immune cells lacking CAR. The proliferation of immune cells comprising CAR may be about 5 times to about 10 times, about 10 times to about 20 times, about 20 times to about 30 times, about 30 times to about 40 times, about 40 times to about 50 times, about 50 times to about 60 times, about 60 times to about 70 times, about 70 times to about 80 times, about 80 times to about 90 times, about 90 times to about 100 times, about 100 times to about 200 times, about 200 times to about 300 times, about 300 times to about 400 times, about 400 times to about 500 times, about 500 times to about 600 times, about 600 times to about 700 times more than the proliferation of comparable immunity. Cells lacking CAR, and wherein the proliferation is determined at least about 12, 24, 36, 48, 60, 72, 84 or 96 hours after contacting B cells with B cell surface proteins. Enhanced proliferation can be determined in vitro or in vivo. In some embodiments, proliferation may include the number of quantitative immune cells. Quantifying a number of immune cells may include flow cytometry, trypan blue exclusion, and / or hemocytometery. Proliferation may also be determined by phenotyping of the immune cells.

[0051] In some cases, the CAR structure includes TET-ON or any other element of a CAR or CAR-T cell expression regulator and a switch that causes CAR-T or CAR-NK cells to commit suicide or apoptosis.

[0052] In one aspect, the present disclosure provides modified tumor infiltrating lymphocytes (TIL) that specifically bind to tumor-associated antigens, including but not limited to neoantigens. The modified TIL may comprise a chimeric stimulatory molecule. The chimeric stimulatory molecule may comprise a polypeptide extracellular domain (PED) that binds to a neoantigen. The PED may be fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal. The binding of the chimeric stimulatory molecule to the neoantigen may produce an immune cell activation signal in the modified TIL. In some embodiments, the PED may be an extracellular domain of a surface protein of an unmodified TIL. In some embodiments, examples of PED include antibodies, and derivatives, variants, and fragments thereof.

[0053] In one aspect, the present disclosure provides modified T cells having dual recognition properties, wherein one aspect of the dual recognition is the recognition of target cells, i.e., the T cells can recognize and kill target cells, which are cells harmful to the human body, such as tumor cells, or non-tumor cells such as viruses and bacteria; and the other aspect is the recognition of booster antigens, i.e., the immune cells can independently recognize booster antigens or adjuvants with booster antigens and be activated to increase their number.

[0054] In one aspect, the present disclosure provides T cells modified by aBA-CAR, which have dual recognition characteristics. One aspect of the dual recognition refers to the recognition of target cells, that is, the T cells can recognize and kill target cells on the one hand, and the target cells are cells harmful to the human body, such as tumor cells, or non-tumor cells such as viruses and bacteria; on the other hand, it refers to the recognition of booster antigens, that is, the immune cells can be activated by independently recognizing booster antigens or adjuvants with booster antigens with the help of aBA-CAR, thereby expanding their number.

[0055] In one aspect, the present disclosure provides T cells modified by aBA-CAR, which have dual recognition characteristics. One aspect of the dual recognition refers to the recognition of target cells, that is, the T cells carry artificial (or exogenous) TCR or CAR (another CAR other than aBA-CAR), and can recognize and kill target cells with the help of artificial TCR or CAR. The target cells are cells harmful to the human body, such as tumor cells, or non-tumor cells such as viruses and bacteria; on the other hand, it refers to the recognition of booster antigens, that is, the immune cells can be activated with the help of aBA-CAR to independently recognize booster antigens or adjuvants with booster antigens and then expand their number.

[0056] In one aspect, the present disclosure provides tumor-recognizing T cells or tumor antigen-reactive T cells modified by aBA-CAR, which have dual recognition properties. One aspect of the dual recognition refers to the recognition of tumor cells, that is, the T cells can recognize and kill tumor cells on the one hand; on the other hand, it refers to the recognition of booster antigens, that is, the immune cells can be activated by independently recognizing booster antigens or adjuvants with booster antigens with the help of aBA-CAR, thereby expanding their number.

[0057] Tumor recognition T cells (or tumor antigen reactive T cells) can recognize tumors by using natural unmodified TCR to recognize tumor neoantigens (neoantigens), tumor-associated antigens (TAA), cancer testis antigens, or viral antigens (such as viral antigens on the surface of cells infected with HPV or EBV and transformed into cancer), etc. This type of tumor recognition T cell can be obtained by vaccine induction, or from tumor infiltrating lymphocytes (TIL), or by positive or negative screening of certain markers in peripheral blood (such as PD1, TIM3, CD137, CD39, CD28, etc.).

[0058] Tumor recognition T cells can also recognize tumors with the help of artificially modified CAR or TCR to form CAR-T or TCR-T to recognize related tumor cells.

[0059] Tumor infiltrating lymphocytes (TIL) can be any cells obtained from a tumor. For example, TIL can be cells that have migrated to a tumor. TIL can be cells that have infiltrated a tumor. In some embodiments, TIL is a leukocyte that has migrated from the bloodstream of a subject into a tumor. TIL can be, for example, a T cell, a B cell, a monocyte, or a natural killer (NK) cell. In some cases, the modified TIL comprises CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17CD4+T cells, natural killer cells, dendritic cells, or M1 macrophages. The immune cell population comprising TIL can be a mixed cell population. TIL populations can include cells of different phenotypes, cells of different degrees of differentiation, cells of different lineages, or any combination thereof. TIL can generally be defined functionally by biochemistry, using cell surface markers, or by its ability to penetrate tumors and affect treatment. TIL can be classified based on the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD25, CD27, CD28, CD39, CD56, CD137, CCR7, CD45Ra, CD95, PD-1 and TIM-3. In some embodiments, the modified TIL expresses at least one of PD-1, CD39, CD137 and TIM-3. In some cases, TIL can be functionally defined by its ability to infiltrate solid tumors when reintroduced into patients. In some cases, the modified TIL includes "primary TIL", which refers to TIL obtained from a patient tissue sample. In some cases, the modified TIL includes "secondary TIL", which refers to TIL that has been amplified or proliferated. TIL can show specific binding to new antigens. In some cases, the TCR complex of TIL confers antigen binding specificity (e.g., new antigen binding).

[0060] In some embodiments, immune cells are tumor infiltrating lymphocytes (TIL). TIL can be, for example, T cells, B cells, monocytes or natural killer (NK) cells. In some cases, TIL includes CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells or M1 macrophages. In some embodiments, TIL can express at least one of PD-1, CD137 and TIM-3. In some cases, the modified TIL includes "primary TIL", which refers to the TIL obtained from a patient tissue sample. In some cases, the modified TIL includes "secondary TIL", which refers to the TIL that has been amplified or proliferated.

[0061] In some cases, TILs can leave the tumor tissue and exist in the peripheral blood, which can be obtained by apheresis of peripheral blood mononuclear cells (PBMCs), or further obtained by screening and enrichment through one or more markers such as PD1, TIM3, CD137, CD39, etc.

[0062] In one aspect, the present disclosure provides modified T cells that specifically bind to neoantigens, the modified T cells not only comprising aBA-CAR, but also comprising enhanced receptors. The enhanced receptor may comprise an extracellular domain (ECD) of a protein. ECD may be fused with an intracellular domain (ICD) of a co-stimulatory molecule that mediates immune cell activation signals. The binding of enhanced receptors to ligands may produce immune cell activation signals in modified T cells, rather than immune cell inactivation signals.

[0063] The modified T cell may include a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen. In some embodiments, the TCR complex is an endogenous TCR complex. In some embodiments, the TCR is an exogenous TCR complex. The TCR complex of the modified immune cell (e.g., endogenous or exogenous) can confer antigen binding specificity (e.g., neoantigen binding) to the immune cell. In some embodiments, the present disclosure provides a modified T cell comprising an endogenous TCR complex that specifically binds to a neoantigen, the modified T cell comprising a chimeric stimulatory molecule, wherein the chimeric stimulatory molecule comprises: a bound polypeptide extracellular domain (PED) including, but not limited to, a membrane protein on a cell of a tumor cell, wherein the PED is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, wherein the chimeric stimulatory molecule is bound to the chimeric stimulatory molecule. The membrane protein produces the immune cell activation signal in the modified T cell.

[0064] The combination of modified immune cells (e.g., modified T cells or modified TILs provided herein) with neoantigens can activate immune cells. The enhanced receptors of modified cells can be used to provide further control of immune cell activity, such as, but not limited to, immune cell activation and amplification. The combination of enhanced receptors with ligands in modified immune cells (e.g., modified T cells or modified TILs) can trigger immune cell activation signals rather than immune cell inactivation signals in modified immune cells. Inducing immune cell activation signals rather than immune cell inactivation signals in modified immune cells can minimize the immunosuppression in immune cells. Minimizing the immunosuppression in immune cells can increase the effectiveness of immune cells in immune responses, such as by increasing the immune cell cytotoxicity against target cells (e.g., tumor cells).

[0065] The enhancing receptor may comprise an extracellular domain (ECD) of a protein, which in an unmodified immune cell triggers an immune cell signal after binding to its ligand, which signal may be an inactivation signal, an activation signal, or neither an activation nor an inactivation signal. The protein may be a signaling receptor or any functional fragment, derivative, or variant thereof. In some cases, the signaling receptor may be a membrane-bound receptor. In response to ligand binding, the signaling receptor may induce one or more signaling pathways in the cell. In some cases, the signaling receptor may be a non-membrane-bound receptor. The enhancing receptor may comprise a fragment of a receptor selected from a G protein-coupled receptor (GPCR), such as an extracellular domain; an integrin receptor; a cadherin receptor; a catalytic receptor (e.g., a kinase); a death receptor; a checkpoint receptor; a cytokine receptor; a chemokine receptor; a growth factor receptor; a hormone receptor; and an immune receptor.

[0066] In some embodiments, the enhancing receptor comprises a fragment of an immune checkpoint receptor, which can participate in the regulation of the immune system. Non-limiting examples of such receptors include, but are not limited to, programmed cell death 1 (PD-1, or PD1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), B and T lymphocyte attenuator (BTLA), killer immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T cell immunoglobulin mucin 3 (TIM-3), T cell immune receptor with Ig and ITIM domains (TIGIT), SIRP α, NKG2D.

[0067] In some embodiments, the enhancing receptor comprises at least an extracellular fragment of a TCR that can participate in recognizing a new antigen of a target cell (e.g., a cancer cell antigen or a tumor antigen). In some examples, the enhancing receptor can comprise an extracellular variable region of a TCR alpha and / or beta chain.

[0068] The enhancing receptor may comprise an immune checkpoint receptor or any derivative, variant or fragment thereof. The enhancing receptor may bind to an antigen comprising any suitable immune checkpoint receptor ligand or any derivative, variant or fragment thereof. Non-limiting examples of such ligands include, but are not limited to, B7-1, B7-H3, B7-H4, HVEM (herpes virus entry mediator), AP2M1, CD80, CD86, SHP-2, PPP2R5A, MHC (e.g., class I, class II), CD47, CD70, PD-L1 (or PDL1) and PD-L2. The region to which the enhancing receptor binds to such ligands may be a natural receptor for such ligands or a monoclonal antibody for such ligands.

[0069] In some embodiments, the enhanced receptor comprises a fragment of a cytokine receptor.Cytokine receptors can play a variety of functions, and its non-limiting examples include immune cell regulation and mediated inflammation.In some embodiments, the enhanced receptor comprises a cytokine receptor, such as type I cytokine receptor or type II cytokine receptor, or any derivative, variant or fragment thereof.In some embodiments, the enhanced receptor comprises an interleukin receptor (e.g., IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-11R, IL-12R, IL-13R, IL-15R, IL-21R, IL-23R, IL-27R and IL-31R), a colony stimulating factor receptor (e.g., erythropoietin receptor, CSF-1R, CSF-2R, GM-CSFR and G-CSFR), hormone receptor / neuropeptide receptor (e.g., growth hormone receptor, prolactin receptor and leptin receptor), or any derivative, variant or fragment thereof. In some embodiments, the enhancing receptor comprises a type II cytokine receptor, or any derivative, variant or fragment thereof. In some embodiments, the enhancing receptor comprises an interferon receptor (e.g., IFNAR1, IFNAR2 and IFNGR), an interleukin receptor (e.g., IL-10R, IL-20R, IL-22R and IL-28R), a tissue factor receptor. (also known as platelet tissue factor), or any derivative, variant or fragment thereof.

