Fusion protein and application thereof

Abstract

The invention provides a fusion protein and application thereof, and particularly relates to an antigen binding protein or an antibody binding protein. Immune cells expressing the antigen-binding protein or antibody-binding protein may exhibit enhanced cytotoxicity. The invention also provides application of the antigen binding protein or the antibody binding protein.

Description

A fusion protein and its application Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a fusion protein and an application thereof. Background Art

[0002] Chimeric antigen receptor (CAR) guides CAR-expressing immune cells to clear tumors by specifically recognizing antigens expressed on the surface of tumor cells. For example, when antigen-positive cells targeted by CAR are present, cells expressing the CAR can recognize and kill these antigen-positive cells. However, existing CARs in the art still have the problem of low activity and weak killing ability of cells expressing the CAR. Therefore, the art needs a CAR with a further optimized structure to achieve the effect of improving the killing ability of cells expressing the CAR.

[0003] Summary of the Invention

[0004] The present invention provides an antigen-binding protein with an improved structure, which may be in the form of a chimeric antigen receptor (CAR). The antigen-binding protein may have one or more effects selected from the following groups: (1) excellent expression efficiency; (2) cells expressing the antigen-binding protein have significant anti-tumor activity; (3) cells expressing the antigen-binding protein have sustained anti-tumor activity; (4) cells expressing the antigen-binding protein have significant cell activation ability; and (5) cells expressing the antigen-binding protein have significant cytokine secretion ability.

[0005] On the one hand, the present invention provides an antigen-binding protein, which comprises an intracellular domain, wherein the intracellular domain comprises a mutated intracellular domain of DNAM-1, wherein the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

[0006] On the other hand, the present invention provides a use of an intracellular domain in preparing an antigen-binding protein, wherein the intracellular domain comprises a mutated intracellular domain of DNAM-1, wherein the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

[0007] On the other hand, the present invention provides an antibody-binding protein, comprising an extracellular antibody-binding domain and an intracellular domain, wherein the intracellular domain comprises a mutated DNAM-1 intracellular domain, wherein the mutated DNAM-1 intracellular domain lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

[0008] On the other hand, the present invention provides a use of an intracellular domain in preparing an antibody-binding protein, wherein the intracellular domain comprises a mutated intracellular domain of DNAM-1, wherein the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

[0009] In another aspect, the present invention provides a polypeptide comprising the antigen-binding protein of the present invention and / or the antibody-binding protein of the present invention.

[0010] In another aspect, the present invention provides a nucleic acid encoding an antigen binding protein of the present invention, an antibody binding protein of the present invention and / or a polypeptide of the present invention.

[0011] In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention.

[0012] In another aspect, the present invention provides a cell comprising the antigen-binding protein of the present invention, the antibody-binding protein of the present invention, the polypeptide of the present invention, the nucleic acid of the present invention and / or the vector of the present invention.

[0013] On the other hand, the present invention provides a method for preparing the antigen-binding protein of the present invention, the antibody-binding protein of the present invention and / or the polypeptide of the present invention, which method comprises culturing the cells of the present invention under conditions such that the antigen-binding protein, the antibody-binding protein of the present invention and / or the polypeptide are expressed.

[0014] In another aspect, the present invention provides a composition comprising an antigen-binding protein of the present invention, an antibody-binding protein of the present invention, a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, and / or a cell of the present invention, and optionally a pharmaceutically acceptable carrier.

[0015] In another aspect, the present invention provides a kit comprising an antigen-binding protein of the present invention, an antibody-binding protein of the present invention, a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, and / or a composition of the present invention.

[0016] In another aspect, the present invention provides a method for stimulating an immune response comprising administering an antigen binding protein of the present invention, an antibody binding protein of the present invention, a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, a composition of the present invention and / or a kit of the present invention.

[0017] On the other hand, the present invention provides the use of the antigen-binding protein of the present invention, the antibody-binding protein of the present invention, the polypeptide of the present invention, the nucleic acid of the present invention, the vector of the present invention, the cell of the present invention, the composition of the present invention and / or the kit of the present invention in the preparation of a medicament for preventing, alleviating and / or treating tumors.

[0018] On the other hand, the present invention provides a method for preventing, alleviating and / or treating tumors, comprising administering the antigen-binding protein of the present invention, the antibody-binding protein of the present invention, the polypeptide of the present invention, the nucleic acid of the present invention, the vector of the present invention, the cell of the present invention, the composition of the present invention and / or the kit of the present invention.

[0019] On the other hand, the present invention provides the antigen-binding protein of the present invention, the antibody-binding protein of the present invention, the polypeptide of the present invention, the nucleic acid of the present invention, the vector of the present invention, the cell of the present invention, the composition of the present invention and / or the kit of the present invention, which are used to prevent, alleviate and / or treat tumors.

[0020] Those skilled in the art will readily appreciate other aspects and advantages of the present invention from the detailed description below. The detailed description below only shows and describes exemplary embodiments of the present invention. As will be appreciated by those skilled in the art, the disclosure of the present invention enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the invention to which the present invention relates. Accordingly, the descriptions in the drawings and specification of the present invention are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The features and advantages of the present invention can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. The accompanying drawings are briefly described as follows:

[0022] FIG1 shows the expression results of the antibody-dependent cytotoxicity protein structure comprising the intracellular domain of DNAM-1 according to the present invention.

[0023] FIG2 shows the expression results of the CAR structure comprising the DNAM-1 intracellular domain of the present invention in NK92 cells.

[0024] FIG3 shows the cell killing results of NK92 cells expressing CAR comprising the DNAM-1 intracellular domain (GTCAR 38, GTCAR 44) of the present invention.

[0025] FIG4 shows the expression results of the CAR structure comprising the DNAM-1 intracellular domain of the present invention in PBMC-derived primary NK cells (PB-NK).

[0026] FIG5 shows the cell killing results of PB-NK cells expressing CAR comprising the DNAM-1 intracellular domain of the present invention (GTCAR 38, GTCAR 44).

[0027] FIG6 shows the expression results of the CAR structure comprising the CRACC intracellular domain of the present invention in the NK92 cell line.

[0028] FIG7 shows the cell killing results of NK92 cells expressing the CAR (GTCAR37) comprising the CRACC intracellular domain of the present invention and a control CAR (2B4 CAR in which GTCAR is replaced with the 2B4 intracellular domain).

[0029] FIG8 shows the expression results of the CAR structure comprising the CRACC intracellular domain of the present invention in PBMC-derived primary NK cells (PB-NK).

[0030] FIG9 shows the cell killing results of PB-NK cells expressing the CAR (GTCAR37) comprising the CRACC intracellular domain of the present invention and a control CAR (2B4 CAR in which GTCAR is replaced with the 2B4 intracellular domain). DETAILED DESCRIPTION

[0031] The following describes the embodiments of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0032] Definition of terms

[0033] In the present invention, the term "antigen binding protein" generally refers to a protein or a functionally active fragment thereof that is capable of binding to one or more antigens. For example, exemplary antigen binding proteins include chimeric antigen receptors and other various molecules known in the art that include a domain capable of recognizing an antigen.

[0034] In the present invention, the term "basic amino acid" generally refers to an amino acid or salt thereof having a basic side chain at neutral pH. The pKa of a basic amino acid is high enough that it tends to bind a proton, thereby acquiring a positive charge in the process. For example, the basic amino acid includes a side chain containing a nitrogen that either binds to a proton (and is protonated) or releases the binding to the proton (and is deprotonated). For example, the basic amino acid can be in the form of NH2 (deprotonated) and NH3 + (protonated) form or between NH (deprotonated) and NH2 + (protonated) form or between N (deprotonated) and NH+ (protonated) forms are balanced. For basic amino acids at physiological pH, such as about pH 7.0, the protonated form is dominant. In exemplary aspects, the basic amino acid is arginine (Arg; R) or lysine (Lys, K) or histidine (His, H). Although the basic amino acid can be a D-isomer or an L-isomer, in exemplary embodiments, the basic amino acid can be an L-isomer of an amino acid, such as L-Arg, L-Lys, or L-His. For example, the basic amino acid is arginine. For example, the basic amino acid is histidine. For example, the basic amino acid is lysine. For example, the basic amino acid is a derivative of arginine, such as L-2-amino-3-guanidinopropionic acid, 4-guanidinobutyric acid. For example, the basic amino acid is a derivative of lysine, such as 5-hydroxylysine, ornithine, N-acetyl-L-lysine, 2,4-diaminobutyric acid. For example, the basic amino acid is a derivative of histidine, such as desaminohistidine, hydroxy-histidine, acetyl-histidine, homo-histidine, N-methylhistidine, α-methylhistidine, imidazoleacetic acid, or α,α-dimethylimidazoleacetic acid (DMIA).

[0035] In the present invention, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one transmembrane domain. CAR can be a core component of a chimeric antigen receptor cell, which may include an antigen (e.g., a tumor-specific antigen and / or tumor-associated antigen) binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0036] In the present invention, the term "signal peptide" generally refers to a propeptide present as an N-terminal peptide on a protein precursor. The function of the signal peptide is to promote the translocation of the expressed polypeptide connected to the endoplasmic reticulum and to express the target protein on the cell membrane. The signal peptide can generally be removed during this process. For example, the antigen-binding protein of the present invention may not have a signal peptide and its biological activity may not be affected. The signal peptide can be heterologous or homologous to the organism used to produce the polypeptide.

[0037] In the present invention, the term "antigen binding domain" generally refers to a domain that (specifically) binds to a given target epitope or a given target site on a target molecule (antigen), or interacts with the given target epitope or a given target site, or recognizes the given target epitope or a given target site.

[0038] In the present invention, the term "specific binding" generally refers to a measurable and / or reproducible interaction, such as binding between a target and an antibody, which can be determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which can be an epitope) is one that binds to the target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0039] In the present invention, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. The term "antibody" may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function.

[0040] In the present invention, the term "single-chain antibody (scFv)" may refer to an antibody composed of the heavy chain variable region and the light chain variable region of the antibody or an antibody connected by a linker.

[0041] In the present invention, the term "CD19" generally refers to lymphocyte antigen CD19. CD19 can be found under UniProt number P15391. The term includes variants, homologs, and functionally active fragments thereof. In the present invention, the term "Claudin 18.2" generally refers to "CLDN18.2," "CLD18.2," "Claudin18.2," "claudin18.2," or "claudin 18.2," including type 2 Claudin 18. Claudin 18.2 can be found under UniProt number P56856. The term includes variants, homologs, and functionally active fragments thereof. In the present invention, the term "GPC3" generally refers to Glypican-3. GPC3 can be found under UniProt number P51654. The term includes variants, homologs, and functionally active fragments thereof. In the present invention, the term "Mesothelin" generally refers to MSLN. Mesothelin can be found under UniProt number Q13421. This term includes variants, homologs, and functionally active fragments thereof. In the present invention, the term "ROR1" generally refers to the inactive tyrosine-protein kinase transmembrane receptor ROR1. ROR1 can be found under UniProt number Q01973. This term includes variants, homologs, and functionally active fragments thereof.

[0042] In the present invention, the term "hinge domain" generally refers to the connecting region between the antigen binding domain and the transmembrane region. Typically, the hinge domain has a certain degree of flexibility, and the CAR molecule can optionally include or not include a hinge domain.

