A cell expressing a multispecific antigen-binding protein and its applications

Abstract

A cell expressing a multispecific antigen-binding protein and its applications are provided. This cell possesses enhanced capabilities, such as cell proliferation, cytokine secretion, and tumor cell killing.

Description

A cell expressing multispecific antigen-binding protein and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a cell expressing a multispecific antigen-binding protein and applications thereof. Background Art

[0002] Currently, immunotherapy is an effective treatment for patients with poor prognosis. However, the immune cells used in immunotherapy suffer from poor function, proliferation, and survival after infusion. Therefore, developing modified immune cells and robust and reliable immune cell culture methods are urgent challenges.

[0003] Therefore, there is an urgent need in the art for a genetically modified cell to successfully achieve genetic modification and improve cell function through genetic modification.

[0004] Summary of the Invention

[0005] The present invention provides an isolated cell comprising a cell expressing a multispecific antigen-binding protein. The cell expressing the multispecific antigen-binding protein of the present invention has one or more of the following characteristics: (1) increased cell proliferation ability; (2) enhanced cytokine secretion ability; (3) increased tumor cell killing ability; (4) increased or stabilized exogenous gene transduction efficiency; (5) increased downstream signaling pathway strength; and / or (6) improved cell subpopulation ratios, such as an increased proportion of activated cells, a decreased proportion of regulatory cells, a decreased proportion of exhausted cells, an increased proportion of central memory cells and / or naive cells, a decreased proportion of apoptotic cells, and an increased proportion of stem-like cells.

[0006] In another aspect, the invention provides a method of producing a cell of the invention, comprising causing the cell to artificially express a multispecific antigen-binding protein.

[0007] In another aspect, the present invention provides a composition comprising the cells of the present invention, and optionally a pharmaceutically acceptable carrier.

[0008] In another aspect, the present invention provides a kit comprising the cells of the present invention and / or the composition of the present invention.

[0009] In another aspect, the present invention provides a use of the cell of the present invention, the composition of the present invention and / or the kit of the present invention in preparing a medicament for preventing, alleviating and / or treating a disease, preferably a tumor.

[0010] In another aspect, the present invention provides a medicament for preventing and / or treating a disease and / or symptom, preferably a tumor, comprising the cell of the present invention, the composition of the present invention and / or the kit of the present invention.

[0011] In another aspect, the present invention provides a method for preventing and / or treating a disease and / or condition, preferably a tumor, comprising administering the cell of the present invention, the composition of the present invention and / or the kit of the present invention to a subject in need thereof.

[0012] In another aspect, the present invention provides the cell of the present invention, the composition of the present invention and / or the kit of the present invention for use in preventing and / or treating a disease and / or symptom, preferably a tumor.

[0013] 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 present invention. Accordingly, the descriptions in the drawings and specification of the present invention are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The specific features of the present invention are shown in the appended claims. 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 drawings are briefly described as follows:

[0015] Figures 1A-1B show the transduction results of TIL cells from different donors.

[0016] 2A-2D show the results of the killing ability test of TIL cells transduced with the target polypeptide of the present invention.

[0017] Figures 3A-3E show the concentrations of various cytokines in the supernatant after each group of TIL cells were co-incubated with A375 or Caski cells for 24 hours.

[0018] Figures 4A-4D show the concentrations of various cytokines in the supernatant after each group of TIL cells were co-incubated with A375 cells for 24 hours. DETAILED DESCRIPTION

[0019] The following describes the embodiments of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0020] Definition of terms

[0021] In the present invention, the term "EGFR" generally includes epidermal growth factor receptor. For example, the UniProt number of EGFR may be P00533. The EGFR of the present invention may also encompass functionally active fragments thereof, not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or variants comprising the functionally active fragments thereof produced after processing and / or modification in cells.

[0022] In the present invention, the term "BCMA" generally encompasses B cell maturation antigens. For example, BCMA and TNFRSF17 can be used interchangeably, and the UniProt number for BCMA may be Q02223. The BCMA of the present invention also encompasses functionally active fragments thereof, including but not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants thereof, active fragments thereof, and variants comprising such functionally active fragments generated after cellular processing and / or modification.

[0023] In the present invention, the term "DLL3" generally encompasses Delta-like protein 3. For example, the UniProt number for DLL3 may be Q9NYJ7. The DLL3 of the present invention also encompasses functionally active fragments thereof, including but not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants thereof, active fragments thereof, and variants thereof comprising such functionally active fragments resulting from cellular processing and / or modification.

[0024] In the present invention, the term "FAP" generally encompasses fibroblast activation protein. For example, the UniProt number for FAP may be Q12884. The FAP of the present invention may also encompass functionally active fragments thereof, including but not limited to human and mammalian forms, conservative amino acid substitutions, glycoform modifications or variants, active fragments thereof, or variants thereof comprising such functionally active fragments after processing and / or modification in cells.

[0025] In the present invention, the term "peptide linker" is generally used interchangeably with the term "linker." For example, a peptide linker is a peptide that covalently or non-covalently links two or more molecules or peptides to form a larger complex consisting of all molecules or peptides including the peptide linker.

[0026] In the present invention, the term "fusion protein" generally refers to a protein composed of two or more molecules or peptides that are not normally bound in their native state, but whose respective head and terminal ends can be directly or indirectly bound together to form a continuous polypeptide. For example, one portion of a fusion protein can be referred to as a "first polypeptide." For example, another portion of the fusion protein can be referred to as a "second polypeptide." For example, a fusion protein can contain two or more parts, and the "first polypeptide" or "second polypeptide" does not limit the number of components of the fusion protein.

[0027] In the present invention, the term "antigen binding protein" is used in its broad sense and means a protein comprising a portion that binds to an antigen or target and optionally comprising a framework or framework portion that allows the antigen binding portion to adopt a configuration that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof. Antigen binding proteins may include, for example, alternative protein frameworks or artificial frameworks with transplanted CDRs or CDR derivatives. Such frameworks include, but are not limited to: antibody-derived frameworks comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein; and fully synthetic frameworks comprising, for example, biocompatible polymers.

[0028] In the present invention, the term "antibody" is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies and camelized single domain antibodies (e.g., heavy chain variable domain antibodies).

[0029] In the present invention, the term "variable" generally refers to the fact that certain parts of the sequence of the variable domain of an antibody vary strongly, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the entire variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, referred to as complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved parts in the variable domain are referred to as frameworks (FRs). The variable domains of native heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration, connected by three CDRs, forming loops connected, and in some cases forming a part of the β-sheet structure. The CDRs in each chain are closely together through the FR region and form the antigen binding site of the antibody together with the CDRs from the other chain. The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's cytotoxicity that depends on the antibody. In the art, the CDRs of an antibody can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability.

[0030] In the present invention, the term "Fab" refers to the antigen-binding fragment of an antibody. As mentioned above, papain can be used to digest intact antibodies. Papain digestion of an antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, as described above). The Fab fragment consists of an intact L chain with the variable region of a heavy chain and the H chain (V H ) of the first constant region (C H 1) Composition.