[0070] In some embodiments, the extracellular binding domain of the enhanced receptor can be any antibody or antibody fragment, and the antigen bound by the antibody can be any membrane protein widely expressed by human cells, or a membrane protein expressed by tumor cells, including class I RTK (e.g., epidermal growth factor (EGF) receptor family including EGFR; ErbB family including ErbB-2, ErbB-3 and ErbB-4), class II RTK (e.g., insulin receptor family including INSR, IGF-1R and IRR), class III RTK (e.g., platelet-derived growth factor (PDGF) receptor family including PDGFR- α, PDGFR-β, CSF-1R, KIT / SCFR and FLK2 / FLT3), Class IV RTKs (e.g., the fibroblast growth factor (FGF) receptor family, including FGFR-1, FGFR-2, FGFR-3 and FGFR-4), Class V RTKs (e.g., the vascular endothelial growth factor (VEGF) receptor family, including VEGFR1, VEGFR2 and VEGFR3), Class VI RTKs (e.g., the hepatocyte growth factor (HGF) receptor family, including hepatocyte growth factor receptor (HGFR / MET) and RON), a Class VII RTKs (e.g., the tropomyosin receptor kinase (Trk) receptor family, including TRKA, TRKB and TRKC), Class VIII RTKs (e.g., the ephrin (Eph) receptor family, including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5 and EPHB6), Class IX RTK (e.g., AXL receptor family such as AXL, MER and TRYO3), Class X RTK (e.g., LTK receptor family such as LTK and ALK), Class XI RTK (e.g., TIE receptor family such as TIE and TEK), Class XII RTK (e.g., ROR receptor family ROR1 and ROR2), Class XIII RTK (e.g., discoidin domain receptor (DDR) family such as DDR1 and DDR2), Class XIV RTK (e.g., RET receptor family such as RET), Class XV RTK (e.g., KLG receptor family, including PTK7), Class XVI RTK (e.g., RYK receptor family including Ryk), Class XVII RTK (e.g., MuSK receptor family such as MuSK), CD47, CD70, NKG2D, or any derivative, variant or fragment thereof.

[0071] In some embodiments, the enhancing receptor may comprise at least one extracellular region (e.g., ligand binding domain) of a catalytic receptor (e.g., a receptor tyrosine kinase (RTK)) or any derivative, variant, or fragment thereof, or a fragment comprising an antibody variable region with these fragments as antigens. In some embodiments, the enhancing receptor comprises a class I RTK (e.g., the epidermal growth factor (EGF) receptor family including EGFR; the ErbB family including ErbB-2, ErbB-3, and ErbB-4), a class II RTK (e.g., the insulin receptor family including INSR, IGF-1R, and IRR), a class III RTK (e.g., the platelet-derived growth factor (PDGF) receptor family including PDGFR-α, PDGFR-β, CSF-1R, KIT / SCFR, and FLK2 / FLT3), a class IV RTK (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, 3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6), Class IX RTKs (e.g., AXL receptor family such as AXL, MER, and TRYO3), Class X RTKs (e.g., LTK receptor family such as LTK and ALK), Class XI RTKs (e.g., LTK receptor family such as ALK and LTK), and Class IX RTKs (e.g., AXL receptor family such as AXL, MER, and TRYO3). TIE receptor family such as TIE and TEK), Class XII RTK (e.g., ROR receptor family ROR1 and ROR2), Class XIII RTK (e.g., discoidin domain receptor (DDR) family such as DDR1 and DDR2), Class XIV RTK (e.g., RET receptor family such as RET), Class XV RTK (e.g., KLG receptor family, including PTK7), Class XVI RTK (e.g., RYK receptor family including Ryk), Class XVII RTK (e.g., MuSK receptor family such as MuSK), CD47, CD70, NKG2D, or any derivative, variant or fragment thereof.

[0072] The enhancing receptor comprises an RTK or any derivative, variant or fragment thereof. The enhancing receptor can bind to an antigen comprising any suitable RTK ligand or any derivative, variant or fragment thereof, or a fragment comprising an antibody variable region with these fragments as antigens. Non-limiting examples of RTK ligands include growth factors, cytokines and hormones. Growth factors include, for example, members of the epidermal growth factor family (e.g., epidermal growth factor or EGF, heparin-binding EGF-like growth factor or HB-EGF, transforming growth factor-α or TGF-α, amphiregulin or AR, epiregulin or EPR, epigen, betacellulin or BTC, neuregulin-1 or NRG1, neuregulin-2 or NRG2, neuregulin-3 or NRG3, neuregulin-4 or NRG4), the fibroblast growth factor family (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 / 19, FGF16, FGF17, FGF18, FGF20, FGF21 and FGF23), the vascular endothelial growth factor family (e.g., VEGF) -A, VEGF-B, VEGF-C, VEGF-D and PIGF) and platelet-derived growth factor family (e.g., PDGFA, PDGFB, PDGFC and PDGFD). Hormones include, for example, members of the insulin / IGF / relaxin family (e.g., insulin, insulin-like growth factor, relaxin family peptides, including relaxin 1, relaxin 2, relaxin 3, Leydig cell-specific insulin-like peptide (gene INSL3), early placental insulin-like peptide (ELIP) (gene INSL4), insulin-like peptide 5 (gene INSL5) and insulin-like peptide 6).

[0073] In some embodiments, the enhancing receptor comprises at least the extracellular region (e.g., ligand binding domain) of a catalytic receptor, such as a receptor threonine / serine kinase (RTSK), or any derivative, variant or fragment thereof, or a fragment comprising an antibody variable region with these fragments as antigens. The enhancing receptor may comprise a type I RTSK, a type II RTSK, or any derivative, variant or fragment thereof. The enhancing receptor may comprise a type I receptor, or any derivative, variant or fragment thereof, selected from the group consisting of: ALK1 (ACVRL1), ALK2 (ACVR1A), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGF βR1), ALK6 (BMPR1B) and ALK7 (ACVR1C). The enhancing receptor may comprise a type II receptor, or any derivative, variant or fragment thereof selected from the group consisting of: TGF βR2, BMPR2, ACVR2A, ACVR2B and AMHR2 (AMHR). In some embodiments, the enhancing receptor comprises a TGF-β receptor, or any derivative, variant or fragment thereof.

[0074] The enhancing receptor comprising RTSK or any derivative, variant or fragment thereof can bind to an antigen comprising any suitable RTSK ligand or any derivative, variant or fragment thereof, or a fragment comprising an antibody variable region having these fragments as antigens.

[0075] The enhancing receptor may include the intracellular domain (ICD) of the costimulatory molecule that triggers the immune cell activation signal. The costimulatory molecule can bind to the ligand. In some cases, the costimulatory molecule can be activated by the ligand response protein. In some embodiments, the costimulatory molecule can be used to regulate the proliferation and / or survival signals in immune cells. In some embodiments, ICD is the intracellular domain of the costimulatory molecule, which is selected from MHC class I proteins, MHC class II proteins, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction. Lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA or Toll ligand receptors. In some embodiments, the costimulatory molecule or costimulatory 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, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD3, CD30 ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD5, CD53, CD58 / LFA-3, CD69, CD7, CD8 α, CD8 β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, IL-12R,Integrin α4 / CD49d, Integrin; 4β1, Integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9(CD229), Lymphocyte function-associated antigen-1(LFA-1), Lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, 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, TNFRII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1 and VLA-6. ,

[0076] The ECD and ICD of the enhanced receptor can 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 the 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. Insertion is compared with the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural 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, such as a signal indicating ligand binding. In some embodiments, the ECD comprises a transmembrane domain. In some embodiments, the ICD comprises a transmembrane domain.

[0077] In some embodiments, ICD can mediate the generation of immune cell activation signals (or immune cell activation signals) in immune cells. In some embodiments, immune cell activation signals are mediated by activating factors. Activating factors can be immunomodulatory molecules. Activating factors can bind to, activate or stimulate T cells or other immune cells to regulate their activity. In some embodiments, activating factors can be secreted from immune cells. Activating factors can be, for example, soluble cytokines, soluble chemokines or growth factor molecules. 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.

[0078] The immune cell activation signal may include or result in clonal expansion of the modified immune cells (e.g., modified TIL or modified T cells); release of cytokines by the modified immune cells (e.g., modified TIL or modified T cells); cytotoxicity of the modified immune cells (e.g., modified TIL or modified T cells); proliferation of the modified immune cells (e.g., modified TIL or modified T cells); differentiation, dedifferentiation or transdifferentiation of the modified immune cells (e.g., modified TIL or modified T cells); movement and / or trafficking of the modified immune cells (e.g., modified TIL or modified T cells); exhaustion and / or reactivation of the modified immune cells (e.g., modified TIL or modified T cells); release of other intercellular molecules, metabolites, chemical compounds or a combination thereof by the modified immune cells (e.g., modified TIL or modified T cells).

[0079] In some embodiments, the immune cell activation signal includes or causes the clonal expansion of immune cells. The clonal expansion may include the generation of daughter cells produced by immune cells. The daughter cells produced by clonal expansion may include enhanced receptors. The clonal expansion of modified immune cells can be greater than the clonal expansion of comparable immune cells lacking enhanced receptors. The clonal expansion of modified immune cells can be about 5 times to about 10 times, about 10 times to about 20 times, about 20 times to about 30 times, about 30 times to about 40 times, about 40 times to about 50 times, about 50 times to about 60 times, about 60 times to about 70 times, about 70 times to about 80 times, about 80 times to about 90 times, about 90 times to about 100 times, about 100 times to about 200 times, about 200 times to about 200 times. About 300 times, about 300 times to about 400 times, about 400 times to about 500 times, about 500 times to about 600 times, or about 600 times to about 700 times than comparable immune cells lacking enhanced receptors. In some embodiments, determining clonal expansion can include quantifying a plurality of immune cells, such as in the presence and absence of an enhancing receptor and after binding of a ligand to the enhancing receptor. Quantification of a plurality of 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.

[0080] In some embodiments, immune cell activation signal comprises or causes immune cell to release cytokines. In some embodiments, immune cell activity comprises or causes the release of intercellular molecules, metabolites, chemical compounds or their combinations. The cytokines released by 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, IFNγ, TNFα, CSF, TGFβ, granzymes, etc. In some embodiments, cytokine release can be quantitatively measured using enzyme-linked immunosorbent assay (ELISA), flow cytometry, Western blot, etc. The cytokine release of modified immune cells can be greater than the cytokine release of comparable immune cells lacking enhanced receptors. The modified immune cells provided herein can produce about 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times. , 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, 250 times, or more than 300 times cytokine release, compared to comparable immune cells lacking enhanced receptors. When the enhanced receptor binds to the ligand and the modified immune cell binds to the new antigen present on the target cell, the modified immune cell may show increased cytokine secretion compared to comparable immune cells lacking enhanced receptors (e.g., unmodified). In some embodiments, the secreted cytokine is IFNγ or IL-2. In some embodiments, cytokine release can be quantified in vitro or in vivo.

[0081] In some embodiments, the immune cell activation signal includes or causes the immune cell to produce cytotoxicity. In some cases, the cytotoxicity of the modified immune cells provided herein can be used to kill target cells. The immune cells or immune cell groups expressing the enhanced receptor can induce the death of target cells. Killing target cells can be used for a variety of applications, including but not limited to treating diseases or conditions in which cell groups need to be eliminated or it is desired to inhibit their proliferation. Cytotoxicity can also refer to immune cells releasing cytotoxic cytokines, such as IFNγ or granzymes. In some cases, the modified immune cells provided herein can change the release of (i) cytotoxins, such as perforin, granzymes and granulysin and / or (ii) induce apoptosis by Fas-Fas ligand interaction between T cells and target cells. In some embodiments, cytotoxicity can be quantified by cytotoxicity assays, including co-culture assays, ELISPOT, chromium release cytotoxicity assays, etc. The cytotoxicity of the modified immune cells provided herein can be greater than the cytotoxicity of comparable immune cells lacking enhanced receptors. When the enhancing receptor binds to a ligand and the modified immune cell binds to a neoantigen present on the target cell, the modified immune cell may exhibit increased cytotoxicity against the target cell, compared to a comparable immune cell lacking the enhancing receptor (e.g., unmodified). 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 the target cell compared to a comparable immune cell lacking the enhancing receptor. The modified immune cells of the present invention can induce target cell death, which is 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% molecules higher than comparable immune cells without switches. In some embodiments, the immune cells provided herein can induce apoptosis in target cells, and the target cells display target epitopes (e.g., new antigens) on their surfaces. In some embodiments, cytotoxicity can be measured in vitro or in vivo. In some embodiments, determining cytotoxicity may include determining the disease level compared to the disease level before administration after administering the modified immune cells provided herein. In some embodiments, determining cytotoxicity may include determining the disease level after administering the modified immune cells provided herein and administering the disease level after comparable immune cells lacking enhanced receptors.

