Antibodies or antigen-binding portions thereof and uses thereof

Abstract

The invention provides an antibody or an antigen binding part and application thereof, the antibody or the antigen binding part comprises a heavy chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.1, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.2, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.3, and optionally, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.3. One or more of HCDR1, HCDR2, HCDR3 has one, two, three, or four amino acid substitutions, additions, deletions, or combinations thereof. The invention solves the problem that unsatisfied medical requirements of a large number of malignant tumors require monoclonal antibodies of metastatic solid tumor targets with more expected pharmaceutical characteristics.

Description

[0001] This application claims the priority of a Chinese patent application with an application date of November 30, 2023, an application number of 2023116282863, and an invention title of "Antibody or Its Antigen-Binding Portion and Its Use". Technical Field

[0002] The present invention relates to the field of biotechnology, and particularly to an antibody or its antigen-binding portion and its use. Background Art

[0003] Gastric cancer is the fourth (for men) and fifth (for women) most common cause of cancer-related death in developed countries. Many cancers, especially those in advanced stages, are difficult to cure. For example, the five-year survival rate of gastroesophageal cancer is only 20 - 25%, and although the current standard treatment has positive effects, it itself brings considerable side effects. For pancreatic cancer, patients are usually diagnosed at an advanced stage, so the prognosis is quite poor, with a median survival time of less than 6 months and a five-year survival rate of less than 5.5%.

[0004] Antibody therapies have been approved in many places for the treatment of various cancers and have significantly improved the final outcomes of patients. Once bound to cancer antigens, antibodies can trigger antibody-dependent cell-mediated cytotoxicity, activate the complement system, or prevent receptors from interacting with their ligands, all of which can cause cancer cell death.

[0005] Due to the unmet medical needs of a large number of malignancies, there is a need for monoclonal antibodies against metastatic solid tumor targets with more desirable pharmaceutical characteristics.

[0006] Therefore, it is necessary to develop an antibody or its antigen-binding portion and its use to solve the above problems existing in the prior art. Summary of the Invention

[0007] The object of the present invention is to provide an antibody or its antigen-binding portion and its use, which solves the problem of the unmet medical needs of a large number of malignancies and the need for monoclonal antibodies against metastatic solid tumor targets with more desirable pharmaceutical characteristics.

[0008] To achieve the above object, the present invention provides an antibody or an antigen-binding portion thereof and its use. The antibody or an antigen-binding portion thereof comprises a heavy chain variable region. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3. The amino acid sequence of HCDR1 is as shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.3. Wherein, one or more of HCDR1, HCDR2 and HCDR3 have one, two, three or four or more amino acid substitutions, additions, deletions or combinations thereof.

[0009] The amino acid sequence of HCDR1 is as shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.3. Optionally, the amino acid sequence of HCDR1 is as shown in SEQ ID NO.4, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.6; or the amino acid sequence of HCDR1 is as shown in SEQ ID NO.7, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.8, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.9; or the amino acid sequence of HCDR1 is as shown in SEQ ID NO.10, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.11, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.12. In one embodiment, the amino acid sequence of HCDR1 is as shown in SEQ ID NO.31, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.32, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.33. In one embodiment, the amino acid sequence of HCDR1 is as shown in SEQ ID NO.34, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.35, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.36. In one embodiment, the amino acid sequence of HCDR1 is as shown in SEQ ID NO.37, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.38, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.39. The amino acid sequence of HCDR1 is as shown in SEQ ID NO.40, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.41, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.3.

[0010] Optionally, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 42 or 43, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid shown in SEQ ID NO. 42 or 43.

[0011] Optionally, the antibody or its antigen-binding portion is in the form of a scFv.

[0012] The present invention provides a nanobody, which comprises HCDR1, HCDR2 and HCDR3. The amino acid sequence of HCDR1 is as shown in SEQ ID NO. 1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO. 2, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO. 3. Among them, one or more of HCDR1, HCDR2, and HCDR3 have one, two, three, four or more amino acid substitutions, additions, deletions, or combinations thereof.