[0031] As used herein, the term "Fab' fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0032] As used herein, the term "F(ab')2" refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a portion of the hinge region. The F(ab')2 fragment has divalent antigen-binding activity and is capable of cross-linking antigens.

[0033] As used herein, the term "Fv fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy chain variable region and the light chain variable region, and lacking the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, an Fv fragment comprises all or part of the amino-terminal variable region of approximately 110 amino acids of the heavy and light chains.

[0034] In the present invention, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0035] In the present invention, the amino acid residues may be naturally occurring amino acids and / or artificially modified amino acids. For example, naturally occurring amino acids may be selected from the group consisting of 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).

[0036] In the present invention, the term "immune cell" generally refers to cells that participate in an immune response, such as cells that promote immune effector responses. 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. For example, the immune cells of the present invention may include cells derived from artificial pluripotent stem (iPS) cells, PBMC cells, and / or tumor infiltrating lymphocytes. For example, the immune cells of the present invention may be obtained by differentiation of iPS cells. The term also includes engineered immune cells, such as immune cells that are genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0037] In the present invention, the term "killing ability" refers to the killing of cells by contacting the cells with an effective amount of antibodies, T cells, immunoconjugates, bispecific / multispecific molecules or compositions. For example, the fusion polypeptide of the present invention can enhance the killing ability of cells. For example, cells expressing the fusion polypeptide of the present invention can exhibit enhanced killing ability. For example, a combination of cells expressing the fusion polypeptide of the present invention and other immune cells, antibodies, immunoconjugates (e.g., conjugates of binding molecules targeting immune checkpoints with other active molecules), and / or bispecific / multispecific molecules can exhibit enhanced killing ability. The killing ability after the above-mentioned combination of substances can be manifested as cells expressing positive killing antigens, optionally in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis or by a combination of two or more of these mechanisms.

[0038] In the present invention, the term "directly or indirectly linked" refers to direct linkage via a peptide bond, or indirect linkage via a linker or a non-peptide linkage.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In the present invention, the term "therapeutically effective amount" refers to an amount of a fusion protein of the present invention or an amount of cells expressing a fusion protein of the present invention sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). The therapeutically effective amount depends on the disease being treated, and those skilled in the art can readily determine the actual effective amount.

[0043] 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.

[0044] As used herein, the term "composition" refers to a mixture containing one or more compounds of the present invention, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In the present invention, the terms "about" and "approximately" generally refer to a statistically significant numerical range. 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.

[0049] In the present invention, the terms "above", "below", "at most" and "at least" include the number.

[0050] In the present invention, the terms "include", "comprise" or "contain" are open expressions, while "consisting of" is a closed expression. The former covers the latter, and the latter is a special form of the former.

[0051] Detailed Description of the Invention

[0052] The present invention provides improvements to therapies based on tumor infiltrating lymphocytes ("TIL cells"). For example, the improvements of the present invention relate to targeting the tumor microenvironment. For example, cells expressing multispecific antigen-binding proteins of the present invention have one or more of the following characteristics: (1) increased cell proliferation capacity; (2) enhanced cytokine secretion capacity; (3) increased tumor cell killing capacity; (4) increased or stabilized exogenous gene transduction efficiency; (5) increased downstream signaling pathway strength; and / or (6) improved cell subpopulation ratios, such as an increased proportion of activated cells, a decreased proportion of regulatory cells, a decreased proportion of exhausted cells, an increased proportion of central memory cells and / or naive cells, a decreased proportion of apoptotic cells, and an increased proportion of stem-like cells.

[0053] In one aspect, the invention provides an isolated cell that expresses a multispecific antigen-binding protein, wherein an antigen-binding fragment of the multispecific antigen-binding protein specifically binds to a surface antigen of the cell.

[0054] For example, wherein the cell comprises tumor infiltrating lymphocytes (TIL).For example, wherein the TIL is derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastasis lesions, paracancerous tissue fragments, pleural effusion and / or peritoneal effusion TIL and / or derived from TIL revived after cryopreservation.

[0055] For example, wherein the cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and / or a basophil. For example, wherein the immune cell comprises a monocyte, a macrophage and / or a dendritic cell. For example, wherein the cell comprises a B cell, a T cell, a natural killer cell and / or a natural killer-like T cell (NKT). For example, wherein the cell comprises an αβT cell and / or a γδT cell. For example, wherein the cell is derived from an immune cell differentiated from stem cells, wherein the stem cell comprises an induced pluripotent stem cell (iPSC), an embryonic stem cell, a bone marrow stem cell, an umbilical cord blood stem cell and / or a peripheral blood stem cell.

[0056] For example, wherein the cell comprises an engineered immunoreceptor displayed on the cell surface, wherein the engineered immunoreceptor comprises a T cell receptor. For example, wherein the engineered immunoreceptor specifically binds to an antigen expressed on a target cell. For example, wherein the immune cell comprises a chimeric antigen receptor and / or a T cell receptor.

[0057] For example, the multispecific antigen-binding protein comprises a first antigen-binding fragment that binds to CD3. For example, the first antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, the first heavy chain variable region comprising the HCDR1 set forth in SEQ ID NO: 11, the HCDR2 set forth in SEQ ID NO: 12, and the HCDR3 set forth in SEQ ID NO: 13, and the first light chain variable region comprising the LCDR1 set forth in SEQ ID NO: 14, the LCDR2 set forth in SEQ ID NO: 15, and the LCDR3 set forth in SEQ ID NO: 16. For example, the first antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, the first heavy chain variable region comprising the sequence set forth in SEQ ID NO: 17, and the first light chain variable region comprising the sequence set forth in SEQ ID NO: 18. For example, the first antigen-binding fragment comprises the same HCDR1-3 sequences as the first heavy chain variable region set forth in SEQ ID NO: 17, and the same LCDR1-3 sequences as the first light chain variable region set forth in SEQ ID NO: 18. For example, the first antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 19. For example, the first antigen-binding fragment binds to CD3. For example, the multispecific antigen-binding protein of the present invention may comprise a first antigen-binding fragment as shown in SEQ ID NO: 19, and a second antigen-binding fragment that binds to a tumor-specific antigen.