[0082] In some embodiments, immune cell activation signal comprises or causes the proliferation of immune cells.The proliferation of immune cells may refer to the amplification of immune cells.The proliferation of immune cells may refer to the phenotypic changes of immune cells.The proliferation of modified immune cells disclosed herein may be greater than the proliferation of comparable immune cells lacking enhanced receptors.The proliferation of modified immune cells provided herein may be about 5 times to about 10 times, about 10 times to about 20 times, about 20 times to about 30 times, about 30 times to about 40 times, about 40 times to about 50 times, about 50 times to about 60 times, about 60 times to about 70 times, about 70 times to about 80 times, about 80 times to about 90 times, about 90 times to about 100 times, about 100 times to about 200 times, about 200 times to about 300 times, about 300 times to about 400 times, about 400 times to about 500 times, about 500 times to about 600 times, or about 600 times to about 700 times greater than comparable proliferation immune cells lacking enhanced receptors.In some embodiments, proliferation may be determined by quantitatively many immune cells. Quantifying a number of immune cells may include flow cytometry, trypan blue exclusion, and / or hemocytometery. Proliferation may also be determined by phenotyping of the immune cells.

[0083] In some embodiments, immune cell activation signals may include or result in differentiation, dedifferentiation or transdifferentiation of immune cells. Differentiation, dedifferentiation or transdifferentiation of immune cells can be determined by flow cytometry assessment of the phenotypic expression of markers of differentiation, dedifferentiation or transdifferentiation on the cell surface. In some embodiments, compared with comparable immune cells lacking enhanced receptors, the modified immune cells provided herein have increased differentiation capacity. In some embodiments, compared with comparable immune cells lacking enhanced receptors, the modified immune cells provided herein have increased dedifferentiation capacity. In some embodiments, compared with comparable immune cells lacking enhanced receptors, the modified immune cells provided herein have increased dedifferentiation capacity. In some embodiments, compared with comparable immune cells lacking enhanced receptors, the modified immune cells provided herein have greater transdifferentiation capacity.

[0084] In some embodiments, immune cell activation signals may include or cause the movement and / or transportation of immune cells. In some embodiments, movement can be determined by quantifying immune cell localization to the target site. For example, the modified immune cells provided herein can be quantified at the target site after administration, such as at a site that is not a target site. Quantification can be performed by separating lesions and quantifying many immune cells (such as tumor infiltrating lymphocytes) that contain enhanced receptors. The movement and / or transportation of immune cells containing enhanced receptors can be greater than the movement and / or transportation of comparable immune cells lacking enhanced receptors. In some embodiments, the number of immune cells containing enhanced receptors at target sites (such as tumor lesions) can be about 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times or 40 times of comparable immune numbers. Cells lack enhanced receptors. Trafficking can also be determined in vitro using transwell migration assays. In some embodiments, the number of immune cells containing enhanced receptors at target sites, such as in transwell migration assays, can be about 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times or 40 times of the number. Comparable immune cells lack the enhancer receptor.

[0085] In some embodiments, immune cell activation signals may include or cause exhaustion and / or activation of immune cells. Exhaustion and / or activation of immune cells can be determined by phenotypic analysis of flow cytometry or microscopic analysis. For example, the expression level of exhaustion markers, such as programmed cell death protein 1 (PD1), lymphocyte activation gene 3 protein (LAG3), 2B4, CD160, Tim3 and T cell immune receptor (TIGIT) with immunoglobulin and ITIM domains, are quantitatively and / or qualitatively determined. In some cases, immune cells (such as T cells) lose effector functions in a hierarchical manner and become exhausted. Due to fatigue, functions such as IL-2 production and cytokine expression and high proliferation capacity may be lost. Exhaustion may also be the production of IFNγ, TNF and chemokines and the defect of degranulation. The exhaustion or activation of the modified immune cells provided herein may be greater than the exhaustion or activation of comparable immune cells lacking enhanced receptors. In some embodiments, the 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. , 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 comparable immune cells lacking an enhancing receptor, in exhaustion or activation. In some embodiments, the 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 300 fold or more, depletion or activation is reduced compared to comparable immune cells lacking the enhancing receptor.

[0086] In some embodiments, binding of a target cell to an enhancing receptor can generate an immune cell activation signal in an immune cell modified with the enhancing receptor.

[0087] In some embodiments, upon binding of the enhancing receptor to a ligand, an immune cell modified with the enhancing receptor (e.g., a modified TIL or a modified T cell) exhibits enhanced neoantigen binding compared to an immune cell lacking the enhancing receptor.

[0088] In some embodiments, immune cells modified with enhanced receptors (such as TIL, neoantigen-reactive T cells, CAR-T, TCR-T, NK, etc.) can, to a certain extent, better overcome the inhibitory signals to immune cells from the tumor microenvironment compared to immune cells that have not been modified with enhanced modifications (corresponding to the aforementioned TIL, neoantigen-reactive T cells, CAR-T, TCR-T, NK, etc.), and the inhibitory signals may come from the ligand or antigen of the extracellular domain of the enhanced receptor, or from other tumor microenvironment inhibitory signals to immune cells other than the ligand or antigen.

[0089] In some embodiments, the ECD of the enhanced receptor is a PD1 or PDL1 monoclonal antibody, and the ICD is any co-stimulatory molecule, which can effectively overcome tumor inhibitory signals not limited to PDL1 to a certain extent, but also include inhibitory signals from CD47, TIM-3 ligands such as Galectin-9, TIGIT ligands such as CD155 and CD122 or PVR, and CTLA-4 ligands such as B7 on immune cells;

[0090] In some embodiments, if the ECD of the enhanced receptor is SIRPα or CD47 monoclonal antibody, and the ICD is any co-stimulatory molecule, the tumor suppressor signal that can be effectively overcome to a certain extent is not limited to that from CD47, but can also include inhibitory signals from PDL1, TIM-3 ligands such as Galectin-9, TIGIT ligands such as CD155 and CD122 or PVR, and CTLA-4 ligands such as B77 on immune cells;

[0091] In some embodiments, the ECD of the enhanced receptor is a TIM-3 ligand monoclonal antibody such as Galectin-9 monoclonal antibody or TIM-3 monoclonal antibody, and the ICD is any co-stimulatory molecule, which can effectively overcome the tumor inhibitory signals not only from TIM-3 ligands such as Galectin-9 to a certain extent, but also from PDL1, TIGIT ligands such as CD155 and CD122 or PVR, CTLA-4 ligands such as B77 inhibitory signals to immune cells;

[0092] In some embodiments, the ECD of the enhanced receptor is a monoclonal antibody to TIGIT or its ligands, such as CD155 monoclonal antibody, CD122 monoclonal antibody, or PVR monoclonal antibody, and the ICD is any co-stimulatory molecule, which can effectively overcome tumor inhibitory signals not only from TIGIT ligands such as CD155 and CD122 or PVR to a certain extent, but also from PDL1, TIM-3 ligands such as Galectin-9, and CTLA-4 ligands such as B77 to immune cells.

[0093] In some embodiments, the ECD of the enhanced receptor is VISTA, and the ICD is any co-stimulatory molecule, which can effectively overcome tumor suppression signals not only from VISTA ligands to a certain extent; if the ECD of the enhanced receptor is CTLA-4, the tumor suppression signals that can be effectively overcome to a certain extent are not only from CTLA-4 ligands such as B7, but also from PDL1, CD47, TIM-3 ligands such as Galectin-9, TIGIT ligands such as CD155 and CD122 or PVR, CTLA-4 ligands such as B77 inhibitory signals to immune cells;

[0094] In some embodiments, the T cells modified with aBA-CAR and enhanced receptors also carry an artificial (or exogenous) T cell receptor (TCR) or another chimeric antigen receptor CAR. When the TCR or CAR can recognize target cells, the T cells modified with the enhanced receptors have stronger immune cell activation function than T cells not modified with the enhanced receptors; when neither the artificial TCR nor another CAR can recognize target cells, the T cells modified with the enhanced receptors do not have stronger immune cell activation function than T cells not modified with the enhanced receptors.

[0095] The T cell receptor (TCR) complex that exhibits specific binding to a neoantigen can be an endogenous TCR complex or an exogenous TCR complex. The modified immune cell's TCR complex (e.g., endogenous or exogenous) can confer antigen binding specificity (e.g., neoantigen binding) to the immune cell.

[0096] In one aspect, the present disclosure provides a modified immune cell that specifically binds to a neoantigen, wherein the modified immune cell comprises: (a) a chimeric stimulatory molecule comprising a polypeptide extracellular domain (PED) that binds to a neoantigen, wherein the PED is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and wherein the binding of the chimeric stimulatory molecule to the neoantigen produces an immune cell activation signal in the modified immune cell, and (b) an aBA-CAR comprising (i) an antigen interaction domain that can bind to a booster; (ii) a transmembrane domain; (iii) an intracellular signaling domain. In some embodiments, the PED can be an extracellular domain of a surface protein of an unmodified TIL. In some embodiments, examples of PED include antibodies, as well as derivatives, variants, and fragments thereof.

[0097] In one embodiment, the present disclosure provides a modified immune cell that specifically binds to a new antigen or a tumor-associated antigen (TAA) or a cancer-testis antigen or an antigen derived from a virus (such as HPV, EBV), wherein the modified immune cell comprises: (a) a natural TCR or a genetically engineered exogenous TCR that can specifically bind to a new antigen; (b) an enhanced receptor comprising an extracellular domain (ECD) of a protein, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and wherein the enhanced receptor and the ligand produce an immune cell activation signal rather than an immune cell inactivation signal in the modified immune cell, and (c) an aBA-CAR, which comprises (i) a domain that can bind to a booster antigen interaction domain; (ii) a transmembrane domain; (iii) an intracellular signaling domain; among the above a, b, and c, a is indispensable, and in some embodiments, only one of b or c may exist; in some embodiments, a, b, and c coexist.

[0098] In one embodiment, the present disclosure provides a modified tumor-infiltrating lymphocyte (TIL) that specifically binds to a neoantigen, wherein the modified immune cell comprises: (a) a natural TCR that can specifically bind to a neoantigen; (b) an enhanced receptor comprising an extracellular domain (ECD) of a protein. The ECD is fused to the intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal, and wherein the molecule is converted into a ligand to produce an immune cell activation signal rather than an immune cell inactivation signal in the modified TIL, and (c) an aBA-CAR comprising (i) an antigen interaction domain that can bind to a booster; (ii) a transmembrane domain; (iii) an intracellular signaling domain. Among the above a, b, and c, a is indispensable, and in some embodiments, only one of b or c may exist; in some embodiments, a, b, and c coexist.

[0099] In one aspect, the disclosure provides modified immune cells that overexpress cytokines, such as chemokines, wherein the immune cells are (i) tumor infiltrating lymphocytes (TILs); (ii) interstitial tumor infiltrating lymphocytes (sTILs); or (iii) T cells that exhibit specific binding to antigens. The modified immune cells that overexpress chemokines can be any modified immune cells provided herein.

[0100] 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 can be produced by more than one cell. Cytokines can participate in the production of systemic or local immunomodulatory effects.

[0101] Some cytokines can act as proinflammatory cytokines. Proinflammatory cytokines refer to cytokines that participate in inducing or amplifying inflammatory responses. Proinflammatory cytokines can produce immune responses together with various cells of the immune system (e.g., neutrophils and leukocytes). Some cytokines can act as anti-inflammatory cytokines. Anti-inflammatory cytokines refer to cytokines that participate in reducing inflammatory responses. In some cases, anti-inflammatory cytokines can regulate proinflammatory cytokine responses. Some cytokines can act as proinflammatory cytokines and anti-inflammatory cytokines. Some cytokines, such as chemokines, can play a role in chemotaxis. Chemokines can induce directional chemotaxis in nearby responding cells.

[0102] In some embodiments, the expression of cytokines with proinflammatory and / or chemotactic functions can be upregulated in immune cells. Upregulating the expression of cytokines with proinflammatory and / or chemotactic functions can be used, for example, to stimulate an immune response against target cells in immunotherapy.