[0013] The amino acid sequence of the HCDR1 is as shown in SEQ ID NO.1, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.3. Optionally, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.4, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.5, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.6; or the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.7, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.8, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.9; or the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.10, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.11, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.12; or the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.31, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.32, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.33; or the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.34, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.35, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.36; or the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.37, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.38, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.39.

[0014] Optionally, the nanobody comprises the amino acid sequence shown in SEQ ID NO.42 or 43 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acids shown in SEQ ID NO.42 or 43.

[0015] The present invention provides a chimeric antigen receptor, which comprises the antibody or its antigen-binding part or the nanobody;

[0016] The chimeric antigen receptor further comprises a signal peptide;

[0017] The chimeric antigen receptor further comprises a hinge region;

[0018] The chimeric antigen receptor further comprises a transmembrane domain;

[0019] The chimeric antigen receptor further comprises a co-stimulatory signal domain;

[0020] The chimeric antigen receptor further comprises an intracellular signaling domain;

[0021] The signal peptide is selected from the signal peptides of the following molecules: the α-chain and β-chain of the T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6;

[0022] The hinge region is selected from the hinge regions of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, IL-11 receptor;

[0023] The transmembrane domain is selected from the transmembrane domains of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, CD3ε, CD8α, IL-2 receptor, IL-7 receptor, IL-11 receptor;

[0024] The co-stimulatory signal domain is selected from the co-stimulatory signal domains of the following molecules: 4-1BB, CD28, ICOS, CD27, CD19, CD4, CD8α, CD8β, HVEM, LIGHT, CD40, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, CD278;

[0025] The intracellular signaling domain is selected from the intracellular signaling domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, FYN.

[0026] Optionally, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 17 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid shown in SEQ ID NO. 17. Optionally, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 43 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid shown in SEQ ID NO. 43

[0027] The present invention provides a nucleic acid encoding the antibody or its antigen-binding portion, or the nanobody, or the chimeric antigen receptor.

[0028] The present invention provides an expression vector comprising the nucleic acid.

[0029] The present invention provides a host cell comprising the nucleic acid or the expression vector, and the host cell includes T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof.

[0030] The present invention provides an application of the host cell, and the host cell includes CART cells or SNR CART cells. Among them, the CART cells are co-cultured with the cells of metastatic solid tumors to promote antigen presentation so as to kill T cells, or the SNR CART cells are co-cultured with T cells to promote the formation of memory T cells and reduce exhausted T cells.

[0031] The present invention provides a pharmaceutical composition comprising the antibody or its antigen-binding portion, or the nanobody, or the chimeric antigen receptor, or the nucleic acid, or the expression vector, or the host cell, and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises one or more of Pembrolizumab, Paclitaxel, Ramucirumab, Docetaxel.

[0032] The present invention provides the use of the antibody or its antigen-binding portion, or the anti-Claudin 18.2 nanobody, or the chimeric antigen receptor, or the nucleic acid, or the expression vector, or the host cell, or the pharmaceutical composition in the preparation of a drug for treating metastatic solid tumors.

[0033] Optionally, the metastatic solid tumors include lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, liver cancer, squamous cell carcinoma, peritoneal cancer, brain tumor, glioma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, penile cancer, anal cancer, thyroid cancer, head and neck cancer, skin cancer, osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcoma, carcinoid, eye cancer, mesothelioma, lymphocytic / lymphoblastic leukemia.