[0043] In the present invention, the term "CD8a" generally refers to the T-cell surface glycoprotein CD8 alpha chain. CD8a can be found under UniProt number P01732. The term includes variants, homologs, and functionally active fragments thereof (e.g., the extracellular domain, intracellular domain, or transmembrane domain described in UniProt number P01732). In the present invention, the term "CD28" generally refers to the T-cell-specific surface glycoprotein CD28. CD28 can be found under UniProt number P10747. The term includes variants, homologs, and functionally active fragments thereof (e.g., the extracellular domain, intracellular domain, or transmembrane domain described in UniProt number P10747). In the present invention, the term "CD34" generally refers to the hematopoietic progenitor cell antigen CD34. CD34 can be found under UniProt number P28906. The term includes variants, homologues, and functionally active fragments thereof (eg, the extracellular domain, intracellular domain or transmembrane domain described by UniProt No. P28906).

[0044] In the present invention, the term "transmembrane domain" generally refers to the domain in CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays a role in transmitting signals.

[0045] In the present invention, the term "IL-7RA" generally refers to Interleukin-7 receptor subunit alpha. IL-7 RA can be found under UniProt accession number P16871. The term includes variants, homologs, and functionally active fragments thereof.

[0046] In the present invention, the term "mutation" generally refers to a change in sequence relative to the wild type. For example, at least one existing amino acid residue is replaced, inserted, deleted or repeated by one or more amino acid residues. The amino acid residue can be a "naturally occurring amino acid residue" and is selected from the following group: alanine (three-letter code: ala, single-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0047] In the present invention, the term "mutated IL-7 receptor transmembrane sequence" generally refers to a mutation, insertion, deletion, or duplication of one or more amino acid residues relative to the native IL-7 receptor transmembrane sequence. For example, a mutated IL-7 receptor transmembrane sequence may promote dimer formation.

[0048] In the present invention, the term "Nkp80" generally refers to Killer Cell Lectin Like Receptor F1, KLRF1. Nkp80 can be found under UniProt number Q9NZS2. The term includes variants, homologs, and functionally active fragments thereof (e.g., the extracellular domain, transmembrane domain, or intracellular domain described in UniProt number Q9NZS2).

[0049] In the present invention, the term "NTB-A" generally refers to SLAM Family Member 6, SLAMF6. NTB-A can be found under UniProt number Q96DU3. The term includes variants, homologs, and functionally active fragments thereof (e.g., the extracellular domain, transmembrane domain, intracellular domain, or ITSM domain described in UniProt number Q96DU3).

[0050] In the present invention, the term "intracellular domain" generally refers to the intracellular portion of a molecule. The intracellular domain generates signals that promote the immune effector function of cells containing CAR. For example, these include cell lytic activity, adjuvant activity, and secretion of cytokines. For example, the intracellular domain may contain only a truncated portion that retains the function of intracellular signaling.

[0051] In the present invention, the term "costimulatory signaling domain" generally refers to an intracellular domain that can provide immune co-stimulatory molecules. In the present invention, the term "costimulation" generally refers to the source of lymphocyte activation signals, which are usually generated by the interaction of co-stimulatory molecules and their receptors on the surface of immune cells involved in adaptive immunity (T cell / B cell or antigen presenting cell / T cell).

[0052] In the present invention, the term "DNAM-1" generally refers to DNAX Accessory Molecule-1. DNAM-1 can be found under UniProt number Q15762. The term includes variants, homologs, and functionally active fragments thereof (e.g., the extracellular domain, intracellular domain, or transmembrane domain described under UniProt number Q15762).

[0053] In the present invention, the term "CRACC" generally refers to CD2-Like Receptor Activating Cytotoxic Cell. CRACC can be found under the UniProt number Q9NQ25. The term includes variants, homologs, and functionally active fragments thereof (e.g., the extracellular domain, intracellular domain, or transmembrane domain described in UniProt number Q9NQ25).

[0054] In the present invention, the term "2B4" generally refers to CD244 or Natural killer cell receptor 2B4. 2B4 can be found under the UniProt number Q9BZW8. The term includes variants, homologs, and functionally active fragments thereof (e.g., the extracellular domain, intracellular domain, or transmembrane domain recorded by UniProt number Q9BZW8). In the present invention, the term "DAP10" generally refers to Hematopoietic cell signal transducer. DAP10 can be found under the UniProt number Q9UBK5. The term includes variants, homologs, and functionally active fragments thereof (e.g., the extracellular domain, intracellular domain, or transmembrane domain recorded by UniProt number Q9UBK5).

[0055] In the present invention, the term "signaling domain" generally refers to a domain located inside a cell that can conduct signals. In the present invention, the intracellular signaling domain can conduct signals into the cell.

[0056] In the present invention, the term "CD3ζ" generally refers to CD247 or T-cell surface glycoprotein CD3 zeta chain. "CD3ζ" can be found under UniProt number P20963. The term includes variants, homologs, and functionally active fragments thereof. In the present invention, the term "DAP12" generally refers to Transmembrane Immune Signaling Adaptor. DAP12 can be found under UniProt number O43914. The term includes variants, homologs, and functionally active fragments thereof. DAP12 also has an ITAM functional domain.

[0057] In the present invention, the term "immune cell" generally refers to a cell that participates in an immune response, such as a cell that promotes an immune effector response. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. The term also includes engineered immune cells, such as immune cells that have been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0058] In the present invention, the term "killing ability" refers to killing of cells by contacting the cells with an effective amount of an antibody, immunoconjugate, bispecific / multispecific molecule or composition. The method can include killing cells that express an antigen, optionally in the presence of effector cells, such as by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms.

[0059] In the present invention, the term "directly linked" or "indirectly linked" refers to direct linkage via a peptide bond, or indirect linkage via a linker or a non-peptide linkage.

[0060] In the present invention, the term "PBMC" or "human peripheral blood mononuclear cell" generally refers to cells in peripheral blood that have a single nucleus. For example, any blood cell with a round nucleus (i.e., lymphocyte, monocyte, or macrophage). These blood cells are key components of the immune system to fight infection and adapt to invaders. The lymphocyte population is composed of CD4 + and CD8 + T cells, B cells and natural killer cells, CD14 + Monocytes and basophils / neutrophils / eosinophils / dendritic cells. Usually, FICOLL TM(a hydrophilic polysaccharide that separates blood), these cells are separated from whole blood, where monocytes and lymphocytes form the buffy coat below the plasma layer. For example, "PBMC" refers to a cell population that contains at least T cells, and optionally NK cells, NKT cells, and antigen-presenting cells.

[0061] In the present invention, the term "proliferation" refers to an increase in cell division (symmetrical or asymmetrical division of cells). "Proliferation" can refer to symmetrical or asymmetrical division of T cells. "Increased proliferation" occurs when there is an increase in the number of cells in a treated sample compared to cells in an untreated sample.

[0062] In the present invention, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, and can be mammals, such as non-human primates, sheep, dogs, cats, cows and horses.

[0063] In the present invention, the term "therapeutically effective amount" refers to an amount of the drug of the present invention sufficient to prevent or alleviate the symptoms associated with a disease or condition (e.g., cancer). The therapeutically effective amount is related to the disease being treated, and those skilled in the art can readily determine the actual effective amount.

[0064] In the present invention, the term "drug" generally refers to a chemical compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient.

[0065] In the present invention, the term "composition" refers to a mixture containing one or more compounds of the present invention or its physiologically / pharmaceutically acceptable salt or prodrug and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient and thus exert biological activity. The therapeutic composition should generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersant, liposome or other surface active substance suitable for high drug concentration. The active compound (i.e., antigen binding portion) can be incorporated in a desired amount together with one of the ingredients or combinations of ingredients listed above in a suitable solvent, and then filtered and sterilized as needed to prepare a sterile injectable solution.

[0066] In the present invention, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid connected to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to a viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additive mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome, such as naked RNA polynucleotides that cannot autonomously replicate, naked DNA polynucleotides, polynucleotides composed of DNA and RNA in the same chain, poly-lysine-coupled DNA or RNA, peptide-coupled DNA or RNA, liposome-coupled DNA, etc. In addition, certain vectors can direct the expression of genes effectively connected to them. This type of vector is referred to as a "recombinant expression vector" (or simply "expression vector") in the present invention. Generally speaking, the expression vector used in recombinant DNA technology is typically in the form of a plasmid. In this specification, "plasmid" and "vector" are used interchangeably, as plasmid is the most commonly used form of vector.

[0067] In the present invention, the term "adjuvant" generally refers to any substance that assists or regulates the effects of drugs, including but not limited to immunological adjuvants, which enhance or diversify the immune response to antigens.

[0068] As used herein, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastomas and retinoblastomas), sarcomas (including liposarcoma and synovial cell sarcomas), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, and melanomas. "Tumor" may further include "solid tumors," which refer to tumors selected from the group consisting of gastrointestinal cancer, pancreatic cancer, glioblastomas, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer.

[0069] In the present invention, the term "antigen positive tumor" refers to a tumor cell expressing an antigen protein on its surface. In order to determine whether a cell expresses an antigen protein on the surface, it is believed that antigen mRNA expression is relevant to the expression of the antigen protein on the cell surface, and the expression of the antigen mRNA can be determined by a method selected from in situ hybridization and RT-PCR (comprising quantitative RT-PCR). Alternatively, for example, an antigen binding protein directed against an antigen protein can be used to measure the expression of the antigen protein on the cell surface in methods such as immunohistochemistry, FACS. For example, an antigen positive tumor can be a mammalian implanted tumor. In the present invention, the term "antigen positive tumor cell" refers to a cell expressing an antigen protein on its surface.

[0070] In the present invention, the terms "about" and "approximately" generally refer to a statistically significant range of values. Such a range can be within an order of magnitude of a given value or range, can be included within 50%, preferably included within 20%, more preferably included within 10%, and most preferably included within 5%. The permissible variations encompassed by the terms "about" or "approximately" may depend on the specific system under study and can be readily understood by those of ordinary skill in the art. The terms "above," "below," "at most," and "at least" include the number. The terms "include," "comprise," and "contain" are open-ended expressions, while "consisting of" is a closed-ended expression, the latter being a special form of the former, which covers the latter.

[0071] Detailed Description of the Invention

[0072] Fusion protein containing a mutant intracellular domain of DNAM-1

[0073] In one aspect, the present invention provides a fusion protein comprising an intracellular domain, wherein the intracellular domain comprises a mutated DNAM-1 intracellular domain, wherein the mutated DNAM-1 intracellular domain lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1. For example, the fusion protein can be an antigen-binding protein. The fusion protein comprising the DNAM-1 intracellular domain has the function of increasing expression and / or increasing the level of cell activation in a cell. For example, the fusion protein comprising the mutated DNAM-1 intracellular domain has the function of increasing expression in a cell. For example, the fusion protein comprising the mutated DNAM-1 intracellular domain has the function of increasing the level of cell activation.

[0074] On the one hand, the present invention provides an antigen-binding protein, which comprises an intracellular domain, wherein the intracellular domain comprises a mutated intracellular domain of DNAM-1, wherein the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

[0075] In one aspect, the present invention provides an antibody-binding protein comprising an extracellular antibody-binding domain and an intracellular domain, wherein the intracellular domain comprises a mutated DNAM-1 intracellular domain, wherein the mutated DNAM-1 intracellular domain lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

[0076] For example, the intracellular domain of the wild-type DNAM-1 comprises the sequence of NRRRRRERRDLFTESWDTQKAPNNYRSPISTGQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 9).