[0103] Examples of cytokines that can be overexpressed by immune cells provided herein include, but are not limited to, lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α; Mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-α; platelet growth factor; transforming growth factors (TGFs), such as TGF-α, TGF-β, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-β6, TGF-β7, TGF-β8, TGF-β9, TGF-β10, TGF-β11, TGF-β12, TGF-β13, TGF-β14, TGF-β15, TGF-β16, TGF-β17, TGF-β18, TGF-β29, TGF-β30, TGF-β319, TGF-β40, TGF-β511, TGF-β612, TGF-β72, TGF-β73, TGF-β74, TGF-β75, TGF-β76, TGF-β77, TGF-β78, TGF-β8, TGF-β8, TGF-β8, TGF-β9, TGF-β9, TGF-β10, TGF-β111, TGF- 2 and TGF-β3; insulin-like growth factor-I and -II; erythropoietin (EPO); FLT-3L; stem cell factor (SCF); osteoinductive factors; interferons (IFNs), such as IFN-α, IFN-β, IFN-γ; 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 can be membrane-bound and soluble.

[0104] In some embodiments, the overexpressed cytokine is an interleukin (IL-1) family member (e.g., a ligand), an IL-1 receptor family member, an interleukin-6 (IL-6) family member (e.g., a ligand), an IL-6 receptor, an interleukin-10 (IL-10) family member (e.g., a ligand), an IL-10 receptor, an interleukin-12 (IL-12) family member (e.g., a ligand), an IL-12 receptor, an interleukin-17 (IL-17) family member (e.g., a ligand), or an IL-17 receptor.

[0105] In some embodiments, the overexpressed cytokine is an interleukin-1 (IL-1) family member or related protein; a tumor necrosis factor (TNF) family member or related protein; 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 / CD30L. / TNFSF8, CD40LG, CD70, Fas ligand / FASLG / CD95L / CD178, EDA-A1, TNFSF14 / LIGHT / CD258, TNFA, LTA / TNFB / TNFSF1, LTB / TNFC, CD70 / CD27L / TNFSF7, TNFSF10 / TRAIL / APO-2L(CD253), RANKL / OPGL / TNFSF11(CD254), TNFSF12, TNF-α / TNFA, TNFSF13, TL1A / TNFSF15, OX-40L / TNFSF4 / CD252, CD40L / CD154 / TNFSF5, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5 / IFNaG, IFNω / IFNW1, CLCF1, CNTF, IL11, IL31, IL6, Leptin, LIF, OSM, CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CX CL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4 and FAM19A5.

[0106] Cytokine expression can be assessed using a variety of methods. Cytokine expression can be assessed by measuring the cell culture medium in which the modified immune cells are grown (e.g., in vitro production) or serum obtained from one or more serums obtained from a subject with modified immune cells (e.g., in vivo production). Cytokine levels can be quantified in various suitable units using any suitable assay, including concentration. In some embodiments, cytokine proteins are detected. In some embodiments, mRNA transcripts of cytokines are detected. Examples of cytokine assays include enzyme-linked immunosorbent assays (ELISA), immunoblotting, immunofluorescence assays, radioimmunoassays, antibody arrays allowing parallel detection of various cytokines in samples, arrays based on beads, quantitative PCR, microarrays, etc. Other suitable methods may include proteomic methods (2-D gels, MS analysis, etc.).

[0107] In some embodiments, the cytokine overexpressed by the modified immune cells provided herein is a chemokine. The chemokine can be, for example, a CC chemokine, a CXC chemokine, a C chemokine and a CX3C chemokine. In some embodiments, the chemokine overexpressed by the modified immune cells is a CC chemokine selected from 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 CCL28. The chemokine is a CXC chemokine selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17. In some embodiments, the chemokine overexpressed by the modified immune cells is a C chemokine selected from XCL1 and XCL2. In some embodiments, the chemokine overexpressed by the immune cells is a CX3C chemokine, and the CX3C chemokine is CX3CL1.

[0108] In some cases, an immune cell provided herein is a NK cell that has been genetically modified other than by aBA-CAR modification;

[0109] In some cases, provided herein is a NK cell, which is a CAR-NK cell that has been modified with a CAR gene other than aBA-CAR modification, and this CAR gene modification other than aBA-CAR gives the NK cell the property of specifically recognizing and killing target cells; the CAR gene modification other than aBA-CAR may include a partial structure of NKG2D.

[0110] In some cases, the booster adjuvant is composed of multiple / multi-stage booster cells, each of which expresses a different booster antigen as described in any one of claims 1 to 6, and each of which specifically targets another booster antigen with the help of its own BA-CAR, to form a cascade amplification booster system. For example, for an n-stage booster system, the first-stage booster cells express the first booster antigen (BA1), the second-stage booster cells simultaneously express the second booster antigen (BA2) and aBA1-CAR targeting the first booster antigen, the third-stage booster cells simultaneously express the third booster antigen (BA3) and aBA2-CAR targeting the second booster antigen, ... and so on, the last-stage booster cells simultaneously express the last booster antigen (BAn) and aBAn-1-CAR targeting the previous-stage booster antigen, the last booster antigen BAn is recognized by the therapeutic immune cells carrying the aBA-CAR targeting the last booster antigen Ban, and the total number of stages of the cascade amplification booster system is any natural number greater than or equal to 1;

[0111] In some cases, the number n of the multi-stage booster system of cascade amplification is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9 to 10,000,000;

[0112] In some cases, in a multi-stage booster system of cascade amplification, booster cells starting from the second stage can be activated and expanded in vivo by booster antigens at the previous stage with the help of BA-CAR, and booster cells at different stages can be of the same type (such as all T cells, or all NK cells), or of different types, or a mixture of multiple cells;

[0113] The present invention provides an immune cell therapy adjuvant drug, characterized in that the drug comprises the booster adjuvant described herein;

[0114] The present invention provides an immune cell therapy drug combination, characterized in that the drug combination comprises the boosting adjuvant described herein and the therapeutic immune cells described by the author, and the two are used in combination, specifically, the therapeutic immune cells and the boosting adjuvant are reinfused once or continuously multiple times at appropriate time points, so that the therapeutic immune cells can be activated by the adjuvant on the one hand, and the number is expanded, and on the other hand, the therapeutic immune cells can recognize and kill target cells;

[0115] The present invention provides a method for treating cancer, characterized in that the immune cell therapy adjuvant drug and the immune cell therapy drug combination described herein are used, specifically, the therapeutic immune cells and the immune cell therapy adjuvant drug are reinfused once or continuously multiple times at appropriate time points.

[0116] In one aspect, the present disclosure provides a method for treating cancer in a subject, comprising: (a) administering to the subject a modified TIL, a modified T cell, a modified T cell or a modified immune cell that is positively screened by one or more of PD1, TIM3, CD137, CD39 in peripheral blood PBMC, any one of the embodiments. Aspects herein; (b) contacting a target cell of a cancer expressing a neoantigen with a modified TIL, a modified screened T cell, a modified T cell or a modified immune cell under conditions that induce the cytotoxicity of the modified TIL, the modified T cell, the modified T cell or the modified immune cell against the target cell, thereby inducing the death of the cancer target cell.

[0117] In one aspect, the present disclosure provides a method for expanding a T cell population, the method comprising: (a) providing a T cell population comprising at least the modified immune cell of any one of the various embodiments of the aspects described herein; and (b) exposing the T cell population to a booster antigen to achieve expansion of the T cell population.

[0118] In one aspect, the present disclosure provides a method for expanding a T cell group, comprising: (a) introducing a nucleic acid encoding aBA-CAR into a T cell group, thereby producing a cell or cell group expressing aBA-CAR, wherein aBA-CAR comprises (i) A domain that interacts with an antigen specifically recognized by CAR; (ii) a transmembrane domain; (iii) an intracellular signaling domain; (b) contacting a cell group expressing aBA-CAR with a booster antigen, thereby producing an amplified and / or activated immune cell group. Wherein, the antigen is not specifically expressed by tumor target cells.

[0119] In one aspect, the present disclosure provides a composition comprising one or more polynucleotides encoding one or more of the following: (a) an enhancing receptor, wherein the enhancing receptor comprises an extracellular domain (ECD) of a protein, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory protein that mediates an immune cell activation signal; (b) an antigen-specific T cell receptor complex, or one or more components thereof.

[0120] In one aspect, the present disclosure provides a composition comprising one or more polynucleotides encoding one or more of the following: (a) an antigen-specific T cell receptor complex, or one or more components thereof; (b) an aBA-CAR comprising (i) a domain capable of binding to a booster antigen interaction domain; (ii) a transmembrane domain; (iii) an intracellular signaling domain.

[0121] In one aspect, the present disclosure provides a composition comprising one or more polynucleotides encoding one or more of the following: (a) an enhancing receptor, wherein the enhancing receptor comprises an extracellular domain (ECD) of a protein, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory protein that mediates an immune cell activation signal; (b) an aBA-CAR comprising (i) a domain capable of binding to a booster antigen interaction domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0122] In one aspect, the present disclosure provides a composition comprising one or more polynucleotides encoding one or more of the following: (a) an enhancing receptor, wherein the enhancing receptor comprises an extracellular domain (ECD) of a protein, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory protein that mediates an immune cell activation signal; (b) an antigen-specific T cell receptor complex, or one or more components thereof; (c) an aBA-CAR comprising (i) a domain capable of binding to a booster antigen interaction domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0123] In various embodiments of aspects of the present invention, 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 can be an inducible or constitutively active promoter. Alternatively or additionally, the promoter can be tissue or cell specific.

[0124] 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 retrovirus, human elongation factor-1 promoter (EF1), a hybrid construct comprising a cytomegalovirus (CMV) enhancer fused to a chicken beta active promoter (CAG), a mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK) and mouse metallothionein-I. The promoter may be a fungal promoter. The promoter may be a plant promoter. A database of plant promoters (e.g., PlantProm) may be found. The expression vector may also contain a ribosome binding site and a transcription terminator for translation initiation. The expression vector may also include a suitable sequence for amplifying expression.

[0125] In various embodiments of the present invention, the modified immune cells can specifically bind to neoantigens and / or neoepitopes. Neoantigens and neoepitopes generally refer to tumor-specific mutations, which in some cases trigger anti-tumor T cell responses. For example, these endogenous mutations can be identified using whole exome sequencing methods. Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+ T cells in patients with epithelial cancers," Science 344: 641-644 (2014). Modified immune cells (e.g., modified TILs or modified T cells) comprising enhanced receptors can exhibit specific binding to tumor-specific neoantigens. The neoantigens bound by the immune cells can be expressed on the target cells and, for example, encoded by mutations in endogenous genes. In some cases, the neoantigens or neoepitopes specifically bound by the immune cells can be encoded by mutant genes. The gene may be selected from the group consisting of: ABL1, ACO1 1997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B (encoding NY-ESO-1), DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM111B, FGFR3, FRG1B, GAGE1, GAGE ​​10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB 16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB 17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, NY-ESO, PAGE2, PAGE5, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B 1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL and XPOT. In some embodiments, the neoantigens are selected based on the genetic profile of a tumor sample from an individual. In some embodiments, the neoantigens can be selected based on the somatic mutation profile of a tumor sample from an individual.

[0126] In various embodiments of aspects of the present invention, the modified immune cells also include a kill switch (or suicide switch). In cases of severe toxicity, such as hypercytokinemia, the kill switch can be activated to eliminate immune cells. This can 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, rapid heartbeat, hypotension and difficulty breathing. The kill switch can be a drug-induced kill switch. The kill switch can include inducible caspase 9.

[0127] Various embodiments of aspects of this paper 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 subjects. 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 juices, tears, feces, semen, vaginal fluid, interstitial fluid from tumor tissue, eye fluid, sweat, mucus, cerumen, oil, glandular secretions, spinal fluid, hair, nails, plasma, nasal swabs or nasopharyngeal washings, spinal fluid, cerebrospinal fluid, tissue, throat swabs, biopsies, placental fluid, amniotic fluid, cord blood, force fluid, cavity fluid, sputum, pus, microbiota, meconium, breast milk and / or other excreta or body tissues.

[0128] In some cases, the cell can be a T cell population, NK cell, B cell, etc. obtained from a subject. T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from infection sites, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of techniques, such as FicollTM separation, can be used to obtain T cells from blood units collected from subjects. In one embodiment, cells from individual circulating blood 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 can be washed to remove the plasma portion and the cells are placed in a suitable buffer or culture medium for subsequent processing steps.