[0058] For example, wherein the multispecific antigen-binding protein comprises a second antigen-binding fragment that binds to a tumor-specific antigen. For example, "tumor-associated antigens" (TAAs) are well known in the art and refer to molecules that are differentially expressed on and / or in cancer cells relative to non-cancerous cells of the same cell type. Non-limiting examples of TAAs include CD5, CD19, CD20, CD22, CD23, CD25, CD27, CD30, CD33, CD34, CD37, CD38, CD40, CD43, CD44v6, CD47, CD50, CD52, CD56, CD63, CD72a, CD74, CD78, CD79a, CD79b, CD86, CD134, CD137, CD138, CD248, CD319, αvβ3, α5β1, human epidermal growth factor receptor (EGFR or HER1), HER2, HER3, HER4, vascular endothelial growth factor receptor 1 (VEGFR-1), VEGFR-2, VEGFR-3, TRAIL-R2, carbohydrate antigen 19-9 (CA 19-9), carbohydrate antigen 125 (CA 125), carcinoembryonic antigen (CEA), mucin 1 (MUC1), 1), MUC2, MUC3, MUC4, MUC5, MUC7, ganglioside GD2, ganglioside GD3, ganglioside GM2, carbonic anhydrase IX (CAIX), SHH factor, melanoma chondroitin sulfate proteoglycan (MCSP), chondroitin sulfate proteoglycan 4 (CSPG4), six-transmembrane epithelial antigen of prostate (STEAP), A33 antigen, desmoglein-2 (Dsg2), Dsg3, Dsg4, E-cadherin neoepitope, fetal nicotinic acetylcholine receptor (fnAChR), muellerian inhibitory substance receptor type II (MISIIR), tumor-associated antigen L6 (TAL6), Thomsen-Friedenreich antigen;TF) antigen, EPHA1, EPHA2, EPHA3, EPHA4, EPHA7, EPHA8, EPHA10, EPHB4, testicular cancer antigen (CTA), NY-BR1, tumor-associated glycoprotein 72 (TAG-72), α-fetoprotein (AFP), brother of the regulator of the imprinted site (BORIS), B cell activating factor (BAFF), extradomain-B fibronectin (EDB-FN), glycoprotein A33 (GPA33), tenascin-C (TNC), melanoma-associated antigen (MAGE), GAGE, BAGE, prostate stem cell antigen (PSCA), mesothelin, mucin-associated Tn, Sialyl Tn, globo H, stage-specific embryonic antigen-4 (SSEA-4), epithelial cell adhesion molecule (EpCAM), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), prostate-specific membrane antigen (PSMA), fibroblast activation protein (FAP), DLL3, BCMA, vascular cell adhesion protein 1 (VCAM-1), insulin-like growth factor receptor (IGFR), or hepatocyte growth factor receptor (HGFR).

[0059] For example, the multispecific antigen-binding protein comprises a second antigen-binding fragment, which binds to an antigen selected from the following group: epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), fibroblast activation protein (FAP), DLL3, BCMA, CD19, CD79b, CD37, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), interleukin-13 receptor alpha 2 (IL-13Rα2), ephrin type A receptor 1 (EphA1), human epidermal growth factor receptor 2 (HER2), mesothelin, cell surface-associated mucin 1 (MUC1) or cell surface-associated mucin 16 (MUC16).

[0060] For example, the second antigen-binding fragment of the present invention comprises an anti-TAA binding portion, which in some instances comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some instances, the anti-TAA binding portion is specific for CD20 (e.g., human CD20). In some instances, the anti-TAA binding portion is specific for CD19 (e.g., human CD19). In some instances, the anti-TAA binding portion is specific for EGFR (e.g., human EGFR). In some instances, the anti-TAA binding portion is specific for HER2 (e.g., human HER2). In some instances, the anti-TAA binding portion is specific for PSMA (e.g., human PSMA). In some instances, the anti-TAA binding portion is specific for DLL3. In some instances, the anti-TAA binding portion is specific for BCMA. In some instances, the anti-TAA binding portion is specific for CEA (e.g., human CEA). In some instances, the anti-TAA binding portion is specific for EpCAM (e.g., human EpCAM). In some instances, the anti-TAA binding portion is specific for FAP (e.g., human FAP). In some instances, the anti-TAA binding portion is specific for PDL1 (e.g., human PDL1). In some instances, the anti-TAA binding portion is specific for CD38 (e.g., human CD38). In some instances, the anti-TAA binding portion is specific for CD33 (e.g., human CD33). In some instances, the anti-TAA binding portion is specific for HGFR (cMET) (e.g., human cMET). In some instances, the anti-TAA binding portion is specific for CD47 (e.g., human CD47). In some instances, the anti-TAA binding portion is specific for TRAIL-R2 (e.g., human TRAIL-R2). In some instances, the anti-TAA binding portion is specific for mesothelin (e.g., human mesothelin). In some instances, the anti-TAA binding portion is specific for GD2 (e.g., human GD2).

[0061] For example, the antigen-binding fragments described herein have suitable binding affinities for one or both of the target antigens (e.g., CD3 and TAA) or their antigenic epitopes. As used herein, "binding affinity" refers to the association constant or KA. KA is the reciprocal of the dissociation constant (KD). The binding affinity (KD) for CD3 described herein can be at least 100 nM, 10 nM, 1 nM, 0.1 nM, or less (e.g., less than 1 nM or 0.1 nM). Alternatively, the binding affinity (KD) for TAA described herein can be at least 100 nM, 10 nM, 1 nM, 0.1 nM, or less. Binding affinity (or binding specificity) can also be determined by various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence analysis).

[0062] For example, the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the HCDR1 set forth in SEQ ID NO:21, the HCDR2 set forth in SEQ ID NO:22, and the HCDR3 set forth in SEQ ID NO:23, and the second light chain variable region comprising the LCDR1 set forth in SEQ ID NO:24, the LCDR2 set forth in SEQ ID NO:25, and the LCDR3 set forth in SEQ ID NO:26. For example, the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the sequence set forth in SEQ ID NO:27, and the second light chain variable region comprising the sequence set forth in SEQ ID NO:28. For example, the second antigen-binding fragment comprises the same HCDR1-3 sequences as the second heavy chain variable region set forth in SEQ ID NO:27, and the same LCDR1-3 sequences as the second light chain variable region set forth in SEQ ID NO:28. For example, the second antigen-binding fragment comprises the sequence set forth in SEQ ID NO:29. For example, the second antigen-binding fragment binds to EGFR. For example, the multispecific antigen-binding protein of the present invention may comprise a second antigen-binding fragment represented by SEQ ID NO: 29, and a first antigen-binding fragment that binds to an immune cell surface protein.

[0063] For example, the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the HCDR1 set forth in SEQ ID NO:31, the HCDR2 set forth in SEQ ID NO:32, and the HCDR3 set forth in SEQ ID NO:33, and the second light chain variable region comprising the LCDR1 set forth in SEQ ID NO:34, the LCDR2 set forth in SEQ ID NO:35, and the LCDR3 set forth in SEQ ID NO:36. For example, the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the sequence set forth in SEQ ID NO:37, and the second light chain variable region comprising the sequence set forth in SEQ ID NO:38. For example, the second antigen-binding fragment comprises the same HCDR1-3 sequences as the second heavy chain variable region set forth in SEQ ID NO:37, and the same LCDR1-3 sequences as the second light chain variable region set forth in SEQ ID NO:38. For example, the second antigen-binding fragment comprises the sequence set forth in SEQ ID NO:39. For example, the second antigen-binding fragment binds to EGFR. For example, the multispecific antigen-binding protein of the present invention may comprise a second antigen-binding fragment represented by SEQ ID NO: 39, and a first antigen-binding fragment that binds to an immune cell surface protein.