[0034] In the embodiments herein, the VHH of the antibody or its antigen-binding portion is obtained from sharks, and the nanobody is a shark-derived nanobody. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the structure of the vector of the embodiment of the present invention;

[0036] Figure 2 Schematic diagram of the results of the PCR gel of the lentivirus mycoplasma detection of the embodiment of the present invention;

[0037] Figure 3 Schematic diagram of the expression level of Claudin 18.2 after co-culture of Claudin 18.2 CAR-T cells of the embodiment of the present invention with SW480 (human colon cancer cells), HCT116 (human colon cancer cells), A549 (human non-small cell lung cancer cells) and RKO (colon cancer cells);

[0038] Figure 4 Schematic diagram of the proliferation of HCT116 cells after co-culture of CART cells containing different concentrations of Claudin 18.2 nanobodies of the embodiment of the present invention with HCT116;

[0039] Figure 5 Schematic diagram of the proliferation of HCT116 cells after co-culture of mGFP CAR-T cells and anti-Claudin 18.2 CAR-T cells of the embodiment of the present invention with HCT116;

[0040] Figure 6 Schematic diagram of the proliferation of HCT116 cells after co-culture of anti-Claudin 18.2 CAR-T cells with different ratios of the embodiment of the present invention with HCT116;

[0041] Figure 7 The TCR VDJ sequencing results are shown, and the results show that the CDR sequence and the HCDR sequence completely coincide. The CDR sequence is IVAMG (SEQ ID NO.1).

[0042] Figure 8Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence mostly overlap. The CDR sequence is TITRGGSTYYADSMKG (SEQ ID NO.2).

[0043] Figure 9 Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence partially overlap. The CDR sequence is RVEVPFMQPNDY (SEQ ID NO.3).

[0044] Figure 10 Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence partially overlap. The CDR sequence is IPVMG (SEQ ID NO.4).

[0045] Figure 11 Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence partially overlap. The CDR sequence is GISTGGTTNYGDSVKG (SEQ ID NO.5).

[0046] Figure 12 Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence partially overlap. The CDR sequence is LVVSGIGSTLEV (SEQ ID NO.6).

[0047] Figure 13 Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence partially overlap. The CDR sequence is GSIFNIP (SEQ ID NO.7).

[0048] Figure 14 Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence partially overlap. The CDR sequence is STGGT (SEQ ID NO.8).

[0049] Figure 15 Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence partially overlap. The CDR sequence is LVVSGIGSTLEV (SEQ ID NO.9).

[0050] Figure 16 Displays the TCR VDJ sequencing results, showing that the CDR sequence and the HCDR sequence partially overlap. The CDR sequence is GSIFNIPV (SEQ ID NO.10).

[0051] Figure 17Displays the TCR VDJ sequencing results, showing partial overlap between the CDR sequence and the HCDR sequence. The CDR sequence is ISTGGTT (SEQ ID NO.11).

[0052] Figure 18 Displays the TCR VDJ sequencing results, showing partial overlap between the CDR sequence and the HCDR sequence. The CDR sequence is NVLVVSGIGSTLEV (SEQ ID NO.12).

[0053] Figure 19 Displays the TCR VDJ sequencing results, showing partial overlap between the CDR sequence and the HCDR sequence. The CDR sequence is EVQLVESGGGLVQPGGSLRLSCAASGSFFR (SEQ ID NO.13). Detailed implementation manners

[0054] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts shall fall within the protection scope of the present invention.

[0055] Claudin was first reported by Shorichiro Tsukita et al. in 1998 (Furuse et al., (1998) J Cell Biol 141(7): 1539-1550), and is a family of cell surface proteins that establish paracellular barriers and control the flow of molecules between cells (Singh et al., (2010) J Oncol 2010: 541957). Claudin is an essential component of tight junctions and plays an important role in maintaining epithelial cell polarity, controlling paracellular diffusion, and regulating cell growth and differentiation. The other two major tight junction proteins are occludin and junctional adhesion molecule (JAM). Each Claudin molecule spans the cell membrane four times, with both the N-terminus and C-terminus located in the cytoplasm.

[0056] To date, 24 claudin members have been identified in mammals, among which claudin 13 is absent in humans. Different claudin members are expressed in different tissues, and their altered functions are associated with cancer formation. The expression changes of Claudin 1, Claudin 18, and Claudin 10 are respectively associated with colorectal cancer, gastric cancer, and hepatocellular carcinoma. Therefore, claudin has become a promising target for therapeutic strategies (Swisshelm et al., (2005) Adv Drug Deliv Rev 57(6): 919-928).