[0129] Any of a variety of immune cells can be used in the aspects of this article. In some embodiments, immune cells include granulocytes, such as asophils, eosinophils and neutrophils; mast cells; monocytes can 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, immune cells are immune effector cells. Immune effector cells refer to immune cells that can respond to stimulation and perform specific functions. In some embodiments, immune cells are immune effector cells that can induce cell death. In some embodiments, immune cells are lymphocytes. In some embodiments, lymphocytes are NK cells. In some embodiments, lymphocytes are T cells. In some embodiments, T cells are activated T cells. 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 CTL 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), αβT cells, γδT cells and similar unique categories of T cell lineages. T cells can be divided into two major 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 (CTL). CTLs expressing the subject system can participate in the recognition and removal of virus-infected cells and cancer cells. CTLs have specialized compartments, or granules, that contain cytotoxins that cause apoptosis, i.e. programmed cell death. CD4+ T cells can be subdivided into four subgroups - Th1, Th2, Th17, and Treg, with "Th" referring to "T helper cells," although other subgroups may exist. Th1 cells can coordinate immune responses against intracellular microorganisms, particularly bacteria. They can produce and secrete molecules that alarm and activate other immune cells, such as bacteria that are ingested by macrophages. Th2 cells are involved in coordinating immune responses against extracellular pathogens, such as helminths (parasites), by alerting B cells, granulocytes, and mast cells. Th17 cells can produce interleukin 17 (IL-17), a signaling molecule that activates immune and non-immune cells. Th17 cells are important for recruiting neutrophils.

[0130] In some embodiments, the immune cell populations provided herein can be heterogeneous. In some embodiments, the cells used can be composed of a heterogeneous mixture of CD4 and CD8 T cells. CD4 and CD8 cells can have phenotypic characteristics of circulating effector T cells. The CD4 and CD8 cells can also have phenotypic characteristics of effector memory cells. In some embodiments, the cells can be central memory cells.

[0131] In some embodiments, cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other blood cell subsets, 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, cells can be any immune cells, including any T cells, such as tumor infiltrating cells (TIL), such as CD3+T cells, CD4+T cells, CD8+T cells or any other type of T-cells. Cells. 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 number of populations, for example, selecting T cells from whole blood. T cells may also be amplified from a large number of populations. T cells may also be biased toward specific populations and phenotypes. For example, T cells can be skewed to a phenotype including CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+). Suitable cells can be selected comprising one or more markers selected from the following list: CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+). Cells also include stem cells, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells. Cells may include any number of primary cells, such as human cells, non-human cells and / or mouse cells. Cells may be progenitor cells. Cells may be from a subject to be treated (e.g., a patient). Cells may be derived from human donors. The host cell can be a stem memory TSCM cell composed of CD45RO(-), CCR7(+), CD45RA(+), CD62L+(L-selectin), CD27+, CD28+ and IL-7Rα+, which can also express CD95, IL-2Rβ, CXCR3 and LFA-1, and show many functional attributes different from the stem storage cells. The host cell can be a central memory TCM cell containing L-selectin and CCR7, which can secrete, for example, IL-2, but not IFNγ or IL-4. The cell can also be an effector memory TEM cell containing L-selectin or CCR7, and produces, for example, effector cytokines such as IFNγ and IL-4.

[0132] In various embodiments of aspects of this paper, immune cells include lymphocytes. In some embodiments, lymphocytes are natural killer cells (NK cells). In some embodiments, lymphocytes are T cells. T cells can be obtained from many 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 can be used. Immune cells such as lymphocytes (e.g., cytotoxic lymphocytes) can preferably be autologous cells, but heterologous cells can also be used. Any number of techniques, such as Ficoll separation, can be used to obtain T cells from blood units collected from subjects. Cells from individual circulating blood can be obtained by apheresis or leukocyte removal. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. Cells collected by apheresis can be washed to remove the plasma portion, and the cells are 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 PBS without Ca and Mg. Alternatively, the undesired components in the single blood component sample can be removed and the cells can be resuspended directly in the culture medium. The sample can be provided directly by the subject, or indirectly through one or more intermediates, such as a sample collection service provider or a medical provider (e.g., a doctor or nurse). In some embodiments, separation of T cells from peripheral blood leukocytes can include lysing red blood cells and separating monocytes from peripheral blood leukocytes by, for example, centrifugation through a PERCOL™ gradient.

[0133] Specific subpopulations of T cells, such as CD4+ or CD8+ T cells, can be further separated by positive or negative selection techniques. For example, with a combination of antibodies for surface markers specific to negatively selected cells, negative selection of T cell populations can be achieved. A suitable technique includes cell sorting by negative magnetic immunoadhesion, which utilizes a mixture of monoclonal antibodies for cell surface markers present on negatively selected cells. For example, in order to separate CD4+ cells, a mixture of monoclonal antibodies may include antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD8. Negative selection processes can be used to produce a mainly homogeneous desired T cell population. In some embodiments, the composition comprises a mixture of two or more (e.g., 2,3,4,5 or more) different types of T cells.

[0134] In some embodiments, immune cells are members of enriched cell populations. One or more desired cell types can be enriched by any suitable method, non-limiting examples of which include treating cell populations to trigger amplification and / or differentiation into desired cell types, treatment to prevent the growth of unwanted cell types (s), killing or cracking the treatment of unwanted cell types, purifying desired cell types (e.g., purifying on affinity columns to retain desired or unwanted cell types based on one or more cell surface markers). In some embodiments, the enriched cell population is a cell population rich in cytotoxic lymphocytes, which are selected from 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.

[0135] In order to separate the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be changed. In certain embodiments, it may be necessary to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, a concentration of 2 billion cells / mL can be used. In some embodiments, a concentration of 1 billion cells / mL is used. In some embodiments, more than 100 million cells / mL are used. A cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / mL can be used. In another embodiment, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells / mL can be used. In a further embodiment, a concentration of 125 or 150 million cells / mL can be used. Using high concentrations can lead to increased cell yield, cell activation and cell expansion.

[0136] 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 can be in vitro. The target cell can be in vivo. The target cell can be ex vivo. The target cell can be an isolated cell. The target cell can be a cell in an organism. The target cell can be an organism. The target cell can be a cell in a cell culture. The target cell can be one of a collection of cells. The target cell can be a mammalian cell or derived from a mammalian cell. The target cell can be a rodent cell or derived from a rodent cell. The target cell can be a human cell or derived from a human cell. The target cell can be a prokaryotic cell or derived from a prokaryotic cell. The target cell can be a bacterial cell or can be derived from a bacterial cell. The target cell can be an archaeal cell or derived from an archaeal cell. The target cell can be a eukaryotic cell or derived from a eukaryotic cell. The target cell can be a pluripotent stem cell. The target cell can be a plant cell or derived from a plant cell. The target cell can be an animal cell or derived from an animal cell. The target cell can be an invertebrate cell or derived from an invertebrate cell. The target cell can be a vertebrate cell or derived from a vertebrate cell. The target cell can 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 cell may be derived from a specific organ or tissue.

[0137] The target cell can be a stem cell or a progenitor cell. The target cell 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.). The target cell can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. The cloned cell can include the offspring of the cell. The target cell can include the target nucleic acid. The target cell can be in a living organism. The target cell can be a genetically modified cell. The target cell can be a host cell.

[0138] The target cell can be a primary cell. For example, a culture of a primary cell can be passaged 0, 1, 2, 4, 5, 10, 15 or more times. The cell can be a unicellular organism. The cell can be grown in a culture medium.

[0139] The target cell may be a diseased cell. The diseased cell may have altered metabolic, gene expression and / or morphological characteristics. The diseased cell may be a cancer cell, a diabetic cell, and an apoptotic cell. The diseased cell may be a cell from a diseased subject. Exemplary diseases may include blood disorders, cancer, metabolic disorders, eye diseases, organ disorders, musculoskeletal disorders, heart disease, etc.

[0140] If the target cells are primary cells, they can be harvested from individuals by any method. For example, leukocytes can be harvested by apheresis, leukocyte removal, density gradient separation, etc. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. Suitable solutions can be used to separate or suspend harvested cells. Such solutions can generally 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 acceptable low concentrations of buffer. Buffers can include HEPES, phosphate buffers, lactate buffers, etc. Cells can be used immediately, or they can be stored (e.g., by freezing). Frozen cells can be thawed and can be reused. Cells can be frozen in DMSO, serum, culture medium buffer (e.g., 10% DMSO, 50% serum, 40% buffered culture medium) and / or some other such commonly used solutions for preserving cells at freezing temperatures.

[0141] 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., US20080241194); myeloid cells, such as granulocytes (basophils, eosinophils, neutrophils / excessive neutrophils), 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 (pinealocytes) cells; nervous system cells, including glial cells (astrocytes, microglia), giant cell neurosecretory cells, stellate cells, Boettcher cells and pituitary (gonadotropins, corticosteroids, thyroid hormones, growth hormones, lactate hormones); respiratory system cells, including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells; Cells of the circulatory system, including myocardial cells, pericytes; cells of the digestive system, including stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, teeth (osteoblasts, ameloblasts); cartilage cells, including chondrocytes, chondrocytes; skin cells, including hair cells, keratinocytes, melanocytes (nevus cells); muscle cells, including muscle cells; urinary system cells, including podocytes, juxtaglomerular cells, mesangial cells / glomerular mesangial cells, renal proximal tubule brush border cells, macular cells; reproductive system cells, including sperm, supporting cells, Leydig cells, eggs; and other cells, including adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail basal cells (stem cells), medullary hair cells, cortical hair shaft cells, keratin hair stem cells, keratin hair root sheath cells, Huxley Hair root sheath cells of the stratum henle, Hair root sheath cells of the stratum henle, Outer hair sheath cells, Hair matrix cells (stem cells), Wet stratified barrier epithelial cells, Stratified squamous surface epithelial cells of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Basal cells (stem cells) of the corneal epithelium, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urothelial cells (urinary bladder and ureter), Exocrine secretory epithelial cells, Salivary mucous cells (polysaccharide-rich secretions), Salivary serous cells (glycoprotein-rich secretions),Von Ebner's glandular cells in the tongue (washing taste buds), milk gland cells (milk secretion), lacrimal gland cells (tear secretion), carmine gland 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 (smell secretion, sex hormone sensitive), Moll cell glands of the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Bowman gland cells in the nose (cleans the olfactory epithelium), Brunner gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secrete seminal fluid components, including fructose for swimming sperm), prostate cells (secrete seminal fluid components), Bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubricant secretion), Littre cell glands (mucus secretion), endometrial cells (carbohydrate secretion), goblet cells of the respiratory and digestive tracts (mucus secretion), gastric mucosal cells (mucus secretion), gastric gland fermentation cells (pepsinogen secretion), gastric gland oxyntic cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Paneth cells of the small intestine (lysozyme secretion), Type II lung pneumocytes (surfactant secretion), Clara cells of the lung, Hormone secreting cells, Cells of the anterior pituitary, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary cells, Magnocellular neurosecretory cells, Cells of the intestinal and respiratory tract, Thyroid cells, Thyroid epithelial cells, Parafollicular cells, Parathyroid cells, Parathyroid chief cells, Oxyphil cells, Adrenal cells, Chromaffin cells, Testicular Ley dig cells, Ovarian follicle endometrial cells, Ruptured ovarian follicle luteal cells, Granulosa cell lutein cells, Theca lutein cells, Juxtaglomerular cells (renin secretion), Kidney Macula densa cells, metabolic and storage cells,, barrier function cells (lung, intestine, exocrine glands and urogenital tract), kidney, type I pneumocytes (lining the air spaces in the lungs), pancreatic duct cells (central cells), non-circular duct cells (sweat glands, salivary glands, mammary glands, etc.), ductal cells (spermatic vesicles, prostate, etc.), epithelial cells lining the closed body cavities, ciliated cells with propulsion function, extracellular matrix secreting cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (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 progenitor cells for blood and immune system (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 supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, Oogonium / Oocyte, Spermatid, Spermatocyte, S permatogonium cells (stem cells of spermatocytes), sperm, nurse cells, ovarian follicle cells, supporting cells (testis), thymic epithelial cells, interstitial cells and interstitial kidney cells, or processed or irradiated non-human cells such as K562, NK92, etc. Target cells can be natural cells or modified cells. ,