[0077] For example, the intracellular domain of the mutant DNAM-1 has a deletion of basic amino acids at the N-terminus and / or C-terminus, a truncated sequence containing one or more basic amino acids, and / or a substitution of basic amino acids with non-basic amino acids, relative to the intracellular domain of the wild-type DNAM-1. For example, the intracellular domain of the mutant DNAM-1 has a deletion of 1 to 5, for example, 1, 2, 3, 4, or 5 basic amino acids at the N-terminus and / or C-terminus, relative to the intracellular domain of the wild-type DNAM-1. For example, the intracellular domain of the mutant DNAM-1 is truncated by 1 to 40 amino acids at the N-terminus and / or C-terminus relative to the intracellular domain of wild-type DNAM-1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. For example, relative to the wild-type DNAM-1 intracellular domain, 1 to 5, for example 1, 2, 3, 4 or 5 basic amino acids at the N-terminus and / or C-terminus of the mutant DNAM-1 intracellular domain are replaced by non-basic amino acids. For example, the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 40, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, of the N-terminus and / or C-terminus of the intracellular domain of the mutant DNAM-1 relative to the wild-type DNAM-1. For example, the intracellular domain of the mutant DNAM-1 is truncated at amino acids 2 to 4 of the N-terminus relative to the intracellular domain of the wild-type DNAM-1. For example, the intracellular domain of the mutant DNAM-1 of the present invention comprises the sequence of any one of SEQ ID NOs: 10-13.

[0078] For example, the mutated intracellular domain of DNAM-1 of the present invention can be more conducive to the expression of the fusion protein relative to the intracellular domain sequence of wild-type DNAM-1. For example, in cells expressing the fusion protein, the proportion of cells expressing the fusion protein containing the mutated intracellular domain of DNAM-1 of the present invention can be increased by 5% to 85% relative to the expression of the intracellular domain sequence containing wild-type DNAM-1, for example, by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 54%, about 55%, about 60%, about 63%, about 65%, about 70%, about 75%, about 80%, or about 85%. For example, in cells expressing the fusion protein, the proportion of cells expressing the fusion protein containing the mutated intracellular domain of DNAM-1 of the present invention can be detected by flow cytometry.

[0079] For example, the antigen-binding protein may further comprise other intracellular domains besides the mutated intracellular domain of DNAM-1, wherein the intracellular domain is directly or indirectly connected to the transmembrane domain.

[0080] For example, the antigen binding protein may comprise one or more of the other intracellular domains. For example, the antigen binding protein may comprise 1 to 5, such as 1, 2, 3, 4 or 5, other intracellular domains. For example, the other intracellular domain may comprise a co-stimulatory signaling domain and / or a signaling domain. For example, the other intracellular domain may be located at the C-terminus of the transmembrane domain. For example, the other intracellular domain may comprise a signaling domain of a protein or a functional fragment thereof selected from the group consisting of CD28, 2B4, and DAP10. For example, the other intracellular domain may comprise the sequence shown in SEQ ID NO: 8.

[0081] For example, the antigen-binding protein comprises one or more of the other intracellular domains. For example, the intracellular domain may comprise a co-stimulatory signaling domain and / or a signaling domain. For example, the antigen-binding protein comprises 1 to 5, such as 1, 2, 3, 4 or 5 other intracellular domains. For example, the other intracellular domains are located at the N-terminus of the reversely arranged Nkp80 intracellular domain. For example, the other intracellular domains of the present invention comprise intracellular domain structures for chimeric antigen receptors known in the art. For example, the other intracellular domains of the present invention may comprise intracellular domains of proteins or functional fragments thereof selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1, CD2, CD7, LIGHT, NKG2C and B7-H3. For example, the other intracellular domains of the present invention may comprise an intracellular domain of a protein or a functional fragment thereof selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

[0082] For example, the antigen-binding protein further comprises a transmembrane domain, which is directly or indirectly connected to the costimulatory signaling domain. For example, the transmembrane domain is located at the N-terminus of the costimulatory signaling domain. For example, the transmembrane domain of the present invention comprises a transmembrane domain structure for a chimeric antigen receptor known in the art. For example, the transmembrane domain of the present invention comprises a transmembrane domain of a protein or a functional fragment thereof selected from the group consisting of CD4, CD8, CD28, and IL receptor family proteins. For example, the transmembrane domain of the present invention comprises a transmembrane domain of a protein or a functional fragment thereof selected from the group consisting of α, β or ζ chain of a cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C. For example, the transmembrane domain can recruit a protein with a costimulatory effect; the costimulatory protein comprises DAP10 protein. For example, the transmembrane domain comprises a transmembrane domain of a protein or functional fragment thereof selected from the group consisting of NKG2D and CD8a. For example, the transmembrane domain comprises the sequence shown in SEQ ID NO: 6 or 7. For example, the transmembrane domain of the present invention may comprise the transmembrane domain of any protein known in the art.

[0083] For example, the antigen-binding protein further comprises a hinge domain, which is directly or indirectly connected to the costimulatory signaling domain. For example, the hinge domain is located between the transmembrane domain and the antigen-binding domain. For example, the hinge domain of the present invention comprises a hinge domain structure for a chimeric antigen receptor known in the art. For example, the hinge domain of the present invention comprises a hinge domain of a protein or a functional fragment thereof selected from the group consisting of: CD8, CD28, and CD4. For example, the hinge domain of the present invention comprises a hinge domain of a protein or a functional fragment thereof selected from the group consisting of: CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154. For example, the hinge domain comprises the hinge domain of CD8a or a functional fragment thereof. For example, the hinge domain comprises the sequence shown in SEQ ID NO: 5. For example, the hinge domain of the present invention may comprise the hinge domain of any protein known in the art.

[0084] For example, the antigen-binding protein may further comprise a signal peptide, for example, the signal peptide comprises the sequence shown in SEQ ID NO: 3.

[0085] For example, the antigen-binding protein may comprise an antibody or antigen-binding fragment thereof that can recognize an antigen. For example, the antigen may comprise a protein or functional fragment thereof selected from the following group: CD19, BCMA, Claudin 18.2, and GPC3. For example, the antigen of the present invention may be a tumor antigen known in the art. For example, the antigen may comprise a protein or functional fragment thereof selected from the following group: Mesothelin, ROR1, CD19, BCMA, Claudin 18.2, and GPC3. For example, the antigen-binding fragment may comprise a single-chain antibody (scFv). For example, the antigen-binding fragment may comprise the sequence set forth in SEQ ID NO: 4.

[0086] For example, the antigen binding proteins include chimeric antigen receptors.For example, the chimeric antigen receptor (CAR) can include an antigen binding domain, an optional hinge domain, a transmembrane domain, a costimulatory signal domain and / or a signal transduction domain from N-terminal to C-terminal.

[0087] For example, the chimeric antigen receptor (CAR) may comprise, from N-terminus to C-terminus, an antigen binding domain, an optional hinge domain, a transmembrane domain, a mutated intracellular domain of DNAM-1 as a co-stimulatory signaling domain and / or an intracellular domain of CD3ζ as a signal transduction domain.

[0088] For example, the chimeric antigen receptor (CAR) may comprise, from N-terminus to C-terminus, an antigen binding domain, a hinge domain of CD8a, a transmembrane domain of CD8a, an intracellular domain of a mutated DNAM-1 as a co-stimulatory signaling domain, and / or an intracellular domain of CD3ζ as a signal transduction domain.

[0089] For example, the chimeric antigen receptor (CAR) may comprise, from N-terminus to C-terminus, an antigen binding domain, a hinge domain of CD8a, a mutated IL-7 receptor transmembrane sequence as a transmembrane domain, a mutated DNAM-1 intracellular domain as a co-stimulatory signaling domain and / or an intracellular domain of CD3ζ as a signal transduction domain.

[0090] For example, the chimeric antigen receptor (CAR) may comprise, from N-terminus to C-terminus, an antigen binding domain, a hinge domain of CD8a, a transmembrane domain of CD8a, an intracellular domain of a mutated DNAM-1 of any one of SEQ ID NOs: 10-13 as a co-stimulatory signal domain and / or an intracellular domain of CD3ζ as a signal transduction domain.

[0091] For example, the chimeric antigen receptor (CAR) may comprise, from N-terminus to C-terminus, an antigen binding domain, a hinge domain of CD8a, a mutated IL-7 receptor transmembrane sequence shown in SEQ ID NO: 7 as a transmembrane domain, an intracellular domain of a mutated DNAM-1 of any one of SEQ ID NOs: 10-13 as a co-stimulatory signal domain and / or an intracellular domain of CD3ζ as a signal transduction domain.

[0092] For example, the antigen binding protein comprises the sequence shown in SEQ ID NO: 1 or 2.

[0093] For example, the extracellular antibody binding domain comprises an extracellular domain capable of binding to an antibody Fc. For example, the antibody Fc segment can be a fragment comprising a heavy chain constant region (CH). For example, the extracellular antibody binding domain comprises the extracellular domain of CD16, CD32 and / or CD64. For example, the extracellular antibody binding domain comprises the extracellular domain of CD16, its active fragment, and / or its mutant. For example, the extracellular antibody binding domain comprises the extracellular domain of CD32, its active fragment, and / or its mutant. For example, the extracellular antibody binding domain comprises the extracellular domain of CD64, its active fragment, and / or its mutant.

[0094] For example, the extracellular antibody binding domain comprises a mutated extracellular domain of CD16. For example, the extracellular antibody binding domain comprises an extracellular domain of CD16 having a 158V and / or S197P mutation relative to wild-type CD16. For example, the extracellular antibody binding domain comprises the sequence set forth in SEQ ID NO: 15.

[0095] For example, the antibody binding protein comprises the sequence of any one of SEQ ID NOs: 16 to 20. For example, the antibody binding protein can bind to the Fc region of an antibody and trigger an intracellular activation signal in a cell expressing the antibody binding protein through the intracellular domain of the present invention.

[0096] On the one hand, the present invention provides a use of an intracellular domain in preparing an antigen-binding protein, wherein the intracellular domain comprises a mutated intracellular domain of DNAM-1, wherein the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

[0097] On the one hand, the present invention provides a use of an intracellular domain in preparing an antibody-binding protein, wherein the intracellular domain comprises a mutated intracellular domain of DNAM-1, wherein the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

[0098] For example, the intracellular domain of the mutant DNAM-1 has a deletion of basic amino acids at the N-terminus and / or C-terminus, a truncated sequence containing one or more basic amino acids, and / or a substitution of basic amino acids with non-basic amino acids, relative to the intracellular domain of the wild-type DNAM-1. For example, the intracellular domain of the mutant DNAM-1 has a deletion of 1 to 5, for example, 1, 2, 3, 4, or 5 basic amino acids at the N-terminus and / or C-terminus, relative to the intracellular domain of the wild-type DNAM-1. For example, the intracellular domain of the mutant DNAM-1 is truncated by 1 to 40 amino acids at the N-terminus and / or C-terminus relative to the intracellular domain of wild-type DNAM-1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. For example, relative to the wild-type DNAM-1 intracellular domain, 1 to 5, for example 1, 2, 3, 4 or 5 basic amino acids at the N-terminus and / or C-terminus of the mutant DNAM-1 intracellular domain are replaced by non-basic amino acids. For example, the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 40, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, of the N-terminus and / or C-terminus of the intracellular domain of the mutant DNAM-1 relative to the wild-type DNAM-1. For example, the intracellular domain of the mutant DNAM-1 is truncated at amino acids 2 to 4 of the N-terminus relative to the intracellular domain of the wild-type DNAM-1. For example, the intracellular domain of the mutant DNAM-1 of the present invention comprises the sequence of any one of SEQ ID NOs: 10-13.