[0064] For example, the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the HCDR1 set forth in SEQ ID NO:41, the HCDR2 set forth in SEQ ID NO:42, and the HCDR3 set forth in SEQ ID NO:43, and the second light chain variable region comprising the LCDR1 set forth in SEQ ID NO:44, the LCDR2 set forth in SEQ ID NO:45, and the LCDR3 set forth in SEQ ID NO:46. For example, the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the sequence set forth in SEQ ID NO:47, and the second light chain variable region comprising the sequence set forth in SEQ ID NO:48. For example, the second antigen-binding fragment comprises the same HCDR1-3 sequences as the second heavy chain variable region set forth in SEQ ID NO:47, and the same LCDR1-3 sequences as the second light chain variable region set forth in SEQ ID NO:48. For example, the second antigen-binding fragment comprises the sequence set forth in SEQ ID NO:49. For example, the second antigen-binding fragment binds to FAP. For example, the multispecific antigen-binding protein of the present invention may comprise a second antigen-binding fragment represented by SEQ ID NO: 49, and a first antigen-binding fragment that binds to an immune cell surface protein.

[0065] For example, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the HCDR1 set forth in SEQ ID NO:51, the HCDR2 set forth in SEQ ID NO:52, and the HCDR3 set forth in SEQ ID NO:53, and the second light chain variable region comprising the LCDR1 set forth in SEQ ID NO:54, the LCDR2 set forth in SEQ ID NO:55, and the LCDR3 set forth in SEQ ID NO:56. For example, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the sequence set forth in SEQ ID NO:57, and the second light chain variable region comprising the sequence set forth in SEQ ID NO:58. For example, wherein the second antigen-binding fragment comprises the same HCDR1-3 sequences as the second heavy chain variable region set forth in SEQ ID NO:57, and the same LCDR1-3 sequences as the second light chain variable region set forth in SEQ ID NO:58. For example, wherein the second antigen-binding fragment comprises the sequence set forth in SEQ ID NO:59. For example, the second antigen-binding fragment binds to FAP. For example, the multispecific antigen-binding protein of the present invention may comprise a second antigen-binding fragment represented by SEQ ID NO: 59, and a first antigen-binding fragment that binds to an immune cell surface protein.

[0066] For example, the antibodies of the present invention can be divided using the Kabat method, Chothia method, AbM method, Contact method or IMGT method known in the art. For example, the CDR division method in the present invention is the Kabat method.

[0067] For example, wherein the multispecific antigen-binding protein comprises a first antigen-binding fragment and a second antigen-binding fragment, wherein the first antigen-binding fragment and the second antigen-binding fragment are connected by a peptide linker. The first antigen-binding fragment can be located at the N-terminus or the C-terminus of the second antigen-binding fragment.

[0068] For example, the multispecific antigen-binding proteins of the present invention comprise BiTEs. "Bispecific T cell engagers," "BiTE antibody constructs," or "BiTEs" refer to polypeptides each comprising a single-chain variable fragment (scFv) or nanobody (VHH) linked in series. Optionally, the scFvs are linked by a linker (e.g., a glycine-rich linker). One scFv or VHH of the BiTE binds to a T cell receptor (TCR) (e.g., binds to a CD3ε subunit), and the other scFv or VHH binds to a target antigen (e.g., a tumor-associated antigen).

[0069] For example, the multispecific antigen-binding proteins of the present invention comprise BiKEs. "Bispecific NK cell engagers," "BiKE antibody constructs," or "BiKEs" refer to polypeptides each comprising a single-chain variable fragment (scFv) or nanobody (VHH) linked in series. Optionally, the scFvs are linked via a linker (e.g., a glycine-rich linker). For example, one scFv or VHH of a BiKE binds to CD16, and another scFv or VHH binds to a target antigen (e.g., a tumor-associated antigen).

[0070] For example, a multispecific antigen-binding protein of the present invention may comprise a second antigen-binding fragment set forth in SEQ ID NO:29, and a first antigen-binding fragment that binds CD3. For example, a multispecific antigen-binding protein of the present invention may comprise a second antigen-binding fragment set forth in SEQ ID NO:39, and a first antigen-binding fragment that binds CD3. For example, a multispecific antigen-binding protein of the present invention may comprise a second antigen-binding fragment set forth in SEQ ID NO:49, and a first antigen-binding fragment that binds CD3. For example, a multispecific antigen-binding protein of the present invention may comprise a second antigen-binding fragment set forth in SEQ ID NO:59, and a first antigen-binding fragment that binds CD3. For example, the present invention also provides a multispecific antigen-binding protein, wherein the multispecific antigen-binding protein comprises the sequences set forth in SEQ ID NOs:30, 40, 50, and / or 60.

[0071] For example, the multispecific antigen-binding protein can include a known signal peptide. For example, the multispecific antigen-binding protein can include the signal peptide set forth in SEQ ID NO: 1 of the present invention. For example, the multispecific antigen-binding protein can include a tag polypeptide FLAG and / or 8His. For example, the multispecific antigen-binding protein can include the sequence set forth in SEQ ID NO: 3 and / or 4 of the present invention. For example, the multispecific antigen-binding protein can be linked to a peptide segment comprising Thy1.1 or a fragment thereof. For example, the Thy1.1 comprises the amino acid sequence set forth in SEQ ID NO: 6. For example, the Thy1.1 is linked to the multispecific antigen-binding protein via a cleavable peptide.

[0072] For example, the cleavable peptide can be used to connect two or more components, such as molecules or peptides. For example, the cleavable peptide can be used to connect two or more non-peptide molecules. For example, the cleavable peptide can be used to connect two or more peptide molecules. For example, the cleavable peptide can be used to connect different types of components, for example, one or some components are non-peptide small molecules, and another or some components are peptides. For example, the substance connected by the cleavable peptide in the present invention can be called a fusion protein or fusion polypeptide (the two terms are used interchangeably in the present invention). For example, the cleavable peptide contains T2A and / or P2A, and the cleavable peptide optionally also contains Furin peptide. For example, the Furin peptide is extended on the basis of T2A and / or P2A, and when T2A and / or P2A are cleaved, the Furin peptide can act as a buffer to prevent the active ingredient peptide connected to T2A and / or P2A from being cleaved. For example, the amino acid sequence of the cleavable peptide is shown in SEQ ID NO: 5.

[0073] For example, the heavy chain variable region and the light chain variable region in the multispecific antigen-binding protein can be connected to each other via a linker (e.g., a glycine-based linker). For example, the heavy chain variable region and the light chain variable region in the multispecific antigen-binding protein can be connected via the sequence shown in SEQ ID NO: 2 of the present invention.

[0074] In another aspect, the present invention provides a method for producing a cell of the present invention, comprising causing the cell to artificially express the multispecific antigen-binding protein, for example, causing the cell to artificially express a sequence comprising SEQ ID NOs: 30, 40, 50, and / or 60. For example, the methods of the present invention can transduce a nucleic acid sequence encoding a sequence of SEQ ID NOs: 7, 8, 9, and / or 10 into the cell.

[0075] For example, the sequences mentioned herein include sequences that are at least about 80% homologous to the sequence. For example, the sequences mentioned herein include sequences that are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homologous to the sequence.

[0076] In one aspect, the present invention provides a composition comprising the cells of the present invention, and optionally a pharmaceutically acceptable carrier.

[0077] In one aspect, the invention provides a kit comprising a cell of the invention and / or a composition of the invention.