[0142] 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, Acrospiroma, Acute eosinophilic leukemia, Acute lymphocytic leukemia, Acute megakaryocytic leukemia, Acute monocytic leukemia, Mature acute myeloblastic leukemia, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Diamond disease, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatous odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, Alveolar soft tissue sarcoma, Ameloblastoma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid carcinoma, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Adnexal carcinoma, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Bilia rytract cancer, bladder cancer, Blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, cancer of unknown primary, Carcinosarcoma, Castleman disease, embryonal tumor of the central nervous system, cerebellar astrocytoma, brain astrocytoma, cervical cancer, bile duct cancer, chondroma, chondrosarcoma, chordoma, choroid carcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloproliferative disease, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, cutaneous fibrosarcoma, dermoid cyst, hyperplastic small round cell tumor, diffuse large B-cell cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial carcinoma, endometrial carcinoma, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing family tumors, Ewing family sarcoma, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetal fetal, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, glioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme,Glioma, Gliomatosis Cerebri, Glomus Tumor, Glucagonoma, Gonadoblas toma, Granulosa Cell Tumor, Hairy Cell Leukemia, Hairy Cell Leukemia, Head and Neck Cancer, Head and Neck Cancer, Heart Cancer, Hemangioblastoma, Hemangiopericytoma, Angiosarcoma, Heart Cancer, Hemangioblastoma, Hemangiopericytoma, Angiosarcoma, Hematological Malignancies, Hepatocellular Carcinoma, Hepatosplenic T-Cell Lymphoma, Hereditary Breast Cancer, Hodgkin Lymphoma, Hodgkin Lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory Breast Cancer, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Tumor, Juvenile Myeloid Leukemia, Kaposi's Sarcoma, Kaposi's Sarcoma, Kidney Cancer, Klatzkin Tumor, Krukenberg Tumor, Laryngeal Cancer, Laryngeal Cancer, Malignant Melanoma, Leukemia, Leukemia, Liposarcoma, Liposarcoma , lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant tritonoma, MALT lymphoma, mantle cell lymphoma a, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medulloblastoma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell carcinoma, occult primary, metastatic urothelial carcinoma, mixed Mullerian tumor, monocytic leukemia, oral cancer, myxoma, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic syndrome, myelodysplastic syndrome, myeloid leukemia, myeloma, myeloproliferative disorders, myxoma, nasal cancer, nasopharyngeal cancer, nasopharyngeal cancer, tumor, neuroma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology, oligolymphocytic tumor, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer Ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian tumor of low malignant potential, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pineocytoma, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polyembryonic tumor, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei,Rectal cancer, Renal cell carcinoma, Respiratory tract cancer, NUT gene involved on Chromosomal 15, Retinoblastoma, Rhabdoid tumor, Rhabdomyosarcoma, Richter transformation, Sacrococcygeal teratoma, Salivary gland cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary tumors, Seminomas, Serous tumors, Sertoli-Leydig cell tumors, Sex cord-stromal tumors, Sezary syndrome, Signet ring cell carcinoma, Skin cancer, Small blue cell tumor, Small cell carcinoma, Small cell lung cancer, Small cell lymphoma Tumor, Small Intestinal Cancer, Soft Tissue Sarcoma, Somatostatinoma, Moa Warts, Spinal Cord Tumors, Spinal Tumors, Splenic Marginal Zone Lymphoma, Squamous Cell Carcinoma, Gastric Cancer, Superficial Spreading Melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface Epithelial-Mesenchymal Tumor, Synovial Sarcoma, T-cell Acute Lymphocytic Leukemia, T-cell Large Granular Lymphocytic Leukemia, T-cell Leukemia, T-cell Lymphoma, T-cell Prolymphocytic Leukemia, Teratoma, Advanced Lymphoma, Testicular Cancer, Thecoma, Laryngeal Cancer, Thymic Carcinoma, Thymoma, Thyroi d's Carcinoma, Transitional Cell Carcinoma of the Renal Pelvis and Ureter, Transitional Cell Carcinoma, Urachal Carcinoma, Urethral Carcinoma, Genitourinary Tumors, Uterine Sarcoma, Uveal Melanoma, Vaginal Cancer, Verner Morrison Syndrome, Verrucous Carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Warthin's Tumor, Wilms' Tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is of the hematopoietic lineage, such as a lymphoma. The antigen may be a tumor-associated antigen.

[0143] In some embodiments, the target cells form tumors. Tumors treated with the methods herein can result in stable tumor growth (e.g., the volume of one or more tumors increases by no more than 1%, 5%, 10%, 15% or 20%, and / or does 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 to below the detection level. In some embodiments, the subject remains tumor-free (e.g., in 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.

[0144] The death of 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 markers associated with live or dead cells (e.g., live or dead target cells). The degree of cell death can be determined by any suitable method. In some embodiments, the degree 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 circulating target cell of known concentration. In this case, the degree of cell death can be expressed as the ratio of surviving cells to the starting cell population after treatment. In some embodiments, the degree of cell death can be determined by a suitable cell death assay. Various cell death analyses can be used, and a variety of detection methods can be used. Examples of detection methods include but are not limited to the use of cell staining, microscopy, flow cytometry, cell sorting, and combinations of these.

[0145] When the tumor is surgically removed after the treatment period, the efficacy of the treatment in reducing the size of the tumor can be determined by measuring the percentage of resected tissue that is necrotic (i.e., dead). 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.

[0146] Exposing target cells to immune cells or immune cell groups disclosed herein can be performed in vitro or in vivo. Exposing target cells to immune cells or immune cell groups generally refers to contacting target cells with immune cells and / or being close enough so that the antigen of the target cell (e.g., membrane-bound or non-membrane-bound) can be combined with the enhanced receptor expressed in the immune cell. Exposing target cells to immune cells or immune cell groups also generally refers to contacting target cells with immune cells and / or being close enough so that the antigen of the target cell (e.g., membrane-bound or non-bound). Membrane-bound) can be combined with CAR expressed in immune cells. By co-culturing target cells and immune cells, immune cells or immune cell groups can be exposed to target cells in vitro. Target cells and immune cells can be co-cultured, for example, as adherent cells or as suspensions. Target cells and immune cells can be co-cultured in various suitable types of cell culture media, such as co-cultured with supplements, growth factors, ions, etc. Target cells can be exposed to immune cells or immune cell groups in vivo, in some cases, by administering immune cells to subjects (e.g., human subjects), and allowing immune cells to be located in target cells through the circulatory system. In some cases, immune cells can be delivered to the immediate area where target cells are localized, such as by direct injection.

[0147] Exposure can be for any suitable length of time, e.g., 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.

[0148] The various domains of the enhanced receptor and CAR provided herein can be connected by a chemical bond, such as an amide bond or a disulfide bond; 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. The peptide linker can provide the required flexibility to allow the desired expression, activity, and / or conformational positioning of the chimeric polypeptide. The peptide linker can have any suitable length to connect at least two target domains, and is preferably designed to be flexible enough to allow the correct folding and / or function and / or activity of one or two domains connected thereto. The peptide linker may 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 length of 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 some embodiments, the linker sequence may include an endogenous protein sequence. In some embodiments, the linker sequence includes glycine, alanine and / or serine amino acid residues. In some embodiments, the linker may contain a motif of GS, GGS, GGGGS, GGSG or SGGG, such as multiple or repeated motifs. The linker sequence may include any naturally occurring amino acid, non-naturally occurring amino acid or a combination thereof.

[0149] Any suitable delivery method can be used to introduce the compositions and molecules of the present disclosure (e.g., polypeptides and / or nucleic acids encoding polypeptides) into host cells, such as immune cells. The various components can be delivered simultaneously or separated in time. The choice of method can depend on the type of transformed cells and / or the environment in which the transformation occurs (e.g., in vitro, ex vivo, or in vivo).

[0150] The delivery method may include contacting the target polynucleotide or introducing one or more nucleic acids into a cell (or cell population such as an immune cell), the nucleic acid comprising a nucleotide sequence encoding the composition of the present invention. Suitable nucleic acids comprising a nucleotide sequence encoding the composition of the present disclosure may include an expression vector, wherein the expression vector comprising a nucleotide sequence encoding one or more compositions of the present disclosure is a recombinant expression vector.

[0151] Non-limiting examples of delivery methods or transformation include, e.g., viral or bacteriophage 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.

[0152] In some aspects, the present 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 present disclosure also provides cells produced by these methods, and organisms (e.g., animals, plants, or fungi) comprising these cells or produced by these cells.

[0153] Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to apply nucleic acids encoding the disclosed compositions to cells or host organisms in culture. Non-viral vector delivery systems can include DNA plasmids, RNA (transcripts of vectors such as those described herein), naked nucleic acids, and nucleic acids complexed with delivery vectors, such as liposomes. Viral vector delivery systems can include DNA and RNA viruses, which can have an additional or integrated genome after delivery to cells.

[0154] Methods for non-viral delivery of nucleic acids may include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial viral particles, and agents to enhance uptake of DNA. Cationic and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides may be used. Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). Preparations of lipid: nucleic acid complexes may be used, including targeted liposomes, such as immunolipid complexes.

[0155] RNA or DNA virus-based systems can be used to target specific cells in vivo and transport the viral payload to the nucleus. Viral vectors can be administered directly (in vivo), or they can be used to treat cells in vitro, and can optionally be given (ex vivo) to modified cells. Virus-based systems can include retroviruses, slow viruses, adenoviruses, adeno-associated and herpes simplex virus vectors for gene transfer. Using retroviruses, slow viruses and adeno-associated virus gene transfer methods can be integrated in the host genome, which can result in the long-term expression of the inserted transgene. High transduction efficiency can be observed in many different cell types and target tissues.

[0156] Lentivirus can integrate its genome into host cells (such as 293 cells or T cells). Lentivirus can use a three-plasmid system or a four-plasmid system.

[0157] The tropism of retroviruses can be changed by incorporating foreign envelope proteins, thereby expanding the potential target population of target cells. Lentivirus 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, which have packaging capacity of up to 6-10kb exogenous sequences. Minimal cis-acting LTRs may be sufficient for replication and packaging of vectors, which can be used to integrate therapeutic genes into target cells to provide permanent transgenic expression. Retroviral vectors may include vectors based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV) and combinations thereof.

[0158] Adenovirus-based systems can be used. Adenovirus-based systems can result in transient expression of transgenes. Adenovirus-based vectors can have high transduction efficiencies in cells and can eliminate the need for cell division. High titers and expression levels can be obtained with adenovirus-based vectors. Adeno-associated virus ("AAV") vectors can be used to transduce cells with target nucleic acids, for example, to produce nucleic acids and peptides in vitro, and for in vivo and ex vivo gene therapy procedures.

[0159] Packaging cells can be used to form viral particles capable of infecting host cells. Such cells can include 293 cells (e.g., for packaging lentivirus or adenovirus) and Psi2 cells or PA317 cells (e.g., for packaging retrovirus). Viral vectors can be produced by producing cell lines that package nucleic acid vectors into viral particles. The vector may contain the minimum viral sequence required for packaging and subsequent integration into the host. The vector may contain other viral sequences, which are replaced by the expression cassette of the polynucleotide to be expressed. The missing viral function can be provided in trans by the packaging cell line. For example, the AAV vector may include ITR sequences from the AAV genome, which are necessary for packaging and integration into the host genome. Viral DNA can be packaged in a cell line, which may contain helper plasmids encoding other AAV genes, i.e., rep and cap, and lack ITR sequences. The cell line can also be infected with adenovirus as a helper cell. Helper viruses can promote the replication of AAV vectors and the expression of AAV genes from helper plasmids. Adenoviral contamination can be reduced by, for example, heat treatment to which adenovirus is more sensitive than AAV. Other methods for delivering nucleic acids to cells may be used, for example, as described in US20030087817, which is incorporated herein by reference.

[0160] Host cells can be transiently or non-transiently transfected with one or more vectors described herein. Cells can be transfected because they occur naturally in a subject. Cells can be taken from or derived from a subject and transfected. Cells can be derived from cells taken from a subject, such as a cell line. 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 present 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. .

[0161] Any suitable vector compatible with the host cell can 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™).

[0162] Contacting the cells with the composition can occur in any culture medium and under any culture conditions that promote cell survival. For example, the cells can be suspended in any convenient appropriate 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, particularly 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which the cells respond. Growth factors as defined herein are molecules that are capable of promoting cell survival, growth and / or differentiation in culture or intact tissues by specific action on transmembrane receptors. Growth factors may include polypeptide and non-polypeptide factors.