[0099] Fusion protein containing the intracellular domain of mutant CRACC

[0100] On the other hand, the present invention also provides the following technical solutions:

[0101] An antigen binding protein, wherein the antigen binding protein comprises a costimulatory signaling domain, and the costimulatory signaling domain comprises the intracellular domain of CRACC.

[0102] The antigen binding protein as described in technical solution 1, wherein the intracellular domain of CRACC comprises the sequence described in SEQ ID NO: 24.

[0103] The antigen-binding protein as described in any one of technical solutions 1-2, wherein the costimulatory signal domain of the antigen-binding protein does not contain the intracellular domain of 2B4.

[0104] An antigen-binding protein as described in any one of technical solutions 1-3, wherein the antigen-binding protein comprises a signaling domain, and the signaling domain is located at the C-terminus of the co-stimulatory signal domain.

[0105] The antigen-binding protein as described in technical solution 4, wherein the signaling domain comprises the signaling domain of a protein containing an ITAM functional domain.

[0106] An antigen binding protein as described in any one of technical solutions 4-5, wherein the signaling domain comprises the signaling domain of CD3ζ or a functional fragment thereof and / or the signaling domain of DAP12 or a functional fragment thereof.

[0107] The antigen binding protein as described in any one of technical solutions 4 to 6, wherein the signaling domain comprises the sequence shown in SEQ ID NO: 25.

[0108] An antigen-binding protein as described in any one of technical solutions 1-7, wherein the antigen-binding protein comprises a transmembrane domain, and the transmembrane domain is located at the N-terminus of the co-stimulatory signal domain.

[0109] The antigen-binding protein as described in technical solution 8, wherein the transmembrane domain comprises the transmembrane domain of NKG2D or a functional fragment thereof and / or the transmembrane domain of CD8a or a functional fragment thereof.

[0110] The antigen-binding protein as described in any one of technical solutions 8-9, wherein the transmembrane domain comprises the sequence shown in SEQ ID NO: 23.

[0111] An antigen-binding protein as described in any one of technical solutions 1-10, wherein the antigen-binding protein comprises a hinge domain, and the hinge domain is located at the N-terminus of the transmembrane domain.

[0112] The antigen-binding protein as described in technical solution 11, wherein the hinge domain comprises a hinge domain of a protein or a functional fragment thereof selected from the following group: CD8a, CD28, and CD34.

[0113] The antigen-binding protein as described in any one of technical solutions 11-12, wherein the hinge domain comprises the sequence shown in SEQ ID NO: 5.

[0114] An antigen-binding protein as described in any one of technical solutions 1-13, wherein the antigen-binding protein further comprises an antigen-binding domain, and the antigen-binding domain is located at the N-terminus of the hinge domain.

[0115] The antigen-binding protein as described in technical solution 14, wherein the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof that can recognize the antigen.

[0116] An antigen-binding protein as described in any one of technical solutions 14-15, wherein the antigen comprises a protein or a functional fragment thereof selected from the following group: Mesothelin, ROR1, CD19, BCMA, Claudin 18.2 and GPC3, preferably CD19, BCMA, Claudin 18.2 and GPC3.

[0117] An antigen-binding protein as described in any of technical solutions 14-16, wherein the antigen-binding domain comprises a single-chain antibody.

[0118] The antigen binding protein as described in any one of technical solutions 14 to 17, wherein the antigen binding domain comprises the sequence shown in SEQ ID NO: 4.

[0119] The antigen binding protein as described in any one of technical solutions 1-18, wherein the antigen binding protein is a chimeric antigen receptor.

[0120] The antigen binding protein as described in any one of technical solutions 1 to 19, wherein the antigen binding protein comprises the sequence shown in SEQ ID NO: 21.

[0121] A use of a costimulatory signal domain in preparing an antigen binding protein, wherein the costimulatory signal domain comprises the intracellular domain of CRACC.

[0122] The use as described in technical solution 21, wherein the intracellular domain of CRACC comprises the sequence described in SEQ ID NO: 24.

[0123] The use as described in any of technical solutions 21-22, wherein the costimulatory signal domain of the antigen binding protein does not contain the intracellular domain of 2B4.

[0124] The use as described in any one of technical solutions 21-23, wherein the antigen binding protein is a chimeric antigen receptor.

[0125] A polypeptide, wherein the polypeptide comprises the antigen binding protein according to any one of technical solutions 1-20.

[0126] A nucleic acid, wherein the nucleic acid encodes the antigen-binding protein according to any one of technical solutions 1 to 20 and / or the polypeptide according to technical solution 25.

[0127] A vector, wherein the vector comprises the nucleic acid described in technical solution 26.

[0128] A cell comprising the antigen-binding protein according to any one of technical solutions 1 to 20, the polypeptide according to technical solution 25, the nucleic acid according to technical solution 26 and / or the vector according to technical solution 27.

[0129] A cell as described in technical solution 28, wherein the cell comprises an immune cell.

[0130] A cell as described in any of technical solutions 28-29, wherein the cell comprises NK cells, NKT cells and / or T cells.

[0131] A method for preparing the antigen-binding protein of any one of technical solutions 1-20 and / or the polypeptide of technical solution 25, wherein the method comprises culturing the cells of any one of technical solutions 28-30 under conditions such that the antigen-binding protein and / or the polypeptide are expressed.

[0132] A composition, wherein the composition comprises the antigen-binding protein of any one of Technical Solutions 1-20, the polypeptide of Technical Solution 25, the nucleic acid of Technical Solution 26, the vector of Technical Solution 27, and / or the cell of any one of Technical Solutions 28-30, and optionally a pharmaceutically acceptable carrier.

[0133] A kit, wherein the kit comprises the antigen-binding protein according to any one of technical solutions 1-20, the polypeptide according to technical solution 25, the nucleic acid according to technical solution 26, the vector according to technical solution 27, the cell according to any one of technical solutions 28-30, and / or the composition according to technical solution 32.

[0134] A method for stimulating an immune response, comprising administering the antigen-binding protein of any one of technical solutions 1-20, the polypeptide of technical solution 25, the nucleic acid of technical solution 26, the vector of technical solution 27, the cell of any one of technical solutions 28-30, the composition of technical solution 32 and / or the kit of technical solution 33.

[0135] Use of the antigen-binding protein according to any one of technical solutions 1 to 20, the polypeptide according to technical solution 25, the nucleic acid according to technical solution 26, the vector according to technical solution 27, the cell according to any one of technical solutions 28 to 30, the composition according to technical solution 32 and / or the kit according to technical solution 33 in the preparation of a medicament, wherein the medicament is used to prevent, alleviate and / or treat a disease (preferably a tumor).

[0136] A drug for preventing and / or treating a disease (preferably a tumor), comprising the antigen-binding protein according to any one of technical solutions 1 to 20, the polypeptide according to technical solution 25, the nucleic acid according to technical solution 26, the vector according to technical solution 27, the cell according to any one of technical solutions 28 to 30, the composition according to technical solution 32, and / or the kit according to technical solution 33.

[0137] A method for preventing and / or treating a disease (preferably a tumor), comprising administering to a subject in need thereof the antigen-binding protein of any one of Technical Solutions 1-20, the polypeptide of Technical Solution 25, the nucleic acid of Technical Solution 26, the vector of Technical Solution 27, the cell of any one of Technical Solutions 28-30, the composition of Technical Solution 32, and / or the kit of Technical Solution 33.

[0138] The antigen-binding protein according to any one of technical solutions 1 to 20, the polypeptide according to technical solution 25, the nucleic acid according to technical solution 26, the vector according to technical solution 27, the cell according to any one of technical solutions 28 to 30, the composition according to technical solution 32 and / or the kit according to technical solution 33 are used for preventing and / or treating diseases (preferably tumors).

[0139] The use according to technical solution 35, the drug according to technical solution 36, the method according to technical solution 37, and / or the antigen-binding protein, polypeptide, nucleic acid, vector, cell, composition and / or kit according to the use according to technical solution 38, wherein the disease comprises solid tumors and / or hematological tumors.

[0140] The use according to technical solution 35, the drug according to technical solution 36, the method according to technical solution 37, and / or the antigen-binding protein, polypeptide, nucleic acid, vector, cell, composition and / or kit according to the use according to technical solution 38, wherein the disease is selected from the following group: gastric cancer, liver cancer and leukemia.

[0141] In one aspect, the present invention provides a fusion protein. For example, the fusion protein of the present invention can be an antigen-binding protein, wherein the fusion protein comprises a costimulatory signaling domain, wherein the costimulatory signaling domain comprises the intracellular domain of CRACC. For example, the present application provides a fusion protein having the function of increasing expression and / or increasing the level of cell activation in a cell. For example, the fusion protein comprises the intracellular domain of CRACC. For example, the fusion protein comprises the ITSM (immunoreceptor tyrosine-based switch motif) of CRACC. For example, the fusion protein comprises the sequence set forth in SEQ ID NO: 24. For example, the fusion protein can comprise the intracellular domain of CRACC as the costimulatory signaling domain. For example, the fusion protein can comprise a costimulatory signaling domain other than the intracellular domain of CRACC. For example, the fusion protein can comprise a costimulatory signaling domain other than the intracellular domain of CRACC, but not the intracellular domain of 2B4 as the costimulatory signaling domain. For example, the fusion protein of the present invention can comprise only the intracellular domain of CRACC as the costimulatory signaling domain.

[0142] For example, the fusion protein of the present invention can be an antigen binding protein. For example, the antigen binding protein can further comprise an antigen binding domain, and the antigen binding domain and the costimulatory signaling domain can be directly or indirectly connected. For example, the antigen binding domain and the costimulatory signaling domain can be connected via one or more hinge domains and / or transmembrane domains.

[0143] For example, the antigen-binding protein may comprise an antibody or antigen-binding fragment thereof that can recognize an antigen. For example, the antigen may comprise a protein or functional fragment thereof selected from the following group: CD19, BCMA, Claudin 18.2, and GPC3. For example, the antigen of the present invention may be a tumor antigen known in the art. For example, the antigen may comprise a protein or functional fragment thereof selected from the following group: Mesothelin, ROR1, CD19, BCMA, Claudin 18.2, and GPC3. For example, the antigen-binding fragment may comprise a single-chain antibody (scFv). For example, the antigen-binding fragment may comprise the sequence set forth in SEQ ID NO: 4.

[0144] For example, the antigen binding protein may comprise one or more co-stimulatory signaling domains. For example, the antigen binding protein may comprise 1 to 5, such as 1, 2, 3, 4 or 5, co-stimulatory signaling domains. For example, the co-stimulatory signaling domain may be located at the N-terminus and / or C-terminus of the intracellular domain of CRACC. For example, the co-stimulatory signaling domain may comprise an intracellular domain of a protein or a functional fragment thereof selected from the group consisting of CD28 and DAP10.