[0078] In one aspect, the present invention provides a use of a cell, a composition, and / or a kit of the present invention in the preparation of a medicament for preventing, alleviating, and / or treating a disease, for example, a tumor.

[0079] In one aspect, the present invention provides a medicament for preventing and / or treating a disease and / or symptom, comprising the cell of the present invention, the composition of the present invention, and / or the kit of the present invention. For example, the disease comprises a tumor.

[0080] In one aspect, the present invention provides a method for preventing and / or treating a disease and / or symptom, comprising administering a cell of the present invention, a composition of the present invention, and / or a kit of the present invention to a subject in need thereof. For example, the disease comprises a tumor.

[0081] In one aspect, the present invention provides a cell of the present invention, a composition of the present invention, and / or a kit of the present invention for use in preventing and / or treating a disease and / or symptom. For example, the disease comprises a tumor.

[0082] Without intending to be bound by any theory, the following examples are merely intended to illustrate the products, preparation methods and uses of the present invention, and are not intended to limit the scope of the present invention.

[0083] Example

[0084] Example 1 Tumor Infiltrating Lymphocyte (TIL) Cell Culture Method

[0085] 1.1 Tumor tissue receipt and processing

[0086] 1.1.1 Organization reception

[0087] Receive tumor tissue and blood samples from donors, verify and record sample information, and print corresponding sample labels.

[0088] 1.1.2 Tissue processing and culture

[0089] Use 75% alcohol to disinfect the sample tube and blood collection tube and transfer them to a biosafety cabinet. Isolate PBMC cells from the blood sample and freeze them according to the above-mentioned PBMC manual isolation and freezing procedures. Take a culture flask or culture bag with a breathable surface, such as a culture bag (Origen), and add 300 mL of thawed complete culture medium. The complete culture medium can be arbitrarily selected from X-vivo15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and can be supplemented with essential amino acids and antibiotics, and IL-2 at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, such as 6000 IU / mL). Take several 10 cm culture dishes, add an appropriate amount of culture medium, use sterile ophthalmic forceps to remove the tumor tissue from the sample tube into a 10 cm culture dish, wash the tissue and change the culture dish. Use ophthalmic scissors and forceps to perform initial shearing, removing adipose and necrotic tissue. Each tissue block is then minced to approximately 27 cubic millimeters. A non-suspended tumor tissue block is obtained. A 20 mL syringe is used to remove the internal stopcock and connect to a culture bag. Using a pipette, approximately 1 g of tissue is transferred from the syringe into the culture bag. The culture bag is placed in a CO2 incubator for incubation. The scissors and forceps are cleaned and initially disinfected with 75% alcohol. After ultrasonic cleaning, they are sterilized to obtain the first TIL population.

[0090] 1.2 Step (A) In vitro expansion and harvesting

[0091] 1.2.1 Step (A) In vitro amplification

[0092] Depending on the cell growth status, the medium should be replenished or half-replaced every 3-7 days to ensure cell nutrition. Complete culture medium can be arbitrarily selected from X-vivo 15 culture medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi and other brands of T cell culture medium, and essential amino acids and antibiotics can be added, and IL-2 (double heron and / or tetracycline) at a concentration of 300-9000 IU / mL (e.g. 1000-9000 IU / mL, such as 6000 IU / mL) can be added. 3-14 days in step (A), for example, samples can be taken and counted on the 13th or 14th day. If the cell number is between 5×10 5 to 5×10 8 During this time, the harvesting step (A) is entered.

[0093] 1.2.2 Results of Step (A)

[0094] Collect the cells after in vitro expansion in step (A), centrifuge, discard the culture medium, wash the cells once with PBS or normal saline, obtain the TILs (second TIL population) expanded in vitro in step (A), and take samples for counting and retain about 5×10 5 to 2×10 8 cells into the subsequent in vitro expansion step; about 5×10 5 The remaining cells can be added to the cryopreservation medium and cryopreserved as cryopreserved preREP TIL in vitro cells.

[0095] 1.3 Step (B) TIL activation

[0096] Continue to culture the TILs (second TIL population) expanded in vitro in step (A), or recover the frozen preREP TIL cells in vitro and perform TIL activation in step (B).

[0097] Complete culture medium can be selected from X-vivo 15 medium or other commercial T cell culture medium, such as Stem Cell, Lonza, Thermo, Miltenyi Biotech, etc. Essential amino acids and antibiotics can be added to adjust the cell density to 5×10 5 to 2×10 6 Cells are suspended in a 24-well culture plate at a concentration of 1 mL / well and IL-2 is added at a concentration of 300-9000 IU / mL (e.g., 1000-9000 IU / mL, for example, 6000 IU / mL). T cell activators, such as CD3 agonists and / or CD28 agonists, can be added to the culture medium of each TIL population, for example, approximately 30 ng / mL of CD3 antibody (Miltenyi Biotech, OKT3), approximately 30 ng / mL of CD28 antibody (Merck, 15E8), magnetic beads (Dynabeads, approximately 1 to 10 μm diameter, Thermo Fisher) at a ratio of approximately 1:2-2:1 beads to TILs, and / or transACT (Miltenyi, approximately 100 to 500 nm diameter, TILs) at a ratio of approximately 1:100-1:2000. Culture is continued for approximately 0-4 days to obtain a third TIL population.

[0098] 1.4 TIL cell transduction

[0099] Transduce the activated TILs from step (B) above. One day prior to transduction, coat a 24-well suspension culture plate with recombinant human fibrin fragment (Retronectin, Takara) at a final concentration of 15 μg / mL. Protect from light and incubate overnight at 4°C. Remove the coated 24-well plate, discard the coating solution, and block with 2% BSA blocking solution at room temperature for 30 minutes. Discard the blocking solution and wash the plate twice with a plate washer containing 2.5% HEPES, then discard the plate washer solution.

[0100] Each test group was transduced with a retrovirus carrying a nucleic acid encoding a target polypeptide of the present invention (comprising the multispecific binding peptide EGFR-1-CD3 tandem scFv set forth in SEQ ID NO:30 (BiTE1); or the multispecific binding peptide EGFR-2-CD3 tandem scFv set forth in SEQ ID NO:40 (BiTE2); or the multispecific binding peptide FAP-1-CD3 tandem scFv set forth in SEQ ID NO:50 (BiTE3); or the multispecific binding peptide FAP-2-CD3 tandem scFv set forth in SEQ ID NO:60 (BiTE4); for expression testing only, a fusion protein comprising a multispecific binding peptide as set forth in any one of SEQ ID NOs:7-10 and a marker polypeptide can optionally be expressed).

[0101] The retroviral plasmid construction method is as follows: a nucleic acid fragment encoding the target polypeptide of the present invention is synthesized, digested with EcoRI and NotI, and the exogenous fragment is recovered. Plasmid MP71 is digested with EcoRI and NotI, and the vector fragment is recovered. The exogenous fragment and the vector fragment are ligated using T4 Ligase to obtain a retroviral plasmid capable of expressing the target polypeptide of the present invention.