[0163] In many embodiments, the selected delivery system targets a specific tissue or cell type. In some cases, the tissue or cell targeting of the delivery system is achieved by combining the delivery system with a tissue or cell specific marker (e.g., a cell surface protein). Viral and non-viral delivery systems can be customized to target a tissue or cell type of interest.

[0164] Pharmaceutical compositions containing molecules (e.g., polypeptides and / or nucleic acids or proteins encoding polypeptides) or immune cells described herein can be administered for preventive and / or therapeutic treatments. In therapeutic applications, the compositions can be administered to subjects already suffering from a disease or condition in an amount sufficient to cure or at least partially prevent the symptoms of the disease or condition, or to cure, heal, improve or ameliorate the condition. The amount effective for this use can vary depending on the severity and course of the disease or condition, previous treatment, the subject's health, weight and response to the drug, and the judgment of the treating physician.

[0165] The multiple therapeutic agents may be administered in any order or simultaneously. If simultaneous, the multiple therapeutic agents may be provided in a single, unified form or in multiple forms, for example, as multiple individual pills or cell solutions. The molecules or cell solutions may be packaged together or separately in a single package or in multiple packages. One or all of the therapeutic agents may be given in multiple doses. If not simultaneous, the time between multiple doses may vary to about 1 to 24 months.

[0166] The molecules or cells described herein can be administered before, during or after the onset of a disease or condition, and the time of administering a composition containing a compound can vary. For example, a pharmaceutical composition can be used as a prophylactic and can be continuously administered to a subject with a condition or disease tendency in order to prevent the occurrence of a disease or condition. 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 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. Symptom onset. Initial administration can be by any practical route, such as by any route described herein using any formulation described herein. Molecules can be administered as soon as feasible after the onset of a disease or condition is detected or suspected, and the length of time required to treat the disease, for example, from about 1 month to about 3 months. The treatment time for each subject may be different.

[0167] The molecule can be packaged into a biological compartment. The biological compartment containing the molecule can be administered to a subject. The biological compartment can include, but is not limited to, viruses (lentivirus, adenovirus), nanospheres, liposomes, quantum dots, nanoparticles, microparticles, nanocapsules, vesicles, polyethylene glycol particles, hydrogels, and micelles.

[0168] For example, the biological compartment may comprise a liposome. A liposome may be a self-assembled structure comprising one or more lipid bilayers, each of which may comprise two monolayers containing oppositely oriented amphiphilic lipid molecules. The amphiphilic lipid may comprise a polar (hydrophilic) head group covalently linked to one or two or more non-polar (hydrophobic) acyl or alkyl chains. The energetically unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous medium induces the amphiphilic lipid molecules to align themselves so that the polar head group may be oriented toward the bilayer surface and the acyl chains are oriented toward the interior of the bilayer, effectively shielding the acyl chains from contact with the aqueous environment.

[0169] Phosphoglycerides and sphingolipids may be included in the liposomes, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phoasphatidylglycerol, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine, preferably an amphiphilic compound (DPPC example), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine and egg sphingomyelin, or any combination thereof.

[0170] The biological compartment may contain nanoparticles. The diameter of the nanoparticles may be from about 40 nanometers to about 1.5 micrometers, from about 50 nanometers to about 1.2 micrometers, from about 60 nanometers to about 1 micrometer, from about 70 nanometers to about 800 nanometers, from about 80 nanometers to about 600 nanometers, from about 90 nanometers to about 400 nanometers, from about 100 nanometers to about 200 nanometers.

[0171] In some cases, the release rate can be slowed or prolonged as the size of the nanoparticles increases, and the release rate can be increased as the size of the nanoparticles decreases.

[0172] The amount of albumin in the nanoparticles can 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 contain up to 30, 40, 50, 60, 70 or 80% or more nanoparticles. In some cases, the nucleic acid molecules of the present disclosure can be bound to the surface of the nanoparticles.

[0173] The biological compartment may contain a virus. A virus may be a delivery system for the 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). Viruses may be used to deliver the pharmaceutical composition of the present disclosure to cells. Viruses may infect and transduce cells in vivo, ex vivo, or in vitro. In ex vivo and in vitro delivery, the transduced cells may be administered to a subject in need of treatment.

[0174] The pharmaceutical composition can be packaged into a viral delivery system. For example, the composition can be packaged into viral particles by the HSV-1 helper-free virus packaging system.

[0175] Viral delivery systems (e.g., viruses comprising pharmaceutical compositions of the present disclosure) can be administered by direct injection, stereotaxic injection, intraventricularly, by micropump infusion systems, by convection, catheters, intravenous, parenteral, intraperitoneal and / or subcutaneous injection. Injected into cells, tissues or organs of a subject in need. In some cases, cells can be transduced in vitro or ex vivo with a viral delivery system. Transduced cells can be given to a subject with 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.

[0176] In some cases, the dose of cells administered to a subject may be less than 1×10 4 cells / kg, or about 1×10 4 cells / kg, about 2×10 4 cells / kg, about 3×10 4 Cells / kg, about 4×10 4 cells / kg, about 5×10 4 cells / kg, about 6×10 4 cells / kg, about 7×10 4 cells / kg, about 8×10 4 cells / kg, about 9×10 4 cells, about 1×10 5 cells / kg, about 2×10 5 cells / kg, about 3×10 5 Cells / kg, about 4×10 5 cells / kg, about 5×10 5 cells / kg, about 6×10 5 cells / kg, about 7×10 5 cells / kg, about 8×10 5 cells / kg, about 9×10 5 cells, about 1×10 6 cells / kg, about 2×10 6 cells / kg, about 3×10 6 Cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells, about 1×10 7 cells / kg, about 5×10 7 cells / kg, about 1×10 8Cells / kg or more. The cell dose can be calculated based on the number of cells successfully transfected and effectively modified, or it can be calculated based on the total number of cells.

[0177] The biological compartments can be introduced into cells through viral or bacteriophage 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, etc.

[0178] In some embodiments, immune cells expressing the subject's system are administered. Immune cells expressing the subject's system can be administered before, during, or after the disease or condition occurs, and the time of administering 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 subjects with a condition or disease tendency in order to prevent the occurrence of the disease or condition. Immune cells can be administered to the subject during the onset of symptoms or as quickly as possible. Administration can be started within the first 48 hours of the onset of symptoms, within the first 24 hours of the onset of symptoms, within the first 6 hours of the onset of symptoms, or within 3 hours of the onset of symptoms. Symptoms. Initial administration can be by any suitable route, such as by any route described herein using any formulation described herein. After the onset of a disease or condition is detected or suspected, immune cells can be administered as soon as feasible, and it takes a period of time, such as about 1 month to about 3 months, to treat the disease. Months. The treatment time for each subject may be different.

[0179] The molecules described herein (e.g., polypeptides and / or nucleic acids) can be present in the composition in a range of about 1 mg to about 2000 mg; about 5 mg to about 1000 mg, about 10 mg to about 25 mg to 500 mg, about 50 mg to about 250 mg, about 100 mg to about 200 mg, about 1 mg to about 50 mg, about 1 mg to about 50 mg. 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg. mg, about 350 mg to about 400 mg, about 400 mg to about 450 mg, about 450 mg to about 500 mg, about 500 mg to about 550 mg, about 550 mg to about 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700 mg, about 700 mg to about 750 mg, about 750 mg to about 800 mg, about 800 mg to about 850 mg, about 850 mg to about 900 mg. , about 900 mg to about 950 mg, or about 950 mg to about 1000 mg.

[0180] The molecules (e.g., polypeptides and / or nucleic acids) described herein can be present in the composition in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg , about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg.

[0181] 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 can be modulation of gene expression. In some embodiments, the total number of active units of the molecule delivered to a 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 a 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. DETAILED DESCRIPTION

[0182] In order to more fully understand and apply the present invention, the present invention will be described in detail below with reference to examples, which are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The scope of the present invention is specifically defined by the appended claims.

[0183] Example 1. Preparation of super TIL cells from peripheral blood

[0184] In Examples 1 to 7, we used CD19 as a booster antigen and CAR targeting CD19 as aBA-CAR.

[0185] Preparation of human super TIL cells: After collecting monocytes from patients with advanced tumors, PD1+T cells are obtained using magnetic beads, and PD1+T cells are transfected with lentivirus loaded with two genes, CD19CAR and enhanced receptor-PD1-CD28 fusion gene.

[0186] Example 2: Boosting adjuvant 1: Autologous B cells boost super TIL expansion

[0187] B cells were isolated from the patient's apheresis cells and cultured to 10 7 quantity.

[0188] The super TIL cells were divided into two groups, AB, with 10 6 Number, natural growth in group A was used as control; group B:

[0189] 10 6 Autologous B cells with 10 6 The number of super TIL cells was co-cultured for 10 days as one round, and 10 were added to the system in each round. 6 After three rounds of continuous culture of autologous B cells, it can be observed that the super TILs in each round expanded by more than 5 times compared with the previous round. However, the number of super TILs in group A did not increase significantly.

[0190] Example 3: Boosting adjuvant 2: Autologous CD19+ T cells boost super TIL expansion

[0191] T cells were isolated from the patient's single-collected cells, and CD19+T cells were obtained as booster cells by transfecting the T cells with lentivirus loaded with CD19 gene. The cells were cultured with CD3 magnetic beads for 10 7 Quantity level.

[0192] The super TIL cells were divided into two groups, AB, with 10 6 Quantity, natural growth of group A as control; group B:

[0193] 10 6 Autologous CD19+ T cells are the same as 10 6 The number of super TIL cells was co-cultured for 10 days as one round, and 10 were added to the system in each round. 6 After three rounds of continuous culture of autologous CD19+T cells, it can be observed that the number of super TILs in each round increased by more than 5 times compared with the previous round. However, there was no significant increase in the number of super TILs in group A.

[0194] Example 4: Boosting adjuvant 3: Patient's autologous NK cells boost super TIL expansion

[0195] NK cells were isolated from the patient's single-collected cells, and lentivirus loaded with CD19 gene was used to transfect NK cells to obtain autologous CD19+NK cells as booster cells. The cells were cultured with CD3 magnetic beads for 10 7 Quantity level.

[0196] The super TIL cells were divided into two groups, AB, with 10 6 Quantity, natural growth of group A as control; group B:

[0197] 10 6 Autologous CD19+ NK cells are the same as 10 6 The number of super TIL cells was co-cultured for 10 days as one round, and 10 were added to the system in each round. 6After three rounds of continuous culture of autologous CD19+NK cells, it can be observed that the number of super TILs in each round increased by more than 5 times compared with the previous round. However, the number of super TILs in group A did not increase significantly.

[0198] Example 5. Boosting adjuvant 4: Allogeneic B cells boost super TIL expansion

[0199] The B cells of another individual volunteer were cultured to 10 7 quantity.

[0200] The super TIL cells were divided into two groups, AB, with 10 6 Quantity, natural growth of group A as control; group B:

[0201] 10 6 Allogeneic B cells 6 The number of super TIL cells was co-cultured for 10 days as one round, and 10 were added to the system in each round. 6 After three rounds of continuous culture of allogeneic B cells, it can be observed that the super TILs in each round expanded by more than 5 times compared with the previous round. However, the number of super TILs in group A did not increase significantly.

[0202] Example 6. Boosting adjuvant 5: Allogeneic NK cells boost super TIL expansion

[0203] NK cells were isolated from a single collection of cells from another individual volunteer (allogeneic), and the allogeneic NK cells were transfected with lentivirus loaded with CD19 gene to obtain allogeneic CD19+NK cells as booster cells. The cells were cultured for 10 7 Quantity level.

[0204] The patient's super TIL cells were divided into two groups, AB, with 10 6 Quantity, natural growth of group A as control; group B:

[0205] 10 6 Allogeneic CD19+ NK cells are the same as 10 6 The number of super TIL cells was co-cultured for 10 days as one round, and 10 were added to the system in each round. 6 After three rounds of continuous culture of allogeneic CD19+NK cells, it can be observed that the number of super TILs in each round increased by more than 5 times compared with the previous round. However, the number of super TILs in group A did not increase significantly.

[0206] Example 7: Boosting adjuvant 6: Nanoparticles carrying boosting antigens boost super TIL expansion

[0207] Attach CD19 protein to nanoparticles to construct nanoparticles carrying booster antigens.