[0145] For example, the signaling domain may comprise a signaling domain of a protein containing an ITAM domain. For example, the signaling domain may comprise a signaling domain of CD3ζ or a functional fragment thereof, or a signaling domain of DAP12 or a functional fragment thereof. For example, the signaling domain may comprise the sequence shown in SEQ ID NO: 25.

[0146] For example, the antigen-binding protein comprises one or more of the other intracellular domains. For example, the intracellular domain may comprise a co-stimulatory signaling domain and / or a signaling domain. For example, the antigen-binding protein comprises 1 to 5, such as 1, 2, 3, 4 or 5 other intracellular domains. For example, the other intracellular domains are located at the N-terminus of the reversely arranged Nkp80 intracellular domain. For example, the other intracellular domains of the present invention comprise intracellular domain structures for chimeric antigen receptors known in the art. For example, the other intracellular domains of the present invention may comprise intracellular domains of proteins or their functional fragments selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1, CD2, CD7, LIGHT, NKG2C and B7-H3. For example, the other intracellular domains of the present invention may comprise an intracellular domain of a protein or a functional fragment thereof selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

[0147] For example, the antigen binding protein further comprises a transmembrane domain, which is directly or indirectly connected to the costimulatory signal domain. For example, the transmembrane domain is located at the N-terminus of the costimulatory signal domain. For example, the transmembrane domain of the present invention comprises a transmembrane domain structure for a chimeric antigen receptor known in the art. For example, the transmembrane domain of the present invention comprises a transmembrane domain of a protein or a functional fragment thereof selected from the group consisting of: CD4, CD8, CD28, and IL receptor family proteins. For example, the transmembrane domain of the present invention comprises a transmembrane domain of a protein or a functional fragment thereof selected from the group consisting of: α, β or ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA

[0015] The transmembrane domain comprises a protein selected from the group consisting of: -6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C. For example, the transmembrane domain can recruit a protein with a costimulatory effect; the costimulatory protein comprises DAP10 protein. For example, the transmembrane domain comprises a transmembrane domain of a protein or functional fragment thereof selected from the group consisting of: NKG2D and CD8a. For example, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 23. For example, the transmembrane domain of the present invention may comprise the transmembrane domain of any protein known in the art.

[0148] For example, the antigen-binding protein further comprises a hinge domain, which is directly or indirectly connected to the costimulatory signaling domain. For example, the hinge domain is located between the transmembrane domain and the antigen-binding domain. For example, the hinge domain of the present invention comprises a hinge domain structure for a chimeric antigen receptor known in the art. For example, the hinge domain of the present invention comprises a hinge domain of a protein or a functional fragment thereof selected from the group consisting of: CD8, CD28, and CD4. For example, the hinge domain of the present invention comprises a hinge domain of a protein or a functional fragment thereof selected from the group consisting of: CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154. For example, the hinge domain comprises the hinge domain of CD8a or a functional fragment thereof. For example, the hinge domain comprises the sequence shown in SEQ ID NO: 5. For example, the hinge domain of the present invention may comprise the hinge domain of any protein known in the art.

[0149] For example, the antigen-binding protein may further comprise a signal peptide, for example, the signal peptide comprises the sequence shown in SEQ ID NO: 3.

[0150] For example, the antigen binding protein is a chimeric antigen receptor. For example, the chimeric antigen receptor (CAR) can include an antigen binding domain, an optional hinge domain, a transmembrane domain, a costimulatory signaling domain, and / or a signal transduction domain from the N-terminus to the C-terminus.

[0151] For example, the chimeric antigen receptor (CAR) may comprise, from N-terminus to C-terminus, an antigen binding domain, an optional hinge domain, a transmembrane domain, the intracellular domain of CRACC as a co-stimulatory signaling domain and / or the intracellular domain of CD3ζ as a signal transduction domain.

[0152] For example, the chimeric antigen receptor (CAR) may comprise, from N-terminus to C-terminus, an antigen binding domain, a hinge domain of CD8a, a transmembrane domain of NKG2D, an intracellular domain of CRACC as a costimulatory signaling domain, and / or an intracellular domain of CD3ζ as a signal transduction domain.

[0153] For example, the antigen binding protein comprises the sequence shown in SEQ ID NO: 21.

[0154] In one aspect, the present invention provides a use of the intracellular domain of CRACC in preparing a fusion protein. For example, the fusion protein is an antigen-binding protein. In another aspect, the present invention provides a use of the intracellular domain of CRACC in preparing an antigen-binding protein. For example, compared to CARs composed of other intracellular domain sequences, the antigen-binding protein of the present invention has a more significant effect of enhancing cell activation. For example, the intracellular domain of CRACC comprises the sequence described in SEQ ID NO: 24. For example, the intracellular domain of CRACC serves as the costimulatory signaling domain in the fusion protein. For example, the costimulatory signaling domain of the fusion protein does not comprise the intracellular domain of 2B4. For example, the fusion protein comprises a chimeric antigen receptor. For example, compared to CARs composed of other intracellular domain sequence combinations, the antigen-binding protein constructed in the present invention using the combination of the intracellular domain of CRACC and the intracellular domain of CD3ζ, without the intracellular domain of 2B4, has higher expression efficiency.

[0155] For example, in the preparation of a fusion protein, the intracellular portion of the fusion protein comprises, from N-terminus to C-terminus, the transmembrane domain of NKG2D, the intracellular domain of CRACC as a co-stimulatory signaling domain and / or the intracellular domain of CD3ζ as a signal transduction domain.

[0156] For example, the use in preparing a fusion protein, the fusion protein comprises the following sequence, which, from N-terminus to C-terminus, comprises the hinge domain of CD8a, the transmembrane domain of NKG2D, the intracellular domain of CRACC as a co-stimulatory signal domain and / or the intracellular domain of CD3ζ as a signal transduction domain.

[0157] For example, the use in preparing a fusion protein comprises the following sequence, which comprises, from N-terminus to C-terminus, an antigen binding domain, a hinge domain of CD8a as shown in SEQ ID NO: 5, a transmembrane domain of NKG2D as shown in SEQ ID NO: 23, an intracellular domain of CRACC as shown in SEQ ID NO: 24 as a costimulatory signal domain and / or an intracellular domain of CD3ζ as shown in SEQ ID NO: 25 as a signal transduction domain.

[0158] For example, the chimeric antigen receptor of the present invention can be expressed on the surface of an immune cell. For example, the immune cell of the present invention can include an immune effector cell. For example, the immune cell of the present invention can include a T cell, a NKT cell, a NK cell, or a mixture of the above cells. For example, the immune cell of the present invention can include one or more chimeric antigen receptors of the present invention.

[0159] For example, the sequence mentioned in the present invention comprises a sequence that is at least about 95% homologous to the sequence. For example, the sequence mentioned in the present invention comprises a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homologous to the sequence.

[0160] In one aspect, the present invention provides a polypeptide that can comprise an antigen-binding protein of the present invention. For example, the polypeptide of the present invention can also comprise the full length or fragments of one or more cytokines.

[0161] In one aspect, the present invention provides a nucleic acid encoding an antigen binding protein of the present invention and / or a polypeptide of the present invention.

[0162] In one aspect, the invention provides a vector comprising a nucleic acid of the invention.

[0163] In one aspect, the present invention provides a cell comprising the antigen binding protein of the present invention, the polypeptide of the present invention, the nucleic acid of the present invention and / or the vector of the present invention.

[0164] For example, the chimeric antigen receptors of the present invention and the antibody-binding proteins of the present invention can be expressed on the surface of immune cells. For example, the immune cells of the present invention can include immune effector cells. For example, the immune cells of the present invention can include T cells, NKT cells, NK cells, and mixtures of the above cells. For example, the immune cells of the present invention can include one or more chimeric antigen receptors of the present invention and the antibody-binding proteins of the present invention.

[0165] For example, the sequence mentioned in the present invention comprises a sequence that is at least about 95% homologous to the sequence. For example, the sequence mentioned in the present invention comprises a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homologous to the sequence.

[0166] In one aspect, the present invention provides a polypeptide, which may comprise an antigen-binding protein or an antibody-binding protein of the present invention. For example, the polypeptide of the present invention may also comprise the full length or fragments of one or more cytokines.

[0167] In one aspect, the present invention provides a nucleic acid that can encode an antigen binding protein of the present invention, an antibody binding protein of the present invention, and / or a polypeptide of the present invention.

[0168] In one aspect, the present invention provides a vector, which may comprise the nucleic acid of the present invention.

[0169] In one aspect, the present invention provides a cell that can comprise an antigen-binding protein of the present invention, an antibody-binding protein of the present invention, a polypeptide of the present invention, a nucleic acid of the present invention, and / or a vector of the present invention. For example, the cell can comprise an immune cell. For example, the cell can comprise a NK cell, an NKT cell, and / or a T cell.

[0170] In one aspect, the present invention provides a method for preparing an antigen-binding protein of the present invention, an antibody-binding protein of the present invention and / or a polypeptide of the present invention, the method comprising culturing the cells of the present invention under conditions such that the antigen-binding protein, the antibody-binding protein of the present invention and / or the polypeptide are expressed.

[0171] In one aspect, the present invention provides a composition comprising an antigen-binding protein of the present invention, an antibody-binding protein of the present invention, a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, and / or a cell of the present invention, and optionally a pharmaceutically acceptable carrier. For example, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are compatible with the immune effector cells and are generally safe and non-toxic.

[0172] In one aspect, the present invention provides a kit, which may comprise an antigen-binding protein of the present invention, an antibody-binding protein of the present invention, a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, and / or a pharmaceutical composition of the present invention.

[0173] In one aspect, the present invention provides a method for stimulating an immune response, which may comprise administering an antigen-binding protein of the present invention, an antibody-binding protein of the present invention, a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, a pharmaceutical composition of the present invention, and / or a kit of the present invention. For example, the method for stimulating an immune response may be in vitro and / or ex vivo. For example, the method for stimulating an immune response may be a method for non-therapeutic purposes. In one aspect, stimulating an immune response may be to increase the ability to kill target cells, to increase the ability to release cytokines, and / or to increase the level of cell activation.

[0174] In one aspect, the present invention provides uses of the antigen-binding proteins, antibody-binding proteins, polypeptides, nucleic acids, vectors, cells, pharmaceutical compositions, and / or kits of the present invention in the preparation of medicaments, which can be used to prevent, alleviate, and / or treat tumors. For example, the tumors can include solid tumors and / or hematologic tumors. For example, the tumors are selected from the group consisting of gastric cancer, liver cancer, and leukemia. For example, the tumors of the present invention can be selected from pan-cancer types, such as tumors that are tumor antigen-positive or highly express tumor antigens. For example, the tumors are selected from the group consisting of ovarian cancer, gastric cancer, liver cancer, and leukemia.