[0102] Add 0.25-2 mL of retroviral solution to each well and centrifuge at 32°C, 2000 g for 2 hours. The blank control group is not transduced with cells but is transduced with the control virus. Discard the supernatant from the 24-well plate and add the TILs activated in step 1.3 above to each well of the 24-well plate in a volume of 300-500 μL, with a cell concentration of approximately 1×10 6 1000 g for 10 minutes at 30-32°C. After centrifugation, the culture plate was placed in a 37°C, 5% CO2 incubator for approximately 0-4 days to obtain the transduced TIL population and the fourth TIL population.

[0103] 1.5 Step (D) TIL cell culture after transduction

[0104] Feeder cells (irradiated healthy donor PBMC T cells) are added to the fourth TIL cell population for culture. The time for contacting TIL with feeder cells needs to be several times T after the TIL is contacted with IL-2 and T cell activator (such as CD3 antibody or a nanomatrix containing CD3 antibody and CD28 antibody, such as transACT) in step (B). n Afterwards (T n The time period can be from 0 hours to 12 days, for example, 24 hours or 48 hours. First, resuscitate the mixed feeder cells from 1-5 donors; mix the activated TIL cells and feeder cells at a ratio of approximately 1:200, transfer them to a G-Rex100 culture flask or breathable bag, and supplement with complete culture medium. Samples are taken and counted every 1-3 days, and the medium is replenished or half-filled depending on the cell status until the total number of cells is greater than 1×10 9 Alternatively, the in vitro expansion culture of step (D) is carried out for about 5 days to about 14 days, and the in vitro expansion culture of step (D) is terminated.

[0105] 1.6 Harvesting of Tumor-Infiltrating Lymphocytes

[0106] Take the cells amplified in step (D), centrifuge and discard the culture supernatant, and wash three times with PBS or saline or compound electrolyte solution to obtain TILs amplified in step (D) (fifth TIL population). Samples are counted during the third wash. According to the counting results, after the final centrifugation, discard the supernatant and take 3×10 6 The cells were sent for quality control testing; all the remaining cells were used as the final cell product. Optional freezing medium can be added to adjust the cell density to 1-3×10 8 cells / mL for cryopreservation.

[0107] Example 2 TCR T cell preparation

[0108] T cells from various sources such as peripheral blood and stem cell differentiation, preferably PBMC cells, are cultured. Nucleic acids encoding TCRs (e.g., TCRs targeting NY-ESO-1) are transferred into the above-mentioned T cells according to methods known in the art, such as viral transduction, LNP transduction, electroporation, etc., thereby obtaining TCR-T cells. Before or after the TCR is transduced into the above-mentioned T cells, each test group is transduced with a retrovirus carrying a nucleic acid encoding the target polypeptide of the present invention (e.g., BiTE1, BiTE2, BiTE3, or BiTE4) to obtain TCR T cells transduced with the target polypeptide of the present invention. T cells not transduced with the target polypeptide serve as a blank control group (Control).

[0109] Example 3 Cell transduction efficiency detection

[0110] Referring to Example 1 of the present application, the TIL population obtained on the 7th or 8th day after gene editing was used to detect cytokine expression by flow cytometry.

[0111] Testing steps:

[0112] TILs from each experimental group were centrifuged in a 96-well V-bottom plate. Cell surface staining was performed using a cocktail of antibodies (BV421 anti-CD3, Biolegend 317344; APC anti-CD4, Biolegend 300514; FITC anti-CD8, Biolegend 344704; PE anti-Thy1.1, Invitrogen 12-0900-81) at a 1:10000 concentration. Cells were resuspended in 100 μL / well of the antibody cocktail and incubated at 2-8°C in the dark for 30 minutes. Following staining, cells were washed with an appropriate amount of PBS and centrifuged at 600 g for 3 minutes. The supernatant was discarded. Cells were resuspended in 100 μL / well of PBS and analyzed by flow cytometry.

[0113] Figures 1A-1B show the transduction results of TIL cells derived from different donors. The gray unfilled dashed line, gray unfilled solid line, and black filled solid line in Figures 1A and 1B represent the fluorescence subtraction control (FMO), the blank control (untransduced with the target peptide), TIL expressing BiTE1 (SEQ ID NO: 30), and TIL expressing BiTE2 (SEQ ID NO: 40), respectively. Figure 1A shows TIL derived from a cervical tumor, and Figure 1B shows TIL derived from a melanoma tumor.

[0114] By detecting the staining results of the Thy1.1 marker, the results showed that the Thy1.1 marker of the TIL cells from the two donors without gene editing was negative, and the Thy1.1 marker of the TIL cells from the two donors after gene editing was partially positive. Because the target polypeptide of the present invention and the Thy1.1 marker are encoded by the same plasmid and are located upstream of the Thy1.1 marker, the present invention successfully expresses the target polypeptide of the present invention in TIL cells.

[0115] Example 4 Cell Killing Ability Detection

[0116] The TIL population obtained on the 7th or 8th day after gene editing was tested for its ability to kill tumor cells.

[0117] Testing steps:

[0118] Tumor target cells were plated in a 96-well flat-bottom plate one day in advance. The next day, each group of TIL cells were co-cultured with target cells at an effector-target ratio of 1:1, with 100 μL of target cells and TIL cells respectively. Three replicate wells were set up for each group, and a control group containing only target cells was also set up.

[0119] According to the instructions for the apoptosis detection reagent (SuperView™ 488 Caspase-3 Assay Kit, UEL S6007L), 1.5 μL / well of apoptosis detection reagent was added and diluted with culture medium at 18.5 μL / well. Caspase 3 activity was recorded using an Incucyte recorder (Sartorius) every 3 hours to analyze the killing ability of TIL cells against target cells.

[0120] Figures 2A-2D show the results of the killing ability test of TIL cells transduced with the target polypeptide of the present invention. The significance tests in the figures are all compared with untransduced TIL. Figure 2A is a killing curve made based on the activity of Caspase 3 in the killing test of TIL cells against A375 cells. Figure 2B is the cell morphology of each group at each test time point scanned by Incucyte S3 in the killing test of TIL cells against A375. Figure 2C is a killing curve made based on the activity of Caspase 3 in the killing test of TIL cells against Caski cells. Figure 2D is the cell morphology of each group at each test time point scanned by Incucyte S3 in the killing test of TIL cells against Caski. Figures 2A to 2D are TILs derived from cervical tumors.

[0121] Results showed that gene-edited TIL cells demonstrated significantly greater and faster cytotoxicity against A375 or Caski cells compared to untransduced controls, achieving near-complete elimination of target cells approximately 24 hours after co-incubation. Incucyte scans revealed that after two days and 21 hours of co-incubation, tumor target cells were virtually absent from the gene-edited TIL cell group, while they significantly expanded in the untransduced control group and the target cell-only control group.

[0122] Example 5 Detection of cytokine secretion by TIL cells

[0123] 24 hours after the start of the co-incubation for the killing ability test in Example 4 above, the supernatant of each group was collected for the detection of cytokine secretion.