[0208] The super TIL cells were divided into two groups, AB, with 10 6 Quantity, natural growth of group A as control; group B:

[0209] The total amount of CD19 protein was about 1 μg. 6 The number of super TIL cells was co-cultured for 7 days as a round, and the total amount of CD19 protein in the system was added to about 1 microgram of nanoparticles in each round. After 3 rounds of continuous culture, it can be observed that the super TILs in each round expanded by more than 5 times compared with the previous round. However, the number of super TILs in group A did not increase significantly.

[0210] Example 8: Secondary autologous T cell boosting system to boost super TIL expansion

[0211] Use the patient's own T cells to construct two types of booster cells and build a secondary booster system:

[0212] First-level booster cells: Transfect T cells with lentivirus loaded with EGFRviii gene to obtain EGFRviii + T cells were used as booster cells and cultured with CD3 magnetic beads until 10 7 Quantity level.

[0213] Second-level booster cells: T cells are transfected with lentiviruses carrying both the CD19 gene and the CAR gene targeting EGFRviii to obtain CD19 + EGFRviii-CAR-T cells were cultured with CD3 magnetic beads until 10 7 Quantity level.

[0214] The super TIL cells were divided into two groups, ABC, with 10 6 Quantity, natural growth of group A was used as control;

[0215] Group B: 10 6 The number of second-order booster cells (CD19 + EGFRviii-CAR-T cells) were co-cultured with super TILs as a control for single-stage booster cells. 6 Secondary booster cells are the same as 10 6 The number of super TIL cells was co-cultured for 10 days as one round, and 10 were added to the system in each round. 6 The secondary booster cells were cultured for 3 consecutive rounds;

[0216] Group C: The first-stage booster cells (EGFRviii + T cells) and second-level booster cells (CD19 + After 10 days of co-culture with EGFRviii-CAR-T cells, 10 6 number of super TIL cells, and then add 10 6 The number of first-stage booster cells was 10, and the super TILs were co-cultured in this environment for 10 days. 6Number of first-stage booster cells. Three rounds of co-culture.

[0217] It can be observed that each round of super TIL in group B expanded more than 5 times compared with the previous round, while each round of expansion in group C was more than that in group B. However, there was no significant increase in the number of super TIL in group A.

[0218] Example 9: A two-stage booster system of allogeneic NK cells and autologous T cells to boost super TIL expansion

[0219] Use allogeneic NK cells and the patient's own T cells to construct two types of booster cells and build a secondary booster system:

[0220] First-level booster cells: transfect allogeneic NK cells with lentivirus loaded with EGFRviii gene to obtain EGFRviii + NK cells were used as booster cells and cultured with CD3 magnetic beads until 10 7 Quantity level.

[0221] Second-level booster cells: The lentivirus carrying both the CD19 gene and the CAR gene targeting EGFRviii is transfected into autologous T cells to obtain CD19 + EGFRviii-CAR-T cells were cultured with CD3 magnetic beads until 10 7 Quantity level.

[0222] The super TIL cells were divided into two groups, ABC, with 10 6 Quantity, natural growth of group A was used as control;

[0223] Group B: 10 6 The number of second-order booster cells (CD19 + EGFRviii-CAR-T cells) were co-cultured with super TILs as a control for single-stage booster cells. 6 Secondary booster cells are the same as 10 6 The number of super TIL cells was co-cultured for 10 days as one round, and 10 were added to the system in each round. 6 The secondary booster cells were cultured for 3 consecutive rounds;

[0224] Group C: The first-stage booster cells (EGFRviii + NK cells) and second-level booster cells (CD19 + After 10 days of co-culture with EGFRviii-CAR-T cells, 10 6 number of super TIL cells, and then add 10 6 The number of first-stage booster cells was 10, and the super TILs were co-cultured in this environment for 10 days. 6 Number of first-stage booster cells. Three rounds of co-culture.

[0225] It can be observed that each round of super TIL in group B expanded more than 5 times compared with the previous round, while each round of expansion in group C was more than that in group B. However, there was no significant increase in the number of super TIL in group A.

Claims

1. A cascade amplification boosting system comprising two adjuvants, wherein the two adjuvants are two booster cells consisting of a first-stage booster cell and a second-stage booster cell, wherein the first booster cells express a first booster antigen (BA1), The second booster cells simultaneously express a second booster antigen (BA2) and a chimeric antigen receptor (aBA1-CAR) targeting the first booster antigen, the second booster antigen can be recognized by a therapeutic immune cell carrying a chimeric antigen receptor (aBA2-CAR) targeting the second booster antigen, and the first booster antigen is different from the second booster antigen, wherein the first-stage booster cells and the second-stage booster cells are cells of the same type or cells of different types, or a mixture of multiple types of cells, The second-level booster cells can be activated and expanded by the first booster antigen with the help of aBA1-CAR in vivo. The second booster antigen can activate the therapeutic immune cells in vivo and boost the number of the immune cells in the body.

2. The cascade amplification boosting system according to claim 1, characterized in that: The first or second booster antigen is a marker or membrane protein on the surface of peripheral blood cells, tumor cells or viral pathogens. The peripheral blood cells include B cells, T cells, NK cells, granulocytes, and red blood cells. The viral pathogens include HPV virus, Epstein-Barr virus, Helicobacter pylori (HP), hepatitis B virus (HBV), and human immunodeficiency virus (HIV).

3. The cascade amplification boosting system according to claim 1, characterized in that: The first or second booster antigen is a mutant form of a natural human membrane protein.

4. The cascade amplification boosting system according to claim 1, characterized in that: The first or second booster antigen is a human intracellular protein.

5. The cascade amplification boosting system according to claim 1, characterized in that: The first-stage booster cells or the second-stage booster cells are derived from the patient's own or allogeneic peripheral blood mononuclear cells (PBMC) or any one type of cells among B cells, T cells, and NK cells, or a mixture of multiple types of cells.

6. The cascade amplification boosting system according to claim 1, characterized in that: The first-stage booster cells or the second-stage booster cells are immortalized B cells or NK cells.

7. The cascade amplification boosting system according to claim 6, characterized in that: The first-stage booster cells or the second-stage booster cells are NK92 cells.

8. The cascade amplification boosting system according to claim 1, characterized in that: The first-stage booster cells or the second-stage booster cells are K562 cells.

9. The cascade amplification boosting system according to claim 1, characterized in that: The first or second booster antigen is expressed across the membrane on the booster cell, and the booster antigen is a fusion protein consisting of an extracellular binding domain and a transmembrane region, wherein the extracellular binding domain is a structure recognizable by the second-level booster cells or the therapeutic immune cells, and the transmembrane region is the transmembrane domain of a protein naturally expressed by the booster cells.

10. The cascade amplification boosting system according to claim 9, characterized in that: The booster cell is a T cell, the extracellular binding domain is the extracellular domain of EGFRviii or CD19, and the transmembrane region is the transmembrane domain of CD3.

11. The cascade amplification boosting system of claim 1, wherein the chimeric antigen receptor (aBA1-CAR) targeting the first booster antigen comprises a variable region of an antibody that recognizes the first booster antigen, or the chimeric antigen receptor (aBA-2CAR) targeting the second booster antigen comprises a variable region of an antibody that recognizes the second booster antigen.

12. The cascade amplification boosting system according to claim 11, characterized in that: The aBA1-CAR comprises CD3ζ and the variable region of an antibody targeting the first booster antigen, and a co-stimulatory molecule; or the aBA2-CAR comprises CD3ζ and the variable region of an antibody targeting the second booster antigen, and a co-stimulatory molecule.

13. The cascade amplification boosting system according to claim 12, characterized in that: The co-stimulatory molecules include any one or more of CD28, 41BBz, and ICOS.

14. The cascade amplification boosting system according to claim 11, characterized in that: The aBA1-CAR or aBA2-CAR includes TET-ON or any other CAR or CAR-T cell expression regulation and the switch elements that cause CAR-T or CAR-NK cells to commit suicide or apoptosis.

15. The cascade amplification boosting system according to claim 1, characterized in that: It further comprises a modified therapeutic immune cell, wherein the modified therapeutic immune cell has dual recognition characteristics, wherein the dual recognition refers to recognition of target cells, wherein the immune cell can recognize and kill target cells, wherein the target cells are cells harmful to the human body; and recognition of the second booster antigen, wherein the modified therapeutic immune cell transmembrane expresses a chimeric antigen receptor (aBA2-CAR) targeting the second booster antigen, thereby being able to recognize the second booster antigen and be activated to expand its number.

16. The cascade amplification boosting system according to claim 15, characterized in that: The target cells are tumor cells, virus-infected cells or pathogens.

17. The cascade amplification boosting system according to claim 15, characterized in that: The therapeutic immune cells are any one of T cells or NK cells, or a mixture thereof.

18. The cascade amplification boosting system according to claim 15, characterized in that: The therapeutic immune cells are T cells.

19. The cascade amplification boosting system according to claim 18, characterized in that: The T cells are tumor-recognizing T cells or tumor antigen-responsive T cells.

20. The cascade amplification boosting system according to claim 19, characterized in that: The recognition of tumors or tumor antigens by the T cells is achieved by using natural, unmodified TCR to recognize tumor neoantigens, or tumor-associated antigens (TAA), or cancer-testis antigens, or viral antigens.

21. The cascade amplification boosting system according to claim 20, characterized in that: The viral antigens are viral antigens on the surface of cells infected by HPV or EBV and transformed into cancer.

22. The cascade amplification boosting system according to claim 19, characterized in that: The tumor-recognizing T cells or tumor antigen-responsive T cells are obtained by any of the following methods: obtained by vaccine induction, obtained from tumor-infiltrating lymphocytes (TIL), or obtained from peripheral blood by positive or negative screening of markers.

23. The cascade amplification boosting system according to claim 22, characterized in that: The markers are PD1, TIM3, CD137, CD39 and / or CD28.

24. The cascade amplification boosting system according to claim 19, characterized in that: The T cells are derived from tumor-infiltrating T lymphocytes (TIL), or TIL after positive or negative screening of one or more markers such as PD1, TIM3, CD137, CD39, and CD28.

25. The cascade amplification boosting system according to claim 19, characterized in that: The T cells are derived from peripheral blood mononuclear cells (PBMC) and are positive or negative for one or more markers including PD1, TIM3, CD137, and CD39.

26. The cascade amplification boosting system according to claim 15, characterized in that: The immune cells are T cells that have been modified with other genes in addition to the aBA2-CAR modification.

27. The cascade amplification boosting system according to claim 19, characterized in that: The T cell is a CAR-T or TCR-T cell modified with a CAR or exogenous TCR gene other than the aBA2-CAR modification, and the CAR or exogenous TCR gene modification gives the T cell the ability to recognize target cells independently of its natural TCR.

28. The cascade amplification boosting system according to claim 19, characterized in that: The T cells are genetically modified with an enhanced receptor, which is a transmembrane protein composed of an extracellular domain (ECD) and an intracellular domain (ICD) of the T cells. The ECD is a complete sequence structure of any one of the receptors, ligands, antibodies or other protein structures that can bind to the membrane protein of the target cell of the immune cell, or a portion containing its binding domain, and the ICD is the intracellular domain of a co-stimulatory signal protein.

29. The cascade amplification boosting system according to claim 28, characterized in that: The co-stimulatory signal protein is any one of CD28, 41BB or ICOS.

30. The cascade amplification boosting system according to claim 28, characterized in that: When the ECD of the enhancing receptor binds to its ligand, it will generate an inhibitory signal to the T cell, but when the complete enhancing receptor binds to its ligand, it will generate an activation signal to the T cell.

31. The cascade amplification boosting system according to claim 15, characterized in that: The immune cells are NK cells that have been genetically modified in addition to the aBA2-CAR modification.

32. The cascade amplification boosting system according to claim 31, characterized in that: The NK cells are CAR-NK cells that have been modified with a CAR gene other than the aBA2-CAR modification, and this CAR gene modification other than aBA2-CAR gives the NK cells the ability to specifically recognize and kill target cells.

33. The cascade amplification boosting system according to claim 32, characterized in that: The CAR gene modification other than aBA2-CAR includes a partial structure of NKG2D.

34. An immune cell therapy drug, characterized in that: The drug comprises the cascade amplification booster system as described in any one of claims 15-33.

35. Use of the cascade amplification booster system according to any one of claims 15 to 33 in the preparation of a drug for immune cell therapy.