[0175] In one aspect, the present invention provides antigen-binding proteins of the present invention, antibody-binding proteins of the present invention, polypeptides of the present invention, nucleic acids of the present invention, vectors of the present invention, cells of the present invention, pharmaceutical compositions of the present invention, and / or kits of the present invention, which can be used to prevent, alleviate, and / or treat tumors. For example, the tumor can comprise a solid tumor and / or a hematological tumor. For example, the tumor is selected from the group consisting of gastric cancer, liver cancer, and leukemia. For example, the tumor of the present invention can be selected from a pan-cancer group, for example, the tumor can comprise a tumor that is tumor antigen-positive or highly expresses a tumor antigen. For example, the tumor is selected from the group consisting of ovarian cancer, gastric cancer, liver cancer, and leukemia.

[0176] In one aspect, the present invention provides a method for preventing, alleviating, and / or treating tumors, comprising administering an antigen-binding protein of the present invention, an antibody-binding protein of the present invention, a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, a pharmaceutical composition of the present invention, and / or a kit of the present invention. For example, the tumor may comprise a solid tumor and / or a hematological tumor. For example, the tumor is selected from the group consisting of gastric cancer, liver cancer, and leukemia. For example, the tumor of the present invention may be selected from a pan-cancer group, for example, the tumor may comprise a tumor that is tumor antigen-positive or highly expresses a tumor antigen. For example, the tumor is selected from the group consisting of ovarian cancer, gastric cancer, liver cancer, and leukemia.

[0177] Without intending to be bound by any theory, the following examples are merely intended to illustrate the proteins and uses of the present invention and are not intended to limit the scope of the present invention.

[0178] Example

[0179] The sequence information of the present invention is as follows:

[0180] Example 1 Expression detection of DNAM-1 truncated structure

[0181] The present invention selected the natural intracellular region sequence of DNAM-1 (SEQ ID NO: 9) and the intracellular region sequences of various truncated DNAM-1 (SEQ ID NO: 10 to 13) to verify their expression in cells.

[0182] The present invention has designed CD16-based antibody-dependent cytotoxicity protein constructs (SEQ ID NOs: 16 to 20), comprising a CD16 fragment (with the 158V, S197P mutations) and a native or truncated DNAM-1 intracellular domain fragment. These constructs can be used to simulate the expression efficiency of various DNAM-1 fragments in transmembrane fusion proteins. Expression was detected using an anti-CD16 antibody. The flow cytometer used was a Beckman Coulter Cytoflex.

[0183] Figure 1 shows the expression results of fusion protein constructs comprising the intracellular domain of DNAM-1 according to the present invention. The results demonstrate that fusion proteins comprising truncated DNAM-1 intracellular domains according to the present invention exhibit high expression levels. Optionally, selecting a truncated DNAM-1 intracellular domain can result in more efficient expression in NK cells. For example, expression of fusion proteins comprising a truncated DNAM-1 intracellular domain was detected in approximately 76.68%, 87.61%, or 67.09% or more of the total NK cell population.

[0184] Example 2 CAR structure expression detection and killing ability detection

[0185] The cells used in the present invention can be any immune cells, such as NK, NKT and T cells, etc. For example, in the case of NK cells, NK cells can be isolated from peripheral blood mononuclear cells and obtained by in vitro expansion; for example, NK cells can be any type of NK cells known in the art, such as NK92 cells.

[0186] The experiment was conducted in NK92 cells to determine the expression effect and / or activity of the CAR comprising the DNAM-1 intracellular domain of the present invention. NK cells (such as NK92 cells) were transduced with a retrovirus encoding a CAR structure comprising a DNAM-1 intracellular domain of the present invention, such as GTCAR 38 or GTCAR 44, and CAR expression was detected using an anti-FMC63 antibody (Acro Biosystems, FM3-HPY53). The flow cytometer used was Beckman Coulter Cytoflex.

[0187] Figure 2 shows the expression results of CAR structures comprising the intracellular domain of DNAM-1 according to the present invention. The results indicate that the CARs comprising the intracellular domain of DNAM-1 according to the present invention have high expression levels, enabling more efficient CAR expression by NK cells. For example, GTCAR 38 and GTCAR 44 were detected in approximately 94.55% and 75.57% or more of the total number of NK cells, respectively.

[0188] At the same time, an experiment was conducted to determine the cell killing effect of the CAR structure of the present invention in NK92 cells. A 96-well plate was coated with poly-lysine (Sigma, P7890), and after washing with PBS, 1.6e5 / 100μl of NALM-6-mcherry cells were evenly spread in a 96-well plate. After culturing at 37°C for 2 hours, 4e4 / 100μl of NK92 cells expressing GTCAR 38 and GTCAR 44 were taken and added to the corresponding wells. The mcherry fluorescence signal was recorded using Incucyte, with 2 replicates per group. In the multi-round killing experiment, each round of killing lasted about 48 hours, and then a new round of killing began. For each new round of killing experiment, a new 96-well plate was coated with poly-lysine and plated with 1.6e5 / 100ul NALM-6-mcherry cells. After incubation at 37°C for 2 hours, the NK92 cells from the previous round of killing were thoroughly mixed with a pipette and transferred to a new 96-well plate, and the mcherry fluorescence signal was continued to be recorded using Incucyte.

[0189] Figure 3 shows the results of NK92 cells expressing CAR comprising the DNAM-1 intracellular domain of the present invention (GTCAR 38, GTCAR 44) in the fourth round of killing. The results show that cells expressing CAR comprising the DNAM-1 intracellular domain of the present invention have the ability to kill tumors.

[0190] Example 3 CAR structure expression detection and killing ability detection

[0191] NK cells (PB-NK) were isolated from PBMC (peripheral blood mononuclear cell)-derived blood samples using magnetic beads (Miltenyi, 130-050-401).

[0192] PB-NK cells were transduced with retrovirus encoding GTCAR 38 and GTCAR 44. After 7 days of culture, CAR expression was detected using anti-FMC63 antibody (Acro Biosystems, FM3-HPY53). Flow cytometry was performed using a Beckman Coulter Cytoflex instrument.

[0193] Figure 4 shows the expression results of CAR structures comprising the DNAM-1 intracellular domain of the present invention. The results indicate that the CAR comprising the DNAM-1 intracellular domain of the present invention has a high expression level, which can enable NK cells to express the CAR more efficiently. For example, GTCAR 38 and GTCAR 44 were detected in approximately 72.25% and 55.41% or more of the total NK cell population.

[0194] At the same time, an experiment was conducted to determine the cell killing effect of the CAR structure of the present invention in PB-NK cells. A 96-well plate was coated with poly-lysine (Sigma, P7890), and after washing with PBS, 1E5 / 100μl of NALM-6-mcherry cells were evenly spread in a 96-well plate. After incubation at 37°C for 2 hours, 2.5E4 / 100μl of PB-NK cells expressing GTCAR 38 and GTCAR 44 were taken and added to the corresponding wells. After that, the mcherry fluorescence signal was recorded using Incucyte, with 2 replicates per group.

[0195] Figure 5 shows the killing results of PB-NK cells expressing CAR (GTCAR 38, GTCAR 44) comprising the DNAM-1 intracellular domain of the present invention. The results show that cells expressing CAR comprising the DNAM-1 intracellular domain of the present invention have the ability to kill tumor cells.

[0196] Example 4 Detection of CAR structures promoting cell secretion of cytokines

[0197] Experiments can be performed to determine the cytokine secretion assay and cell surface marker staining results of cells expressing CARs of various structures of the present invention.

[0198] To isolate PBMCs from single-blood samples: Take a blood sample and centrifuge it at 2500g for 10 minutes at room temperature. Remove the upper plasma layer, dilute the sample with saline, and mix thoroughly. Place 20ml of lymphocyte separation buffer (TBD science, LTS1077) into a centrifuge tube and gently layer the sample on top of the lymphocyte separation buffer. Centrifuge 25ml per tube at 600g for 30 minutes at room temperature. After centrifugation, discard the upper saline layer and remove the buffy coat to minimize contamination of the lower lymphocyte separation buffer. Dilute with saline and centrifuge at 1000g for 10 minutes at room temperature. After centrifugation, pour the supernatant into a new centrifuge tube and resuspend the cell pellet in saline. Centrifuge again at 1000g for 10 minutes. Discard the supernatant and resuspend the pellet in saline. Combine the pellet with the previously resuspended cell pellet and centrifuge again at 1000g for 10 minutes. Discard the supernatant and resuspend to an appropriate volume in PBS containing 0.5% BSA and 2mM EDTA. CD56+ NK cells (PB-NK cells) were isolated from PBMCs using CD56 microbeads (Miltenyi Biotec, 130-050-401) according to the manufacturer's instructions.

[0199] PB-NK cells were virally transduced to express CARs of various structures of the present invention, and cultured and expanded. Surface marker staining was performed on days 5 and 10 of culture to observe changes in cell activation markers after CAR transduction. Antibodies used, for example, the first group: CD56-BV786, BD 564058; CD16-APC, Biolegend 360706; CD3-BV510, BD 740187; NKG2D-BV421, Biolegend 320822; NKp44-APC-Cy7, Biolegend 325124; DNAM1-PE, Biolegend 338306; the second group: CD56-APC, Biolegend 362504; CD3-APC-Cy7, Biolegend 300426; NKG2D-BV421, Biolegend 320822; NKp30-BV711, Biolegend 325218; CD137-PE, Biolegend 309894; CD28-BV605, Biolegend 302968, all of which can be purchased from commercial channels.

[0200] After 10 days of culture, CAR-expressing PB-NK cells and NALM-6-mcherry cells were mixed at an E:T ratio of 1:8. After 18 hours, the supernatant was collected and the contents of IL-2, IL-4, IL-6, IL-10, TNFα, IFN-γ, and IL-17A in the supernatant were detected using CBA (BD, 560484). The cytokine secretion was then compared with that of the group cultured with PB-NK and NALM-6-mcherry alone.

[0201] The experimental results show that cells expressing CARs of various structures of the present invention can have a higher proportion of cell activation markers and / or a higher cytokine secretion amount.

[0202] Example 5 CAR structure expression detection and killing ability detection

[0203] The cells used in the present invention can be any immune cells, such as NK, NKT and T cells, etc. For example, in the case of NK cells, NK cells can be isolated from peripheral blood mononuclear cells and obtained by in vitro expansion; for example, NK cells can be any type of NK cells known in the art, such as NK92 cells.

[0204] The experiment was conducted in NK92 cells to determine the expression effect and / or activity of the CAR comprising the CRACC intracellular domain of the present invention. NK cells (such as NK92 cells) were transduced with a retrovirus encoding a CAR structure comprising the CRACC intracellular domain of the present invention, such as GTCAR 37, and CAR expression was detected using anti-FMC63 antibody (Acro Biosystems, FM3-HPY53). The flow cytometer used was Beckman Coulter Cytoflex.

[0205] Figure 6 shows the expression results of the CAR structure containing the CRACC intracellular domain of the present invention. The results show that the CAR containing the CRACC intracellular domain of the present invention has a high expression level, which can enable NK cells to express CAR more efficiently. For example, GTCAR 37 was detected in approximately 16.17% or more of the total number of NK cells.