[0124] Testing steps:

[0125] Cytokine secretion assays can be performed according to the instructions for the Cytokine Assay Kit (BD 560484). Reconstitute the lyophilized human Th1 / Th2 / Th17 cytokine standard powder (BD) with 2 mL of Assay Diluent (BD) (the standard stock solution contains 5000 pg / mL of each cytokine). Then, serially dilute the standard solution in the following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024. Label the tube "Standard Tube." Use one tube containing only Assay Diluent as a reference. Add 2 μL / Beads / well of each Capture Bead (BD), followed by 10 μL / well of PE Detection Reagent (BD) and mix to prepare a mixture. Add 24 μL / well to a V-bottom 96-well plate. Then, add 10 μL / well of the supernatant from each standard and experimental group and mix. Incubate at room temperature for 3 hours in the dark. At the end of the incubation, add 200 μL of Wash Buffer (BD) to each well and centrifuge at 500 g for 3 minutes. After centrifugation, resuspend the cells in 100 μL of Wash Buffer (BD) per well for flow cytometry analysis.

[0126] Figures 3A-3E show the concentrations of various cytokines in the supernatant of TIL cells from each group after 24 hours of co-incubation with A375 or Caski cells. Significance tests in these figures are compared with the untransduced control group. Figures 3A to 3E represent cervical tumor-derived TILs.

[0127] Figures 4A-4D show the concentrations of various cytokines in the supernatant of each group of TIL cells after 24 hours of co-incubation with A375 cells. Significance tests in these figures are compared with the untransduced control group. Figures 4A to 4D represent melanoma-derived TILs.

[0128] The results showed that gene-edited TIL cells had higher IL-2, IL-6, IL-10, TNF and IFN-γ secretion capacity after co-incubation with target cells.

[0129] Example 6 Detection of the Proliferation Ability of TIL

[0130] The transduced TIL population obtained in step 1.5 of Example 1 above was plated onto a 96-well plate after transduction and expansion. Untransduced T cells served as a blank control group. For the stimulated groups, stimulation was performed using the TransACT (diameter approximately 100 to 500 nm, Miltenyi). The unstimulated group was labeled "medium," and the stimulated group was labeled "transact." After 2-3 days of stimulation, cell proliferation in each group was analyzed using a CTG kit, using the same assay as in Example 4.

[0131] The results showed that cells expressing the target polypeptide of the present invention can have better proliferation ability.

[0132] Example 7 Detection of the proliferation ability of TCRT cells

[0133] Plate the cells in a 96-well plate with an anti-CD3 antibody (e.g., OKT3) (plates plated with antibodies are marked as CD3 coating, and plates not plated with antibodies are marked as medium), incubate at 37 degrees Celsius for 2 hours, and then plate the PBMC-TCR T cells transduced with the target polypeptide of the present invention obtained in the above example on a 96-well plate after transduction and amplification. After 3 days, each group was analyzed for cell proliferation using a CTG kit. According to the instructions of the CTG kit (CellTiter-Glo Luminescent Cell Viability Assay, Promega), a CTG substrate (CellTiter-Glo Substrate) was mixed with a CTG buffer (CellTiter-Glo Buffer) to prepare a CTG reaction solution. The test cell suspension was added to a 96-well microplate at 50 μL / well, and a well containing only culture medium was set as the background value of the fluorescence. An equal volume of CTG reaction solution was added to each well, shaken on a horizontal shaker for 2 minutes, and allowed to stand at room temperature for 10 minutes to stabilize the fluorescence signal before reading the fluorescence value.

[0134] The results showed that cells expressing the target polypeptide of the present invention can have better proliferation ability.

[0135] Example 8 Detection of cytokine secretion ability of TCRT cells

[0136] Anti-CD3 was plated in a 96-well plate and incubated at 37°C for 2 hours. Then, the PBMC-TCR T cells transduced with the target polypeptide of the present invention obtained in the above example were plated in a 96-well plate after transduction and expansion. After 24-48 hours, the supernatant was collected and the secretion of cytokines IL-2, TNF, and IFN-γ was analyzed using a Cytometric Bead Array (CBA) kit (BD).

[0137] Refer to the cytokine detection kit instructions for specific methods. Specifically, reconstitute the lyophilized powder of the human Th1 / Th2 / Th17 cytokine standards with 2 mL of Assay Diluent (the standard stock solution contains 5000 pg / mL of each cytokine) and serially dilute in the following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024. Label this tube as the "Standard Tube." Remove one tube containing only Assay Diluent as a reference. Add 2 μL / Beads / well of each Capture Beads (BD), then add 10 μL / well of PE Detection Reagent and mix to prepare a mixture. Add 22 μL / well to a V-bottom 96-well plate, then add 10 μL / well of each standard and experimental group supernatant, mix, and incubate at room temperature in the dark for 3 hours. After incubation, add 200 μL Wash Buffer (BD) to each well and centrifuge at 500 g for 3 minutes. After centrifugation, add 100 μL Wash Buffer (BD) to each well and resuspend the cells for flow cytometry analysis.

[0138] The results showed that cells expressing the target polypeptide of the present invention can have enhanced cytokine secretion capacity.

[0139] Example 9 Detection of Tumor Cell Killing Ability of TCRT Cells

[0140] GFP-labeled A375 tumor cells (A375-GFP) were plated in 96-well plates. After the cells adhered, PBMC-TCR T cells transduced with the target polypeptide of the present invention obtained in the above example were added to each well for co-culture. According to the instructions of the cell apoptosis detection reagent (Incucyte Caspase-3 / 7 Green Dye for Apoptosis, Sartorius), 0.2 μL / well was added. Caspase-3 / 7 Green Dye for Apoptosis was added to 25 μL of culture medium per well to dilute Caspase 3 / 7 Green Dye. Caspase 3 / 7 activity was recorded using an Incucyte recorder (Sartorius) every 3 hours for approximately 3 days to analyze tumor cell cytotoxicity.

[0141] The results showed that cells expressing the target polypeptide of the present invention can have enhanced tumor cell killing ability.

[0142] Example 10 In vivo drug efficacy detection

[0143] The revived tumor cell lines were stably expanded in vitro for 3-5 generations under sterile conditions, and the cells were grown to the logarithmic growth phase. The cells were harvested and counted to adjust the cell density. The cells were subcutaneously inoculated into NOG mice in an SPF-grade animal room. The tumor volume was measured regularly until the tumor volume grew and the mice were randomly divided into groups.

[0144] The NT group served as a negative control, with mice intravenously injected with non-gene-edited TIL cells. Mice in each experimental group were intravenously injected with the gene-edited TIL cells of the present invention, and received adjuvant IL-2 intraperitoneal injection. The results demonstrated that cells expressing the target polypeptide of the present invention exhibited the ability to inhibit tumor growth.

[0145] 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 isolated cell comprising and expressing a multispecific antigen-binding protein, wherein an antigen-binding fragment of the multispecific antigen-binding protein specifically binds to a surface antigen of the cell.

2. The cell of claim 1, wherein the cell comprises tumor infiltrating lymphocytes (TIL).

3. The cell of claim 2, wherein the TIL is TIL derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, tumor metastatic lesions, fragments of paracancerous tissue, pleural effusion and / or peritoneal effusion and / or TIL derived from cryopreservation and resuscitation.