[0206] At the same time, an experiment was conducted to determine the cell killing effect of the CAR structure of the present invention in NK92 cells. A 96-well plate was coated with poly-lysine (Sigma, P7890), and after washing with PBS, 1.6e5 / 100μl of NALM-6-mcherry cells were evenly spread in a 96-well plate. After culturing at 37°C for 2 hours, 4e4 / 100μl of NK92 cells expressing GTCAR 37 were taken and added to the corresponding wells. The mcherry fluorescence signal was recorded using Incucyte, with 2 replicates per group. In the multi-round killing experiment, each round of killing lasted for 48 hours, and then a new round of killing began. For each new round of killing experiment, a new 96-well plate was coated with poly-lysine and plated with 1.6e5 / 100ul NALM-6-mcherry cells. After incubation at 37°C for 2 hours, the NK92 cells from the previous round of killing were thoroughly mixed with a pipette and transferred to a new 96-well plate, and the mcherry fluorescence signal was continued to be recorded using Incucyte.

[0207] Figure 7 shows the cell killing results of CAR (GTCAR37) expressing the present invention comprising the CRACC intracellular domain and the control CAR (GTCAR is replaced with 2B4 CAR of the 2B4 intracellular domain). The results show that the killing tumor results of cells expressing the CAR comprising the CRACC intracellular domain of the present invention, as well as the ability to kill tumor cells after multiple rounds of killing, are stronger than cells expressing the control CAR.

[0208] Example 6 CAR structure expression detection and killing ability detection

[0209] NK cells (PB-NK) were isolated from PBMC (peripheral blood mononuclear cell)-derived blood samples using magnetic beads (Miltenyi, 130-050-401).

[0210] PB-NK cells were transduced with a retrovirus encoding GTCAR 37. After 7 days of culture, CAR expression was detected using an anti-FMC63 antibody (Acro Biosystems, FM3-HPY53). Flow cytometry was performed using a Beckman Coulter Cytoflex instrument.

[0211] Figure 8 shows the expression results of the CAR structure containing the CRACC intracellular domain of the present invention. The results show that the CAR containing the CRACC intracellular domain of the present invention has a high expression level, which can enable NK cells to express CAR more efficiently. For example, GTCAR 37 was detected in approximately 17.74% or more of the total number of NK cells.

[0212] Simultaneously, experiments were conducted to determine the cytotoxicity of the CAR construct of the present invention in PB-NK cells. A 96-well plate was coated with polylysine (Sigma, P7890), washed with PBS, and evenly plated with 1e5 / 100μl of NALM-6-mcherry cells in each 96-well plate. After incubation at 37°C for 2 hours, 2.5e4 / 100μl of PB-NK cells expressing GTCAR 37 were added to the corresponding wells and mcherry fluorescence signals were recorded using an Incucyte. Two replicates were performed for each group. In multiple rounds of cytotoxicity experiments, each round of cytotoxicity lasted for 42 hours before a new round of cytotoxicity was initiated. For each new round of cytotoxicity, a new 96-well plate was coated with polylysine and plated with 1e5 / 100μl of NALM-6-mcherry cells. After incubation at 37°C for 2 hours, the NK92 cells from the previous round of cytotoxicity were thoroughly mixed using a pipette and transferred to a new 96-well plate. The mcherry fluorescence signals were then recorded using an Incucyte.

[0213] Figure 9 shows the cell killing results of CAR (GTCAR37) expressing the present invention comprising a CRACC intracellular domain and a control CAR (GTCAR replaced with 2B4 CAR having a 2B4 intracellular domain). The results show that the ability of cells expressing the CAR comprising the CRACC intracellular domain of the present invention to kill tumor cells after multiple rounds of killing is stronger than that of cells expressing the control CAR, and has a sustained multi-round killing ability.

[0214] Example 7 Detection of CAR structures promoting cell secretion of cytokines

[0215] Experiments can be performed to determine the cytokine secretion assay and cell surface marker staining results of cells expressing CARs of various structures of the present invention.

[0216] To isolate PBMCs from single-blood samples: Take a blood sample and centrifuge it at 2500g for 10 minutes at room temperature. Remove the upper plasma layer, dilute the sample with saline, and mix thoroughly. Place 20ml of lymphocyte separation buffer (TBD science, LTS1077) into a centrifuge tube and gently layer the sample on top of the lymphocyte separation buffer. Centrifuge 25ml per tube at 600g for 30 minutes at room temperature. After centrifugation, discard the upper saline layer and remove the buffy coat to minimize contamination of the lower lymphocyte separation buffer. Dilute with saline and centrifuge at 1000g for 10 minutes at room temperature. After centrifugation, pour the supernatant into a new centrifuge tube and resuspend the cell pellet in saline. Centrifuge again at 1000g for 10 minutes. Discard the supernatant and resuspend the pellet in saline. Combine the pellet with the previously resuspended cell pellet and centrifuge again at 1000g for 10 minutes. Discard the supernatant and resuspend to an appropriate volume in PBS containing 0.5% BSA and 2mM EDTA. CD56+ NK cells (PB-NK cells) were isolated from PBMCs using CD56 microbeads (Miltenyi Biotec, 130-050-401) according to the manufacturer's instructions.

[0217] PB-NK cells were virally transduced to express CARs of various structures of the present invention, and cultured and expanded. Surface marker staining was performed on days 5 and 10 of culture to observe changes in cell activation markers after CAR transduction. Antibodies used, for example, the first group: CD56-BV786, BD 564058; CD16-APC, Biolegend 360706; CD3-BV510, BD 740187; NKG2D-BV421, Biolegend 320822; NKp44-APC-Cy7, Biolegend 325124; DNAM1-PE, Biolegend 338306; the second group: CD56-APC, Biolegend 362504; CD3-APC-Cy7, Biolegend 300426; NKG2D-BV421, Biolegend 320822; NKp30-BV711, Biolegend 325218; CD137-PE, Biolegend 309894; CD28-BV605, Biolegend 302968, all of which can be purchased from commercial channels.

[0218] After 10 days of culture, CAR-expressing PB-NK cells and NALM-6-mcherry cells were mixed at an E:T ratio of 1:8. After 18 hours, the supernatant was collected and the contents of IL-2, IL-4, IL-6, IL-10, TNFα, IFN-γ, and IL-17A in the supernatant were detected using CBA (BD, 560484). The cytokine secretion was then compared with that of the group cultured with PB-NK and NALM-6-mcherry alone.

[0219] The experimental results show that cells expressing CARs of various structures of the present invention can have a higher proportion of cell activation markers and / or a higher cytokine secretion amount.

[0220] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments of the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. An antigen-binding protein, wherein the antigen-binding protein comprises an intracellular domain, wherein the intracellular domain comprises a mutated DNAM-1 intracellular domain, wherein the mutated DNAM-1 intracellular domain lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

2. The antigen binding protein of claim 1, wherein the intracellular domain of the wild-type DNAM-1 comprises the sequence of NRRRRRERRDLFTESWDTQKAPNNYRSPISTGQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 9).

3. The antigen-binding protein according to any one of claims 1 to 2, wherein the intracellular domain of the mutant DNAM-1 lacks 1 to 5 basic amino acids at the C-terminus, truncates a sequence containing 1 to 5 basic amino acids, and / or 1 to 5 basic amino acids are replaced by non-basic amino acids relative to the intracellular domain of the wild-type DNAM-1. 4 . The antigen-binding protein according to claim 1 , wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 4 at the C-terminus relative to the intracellular domain of the wild-type DNAM-1.

5. The antigen-binding protein according to any one of claims 1 to 4, wherein the intracellular domain of the mutant DNAM-1 lacks 1 to 5 basic amino acids at the N-terminus, truncates a sequence containing 1 to 5 basic amino acids, and / or 1 to 5 basic amino acids are replaced by non-basic amino acids relative to the intracellular domain of the wild-type DNAM-1. 6 . The antigen binding protein according to claim 1 , wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 2 to 4 of the N-terminus relative to the intracellular domain of the wild-type DNAM-1.

7. The antigen binding protein according to any one of claims 1 to 6, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 40 at the N-terminus relative to the intracellular domain of the wild-type DNAM-1.

8. The antigen binding protein of any one of claims 1-7, wherein the mutated intracellular domain of DNAM-1 comprises the sequence of any one of SEQ ID NOs: 10-13.

9. The antigen-binding protein according to any one of claims 1 to 8, wherein the antigen-binding protein further comprises other intracellular domains other than the intracellular domain of the mutated DNAM-1, and the other intracellular domains are directly or indirectly connected to the intracellular domain of the mutated DNAM-1.

10. The antigen binding protein of claim 9, wherein the other intracellular domain is located at the N-terminus of the intracellular domain of the mutant DNAM-1.

11. The antigen binding protein according to any one of claims 9 to 10, wherein the other intracellular domain comprises an intracellular domain of a protein or a functional fragment thereof selected from the group consisting of 2B4, and DAP10.

12. The antigen binding protein of any one of claims 9 to 11, wherein the other intracellular domain comprises the sequence shown in SEQ ID NO:

8.

13. The antigen binding protein of any one of claims 1-12, wherein the antigen binding protein further comprises a transmembrane domain, which is directly or indirectly connected to the intracellular domain.

14. The antigen binding protein of claim 13, wherein the transmembrane domain is located at the N-terminus of the other intracellular domain.

15. The antigen binding protein according to any one of claims 13 to 14, wherein the transmembrane domain comprises a transmembrane domain of a protein or a functional fragment thereof selected from the group consisting of an IL-7 receptor, and CD8a.

16. The antigen binding protein of any one of claims 13 to 15, wherein the transmembrane domain comprises a mutated IL-7 receptor transmembrane sequence as shown in SEQ ID NO:

7.

17. The antigen binding protein of any one of claims 13 to 15, wherein the transmembrane domain comprises the CD8a transmembrane sequence shown in SEQ ID NO:

6.

18. The antigen binding protein of any one of claims 1-17, wherein the antigen binding protein further comprises a hinge domain, wherein the hinge domain is directly or indirectly connected to the intracellular domain.

19. The antigen binding protein of claim 18, wherein the hinge domain is located at the N-terminus of the transmembrane domain.

20. The antigen binding protein of any one of claims 18-19, wherein the hinge domain comprises the hinge domain of CD8a or a functional fragment thereof.

21. The antigen binding protein of any one of claims 18-20, wherein the hinge domain comprises the sequence shown in SEQ ID NO:

5.

22. The antigen binding protein of any one of claims 1-21, wherein the antigen binding protein further comprises an antigen binding domain, which is directly connected to the intracellular domain or indirectly connected through a hinge domain.

23. The antigen binding protein of claim 22, wherein the antigen binding domain is located at the N-terminus of the hinge domain.

24. The antigen binding protein of any one of claims 22-23, wherein the antigen binding protein comprises an antibody or antigen binding fragment thereof capable of recognizing the antigen.

25. The antigen binding protein of any one of claims 22-24, wherein the antigen comprises a protein or a functional fragment thereof selected from the following group: Mesothelin, ROR1, CD19, BCMA, Claudin 18.2 and GPC3, preferably CD19, BCMA, Claudin 18.2 and GPC3.

26. The antigen binding protein of any one of claims 22-25, wherein the antigen binding fragment comprises a single chain antibody.

27. The antigen binding protein of any one of claims 22-26, wherein the antigen binding fragment comprises the sequence shown in SEQ ID NO:

4.

28. The antigen binding protein of any one of claims 1-27, wherein the antigen binding protein is a chimeric antigen receptor.

29. The antigen binding protein of any one of claims 1-28, wherein the antigen binding protein comprises the sequence shown in any one of SEQ ID NOs: 1 and 2.