4. The cell of any one of claims 1-3, wherein the cell comprises a phagocyte, a lymphocyte, a neutrophil, an eosinophil and / or a basophil.

5. The cell of any one of claims 1-4, wherein the cell comprises a B cell, a T cell, a natural killer cell and / or a natural killer-like T cell (NKT).

6. The cell of any one of claims 1-5, wherein the cell comprises an αβ T cell and / or a γδ T cell.

7. The cell of any one of claims 1-6, wherein the cell is derived from an immune cell differentiated from a stem cell, wherein the stem cell comprises an induced pluripotent stem cell (iPSC), an embryonic stem cell, a bone marrow stem cell, an umbilical cord blood stem cell and / or a peripheral blood stem cell.

8. The cell of any one of claims 1-7, wherein the cell comprises an engineered immune receptor displayed on the cell surface, the engineered immune receptor comprising a T cell receptor.

9. The cell of any one of claims 1-8, wherein the multispecific antigen-binding protein comprises a first antigen-binding fragment that binds CD3.

10. The cell of claim 9, wherein the first antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, the first heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 12, and HCDR3 shown in SEQ ID NO: 13, and the first light chain variable region comprising LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, and LCDR3 shown in SEQ ID NO:

16.

11. The cell of any one of claims 9-10, wherein the first antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, the first heavy chain variable region comprising the sequence shown in SEQ ID NO: 17 or a sequence at least about 80% homologous to the sequence, and the first light chain variable region comprising the sequence shown in SEQ ID NO: 18 or a sequence at least about 80% homologous to the sequence.

12. The cell of any one of claims 9-11, wherein the first antigen-binding fragment comprises the sequence shown in SEQ ID NO: 19 or a sequence at least about 80% homologous thereto.

13. The cell of any one of claims 1-12, wherein the multispecific antigen-binding protein comprises a second antigen-binding fragment that binds a tumor-specific antigen.

14. The cell of claim 13, wherein the second antigen-binding fragment binds to an antigen selected from the group consisting of DLL3, epidermal growth factor receptor (EGFR), BCMA (B cell maturation antigen), epidermal growth factor receptor variant III (EGFRvIII), fibroblast activation protein (FAP), CD19, CD79b, CD37, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), interleukin-13 receptor α2 (IL-13Rα2), ephrin type A receptor 1 (EphA1), human epidermal growth factor receptor 2 (HER2), mesothelin, cell surface-associated mucin 1 (MUC1), or cell surface-associated mucin 16 (MUC16).

15. The cell of any one of claims 13-14, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 21, HCDR2 shown in SEQ ID NO: 22, and HCDR3 shown in SEQ ID NO: 23, and the second light chain variable region comprises LCDR1 shown in SEQ ID NO: 24, LCDR2 shown in SEQ ID NO: 25, and LCDR3 shown in SEQ ID NO:

26.

16. The cell of any one of claims 13-15, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the sequence shown in SEQ ID NO: 27 or a sequence at least about 80% homologous thereto, and the second light chain variable region comprising the sequence shown in SEQ ID NO: 28 or a sequence at least about 80% homologous thereto.

17. The cell of any one of claims 13-16, wherein the second antigen-binding fragment comprises the sequence shown in SEQ ID NO: 29 or a sequence at least about 80% homologous thereto.

18. The cell of any one of claims 13-14, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 31, HCDR2 shown in SEQ ID NO: 32, and HCDR3 shown in SEQ ID NO: 33, and the second light chain variable region comprises LCDR1 shown in SEQ ID NO: 34, LCDR2 shown in SEQ ID NO: 35, and LCDR3 shown in SEQ ID NO:

36.

19. The cell of any one of claims 13-14 and 18, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the sequence shown in SEQ ID NO: 37 or a sequence at least about 80% homologous thereto, and the second light chain variable region comprising the sequence shown in SEQ ID NO: 38 or a sequence at least about 80% homologous thereto.

20. The cell of any one of claims 13-14 and 18-19, wherein the second antigen-binding fragment comprises the sequence shown in SEQ ID NO: 39 or a sequence at least about 80% homologous thereto.

21. The cell of any one of claims 13-14, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising HCDR1 shown in SEQ ID NO:41, HCDR2 shown in SEQ ID NO:42, and HCDR3 shown in SEQ ID NO:43, and the second light chain variable region comprises LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:

46.

22. The cell of any one of claims 13-14 and 21, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the sequence shown in SEQ ID NO: 47 or a sequence at least about 80% homologous thereto, and the second light chain variable region comprising the sequence shown in SEQ ID NO: 48 or a sequence at least about 80% homologous thereto.

23. The cell of any one of claims 13-14 and 21-22, wherein the second antigen-binding fragment comprises the sequence shown in SEQ ID NO: 49 or a sequence at least about 80% homologous thereto.

24. The cell of any one of claims 13-14, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 51, HCDR2 shown in SEQ ID NO: 52, and HCDR3 shown in SEQ ID NO: 53, and the second light chain variable region comprises LCDR1 shown in SEQ ID NO: 54, LCDR2 shown in SEQ ID NO: 55, and LCDR3 shown in SEQ ID NO:

56.

25. The cell of any one of claims 13-14 and 24, wherein the second antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region comprising the sequence shown in SEQ ID NO: 57 or a sequence at least about 80% homologous thereto, and the second light chain variable region comprising the sequence shown in SEQ ID NO: 58 or a sequence at least about 80% homologous thereto.

26. The cell of any one of claims 13-14 and 24-25, wherein the second antigen-binding fragment comprises the sequence shown in SEQ ID NO: 59 or a sequence at least about 80% homologous thereto.

27. The cell of any one of claims 1-26, wherein the multispecific antigen-binding protein comprises a first antigen-binding fragment and a second antigen-binding fragment, wherein the first antigen-binding fragment and the second antigen-binding fragment are connected by a peptide linker.

28. The cell of any one of claims 1-27, wherein the multispecific antigen-binding protein comprises the sequence shown in SEQ ID NO: 30, 40, 50, and / or 60, or a sequence that is at least about 80% homologous thereto.

29. A method of producing the cell of any one of claims 1-28, comprising causing the cell to artificially express the multispecific antigen-binding protein.

30. A composition comprising the cell of any one of claims 1-28, and optionally a pharmaceutically acceptable carrier.

31. A kit comprising the cell of any one of claims 1-28 and / or the composition of claim 30.

32. Use of the cell according to any one of claims 1 to 28, the composition according to claim 30 and / or the kit according to claim 31 in the preparation of a medicament for preventing, alleviating and / or treating a disease (preferably a tumor).

33. A medicament for preventing and / or treating a disease and / or symptom (preferably a tumor), comprising the cell according to any one of claims 1 to 28, the composition according to claim 30 and / or the kit according to claim 31.

34. A method for preventing and / or treating a disease and / or symptom (preferably a tumor), comprising administering the cell of any one of claims 1 to 28, the composition of claim 30, and / or the kit of claim 31 to a subject in need thereof.

35. The cell according to any one of claims 1 to 28, the composition according to claim 30 and / or the kit according to claim 31, for use in preventing and / or treating a disease and / or symptom (preferably a tumor).

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