30. A use of an intracellular domain in preparing an antigen-binding protein, wherein the intracellular domain comprises a mutated intracellular domain of DNAM-1, and the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

31. The use according to claim 30, wherein the intracellular domain of the wild-type DNAM-1 comprises the sequence of NRRRRRERRDLFTESWDTQKAPNNYRSPISTGQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 9).

32. The use according to any one of claims 30-31, wherein the intracellular domain of the mutant DNAM-1 lacks 1 to 5 basic amino acids at the C-terminus, truncates a sequence containing 1 to 5 basic amino acids, and / or 1 to 5 basic amino acids are replaced by non-basic amino acids relative to the intracellular domain of the wild-type DNAM-1.

33. The use according to any one of claims 30 to 32, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 4 at the C-terminus relative to the intracellular domain of the wild-type DNAM-1.

34. The use according to any one of claims 30 to 33, wherein the intracellular domain of the mutant DNAM-1 lacks 1 to 5 basic amino acids at the N-terminus, truncates a sequence containing 1 to 5 basic amino acids, and / or 1 to 5 basic amino acids are replaced by non-basic amino acids relative to the intracellular domain of the wild-type DNAM-1.

35. The use according to any one of claims 30 to 34, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 2 to 4 of the N-terminus relative to the intracellular domain of the wild-type DNAM-1.

36. The use according to any one of claims 30 to 35, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 40 at the N-terminus relative to the intracellular domain of the wild-type DNAM-1.

37. The use according to any one of claims 30 to 36, wherein the intracellular domain of the mutated DNAM-1 comprises the sequence of any one of SEQ ID NOs: 10 to 13.

38. The use of any one of claims 30-37, wherein the antigen binding protein is a chimeric antigen receptor.

39. An antibody binding protein, wherein the antibody binding protein comprises an extracellular antibody binding domain and an intracellular domain, the intracellular domain comprises a mutated intracellular domain of DNAM-1, and the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

40. The antibody binding protein of claim 39, wherein the intracellular domain of wild-type DNAM-1 comprises the sequence of NRRRRRERRDLFTESWDTQKAPNNYRSPISTGQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 9).

41. The antibody-binding protein of any one of claims 39-40, wherein the intracellular domain of the mutant DNAM-1 lacks 1 to 5 basic amino acids at the C-terminus, truncates a sequence containing 1 to 5 basic amino acids, and / or 1 to 5 basic amino acids are replaced with non-basic amino acids relative to the intracellular domain of the wild-type DNAM-1.

42. The antibody binding protein of any one of claims 39-41, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 4 at the C-terminus relative to the intracellular domain of the wild-type DNAM-1.

43. An antibody-binding protein as described in any one of claims 39-42, wherein the intracellular domain of the mutant DNAM-1 lacks 1 to 5 basic amino acids at the N-terminus, truncates a sequence containing 1 to 5 basic amino acids, and / or 1 to 5 basic amino acids are replaced by non-basic amino acids relative to the intracellular domain of the wild-type DNAM-1.

44. The antibody binding protein of any one of claims 39-43, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 2 to 4 at the N-terminus relative to the intracellular domain of the wild-type DNAM-1.

45. The antibody binding protein of any one of claims 39-44, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 40 at the N-terminus relative to the intracellular domain of the wild-type DNAM-1.

46. ​​The antibody binding protein of any one of claims 39-45, wherein the mutated intracellular domain of DNAM-1 comprises the sequence of any one of SEQ ID NOs: 10-13.

47. The antibody-binding proteins of any one of claims 39-46, wherein the extracellular antibody binding domain comprises an extracellular domain capable of binding to antibody Fc.

48. The antibody binding protein of any one of claims 39-47, wherein the extracellular antibody binding domain comprises the extracellular domain of CD16, CD32 and / or CD64.

49. The antibody binding protein of any one of claims 39-48, wherein the extracellular antibody binding domain comprises a mutated extracellular domain of CD16.

50. The antibody binding protein of any one of claims 39-49, wherein the extracellular antibody binding domain comprises an extracellular domain of CD16 having 158V and / or S197P mutations relative to wild-type CD16.

51. The antibody binding protein of any one of claims 39-50, wherein the extracellular antibody binding domain comprises the sequence described in SEQ ID NO:

15.

52. The antibody binding protein of any one of claims 39-51, wherein the antibody binding protein comprises the sequence of any one of SEQ ID NOs: 16-20.

53. A use of an intracellular domain in preparing an antibody-binding protein, wherein the intracellular domain comprises a mutated intracellular domain of DNAM-1, and the mutated intracellular domain of DNAM-1 lacks one or more basic amino acids, truncates a sequence containing one or more basic amino acids, and / or replaces one or more basic amino acids with non-basic amino acids relative to the intracellular domain of wild-type DNAM-1.

54. The use according to claim 53, wherein the intracellular domain of the wild-type DNAM-1 comprises the sequence of NRRRRRERRDLFTESWDTQKAPNNYRSPISTGQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 9).

55. The use according to any one of claims 53-54, wherein the intracellular domain of the mutant DNAM-1 lacks 1 to 5 basic amino acids at the C-terminus, truncates a sequence containing 1 to 5 basic amino acids, and / or 1 to 5 basic amino acids are replaced by non-basic amino acids relative to the intracellular domain of the wild-type DNAM-1.

56. The use according to any one of claims 53 to 55, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 4 at the C-terminus relative to the intracellular domain of the wild-type DNAM-1.

57. The use according to any one of claims 53 to 56, wherein the intracellular domain of the mutant DNAM-1 lacks 1 to 5 basic amino acids at the N-terminus, truncates a sequence containing 1 to 5 basic amino acids, and / or 1 to 5 basic amino acids are replaced by non-basic amino acids relative to the intracellular domain of the wild-type DNAM-1.

58. The use according to any one of claims 53 to 57, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 2 to 4 at the N-terminus relative to the intracellular domain of the wild-type DNAM-1.

59. The use according to any one of claims 53 to 58, wherein the intracellular domain of the mutant DNAM-1 is truncated at amino acids 1 to 40 at the N-terminus relative to the intracellular domain of the wild-type DNAM-1.

60. The use according to any one of claims 53 to 59, wherein the intracellular domain of the mutated DNAM-1 comprises the sequence of any one of SEQ ID NOs: 10 to 13.

61. The use of any one of claims 53-60, wherein the antibody-binding protein comprises an extracellular antibody-binding domain.

62. The method of claim 61, wherein the extracellular antibody binding domain comprises an extracellular domain capable of binding to antibody Fc.

63. The use of any one of claims 61-62, wherein the extracellular antibody binding domain comprises the extracellular domain of CD16, CD32 and / or CD64.

64. The use of any one of claims 61-63, wherein the extracellular antibody binding domain comprises a mutated extracellular domain of CD16.

65. The use of any one of claims 61-64, wherein the extracellular antibody binding domain comprises an extracellular domain of CD16 having 158V and / or S197P mutations relative to wild-type CD16.

66. The use of any one of claims 61-65, wherein the extracellular antibody binding domain comprises the sequence described in SEQ ID NO:

15.

67. The use of any one of claims 61-66, wherein the antibody binding protein comprises the sequence of any one of SEQ ID NOs: 16-20.

68. A polypeptide, wherein the polypeptide comprises the antigen binding protein of any one of claims 1-29 and / or the antibody binding protein of any one of claims 39-52.

69. A nucleic acid, wherein the nucleic acid encodes the antigen binding protein of any one of claims 1-29, the antibody binding protein of any one of claims 39-52, and / or the polypeptide of claim 68.

70. A vector, wherein the vector comprises the nucleic acid of claim 69.

71. A cell comprising the antigen binding protein of any one of claims 1-29, the antibody binding protein of any one of claims 39-52, the polypeptide of claim 68, the nucleic acid of claim 69 and / or the vector of claim 70.

72. The cell of claim 71, wherein the cell comprises an immune cell.

73. The cell of any one of claims 71-72, wherein the cell comprises a NK cell, a NKT cell and / or a T cell.

74. A method for preparing the antigen binding protein of any one of claims 1-29, the antibody binding protein of any one of claims 39-52, and / or the polypeptide of claim 68, wherein the method comprises culturing the cell of any one of claims 71-73 under conditions such that the antigen binding protein, the antibody binding protein, and / or the polypeptide are expressed.

75. A composition, wherein the composition comprises the antigen binding protein of any one of claims 1-29, the antibody binding protein of any one of claims 39-52, the polypeptide of claim 68, the nucleic acid of claim 69, the vector of claim 70, and / or the cell of any one of claims 71-73, and optionally a pharmaceutically acceptable carrier.

76. A kit, wherein the kit comprises the antigen binding protein of any one of claims 1-29, the antibody binding protein of any one of claims 39-52, the polypeptide of claim 68, the nucleic acid of claim 69, the vector of claim 70, the cell of any one of claims 71-73, and / or the composition of claim 75.

77. A method for stimulating an immune response, comprising administering an antigen binding protein as described in any one of claims 1-29, an antibody binding protein as described in any one of claims 39-52, a polypeptide as described in claim 68, a nucleic acid as described in claim 69, a vector as described in claim 70, a cell as described in any one of claims 71-73, a composition as described in claim 75 and / or a kit as described in claim 76.

78. Use of the antigen-binding protein of any one of claims 1-29, the antibody-binding protein of any one of claims 39-52, the polypeptide of claim 68, the nucleic acid of claim 69, the vector of claim 70, the cell of any one of claims 71-73, the composition of claim 75 and / or the kit of claim 76 in the preparation of a medicament, wherein the medicament is used to prevent, alleviate and / or treat a disease (preferably a tumor).

79. A drug for preventing and / or treating a disease (preferably a tumor), comprising the antigen binding protein of any one of claims 1-29, the antibody binding protein of any one of claims 39-52, the polypeptide of claim 68, the nucleic acid of claim 69, the vector of claim 70, the cell of any one of claims 71-73, the composition of claim 75 and / or the kit of claim 76.

80. A method for preventing and / or treating a disease (preferably a tumor), comprising administering to a subject in need thereof an antigen binding protein as described in any one of claims 1-29, an antibody binding protein as described in any one of claims 39-52, a polypeptide as described in claim 68, a nucleic acid as described in claim 69, a vector as described in claim 70, a cell as described in any one of claims 71-73, a composition as described in claim 75 and / or a kit as described in claim 76.

81. The antigen-binding protein of any one of claims 1-29, the antibody-binding protein of any one of claims 39-52, the polypeptide of claim 68, the nucleic acid of claim 69, the vector of claim 70, the cell of any one of claims 71-73, the composition of claim 75 and / or the kit of claim 76, for the prevention and / or treatment of a disease (preferably a tumor).

82. The use of claim 78, the medicament of claim 79, the method of claim 80, and / or the antigen-binding protein, antibody-binding protein, polypeptide, nucleic acid, vector, cell, composition and / or kit of claim 81, wherein the disease comprises a solid tumor and / or a hematological tumor.

83. The use according to claim 78, the medicament according to claim 79, the method according to claim 80, and / or the antigen-binding protein, antibody-binding protein, polypeptide, nucleic acid, vector, cell, composition and / or kit for the use according to claim 81, wherein the disease is selected from the following group: gastric cancer, liver cancer and leukemia.

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