[0057] Claudin 18, also known as CLD18, has two subtypes. Claudin 18.1 is selectively expressed in normal lung and gastric epithelium. The expression of Claudin18.2 is also highly restricted in normal tissues, limited to the differentiated short-lived cells of the gastric epithelium, and particularly absent from the gastric stem cell region. However, Claudin 18.2 is abundantly present in a large proportion of primary gastric cancers and their metastases and plays an important role in their malignancy. For example, frequent ectopic activation of Claudin18.2 has been found in pancreatic, esophageal, ovarian, and lung tumors. Claudin 18.2 contains an exposed extracellular loop that can be used for monoclonal antibody binding, and CLDN18.2 antibodies have been used in cancer treatment research. For example, Claudiximab (IMAB362), a chimeric IgG1 antibody against Claudin 18.2 developed by Ganymed, showed encouraging efficacy in phase I and II clinical trials for the treatment of advanced gastroesophageal cancer (Sahin et al., (2018) Eur J Cancer 100: 17-26).

[0058] The term "Claudin 18.2" refers to Claudin 18 subtype 2. The term "Claudin 18.2" includes variants, homologs, orthologs, and paralogs. For example, an antibody specific for the human Claudin 18.2 protein can, in some cases, cross-react with the Claudin 18.2 protein of species other than humans, such as monkeys. In other embodiments, an antibody specific for the human Claudin 18.2 protein can be completely specific for the human Claudin18.2 protein without showing cross-reactivity with other species or other types, or can cross-react with the Claudin 18.2 of some other species but not all other species.

[0059] The term "human Claudin 18.2" refers to a Claudin 18.2 protein having a human amino acid sequence, such as the amino acid sequence with Genbank accession number NM-001002026.

[0060] As used herein, the term "antibody" includes full-length antibodies and any antigen-binding fragment thereof (i.e., antigen-binding portion) or single chains thereof. A full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being interconnected by disulfide bonds. Each heavy chain consists of a heavy-chain variable region (abbreviated VH) and a heavy-chain constant region. The heavy-chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light-chain variable region (abbreviated VL) and a light-chain constant region. The light-chain constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity-determining regions (CDRs), which are separated by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0061] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., the Claudin 18.2 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, and CH1 domains; (ii) F(ab′) 2Fragment, a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bridge; (iii) Fd fragment consisting of VH and CH1; (iv) Fv fragment consisting of VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementarity determining region (CDR); and (vii) nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked by recombinant methods via a synthetic linker that makes them a single protein chain, where the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single-chain antibodies are also intended to be included in the "antigen-binding portion" of the term antibody. These antibody fragments are obtained by conventional techniques known to those skilled in the art, and the fragments can be screened and applied in the same manner as intact antibodies.

[0062] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule preparation consisting of a single molecule. The monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0063] The term "chimeric antibody" refers to an antibody obtained by combining non-human genetic material with human genetic material. Or more generally, a chimeric antibody refers to an antibody having genetic material of one species and genetic material of another species.

[0064] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to antibody variants naturally occurring in the human body.

[0065] The terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds an antigen".

[0066] As used herein, an antibody that "specifically binds human Claudin 18.2" refers to an antibody that binds to human Claudin 18.2 protein (and possibly Claudin 18.2 proteins from one or more non-human species) but does not substantially bind to non-Claudin 18.2 proteins. Preferably, the antibody binds to human Claudin 18.2 protein with "high affinity", i.e., KD is 1.0x10 -8M or less, more preferably 5.0x10 -9 M or less.

[0067] The term "EC 50 ", also known as the half-maximal effective concentration, refers to the antibody concentration that elicits a 50% response between the baseline and the maximal response after a certain exposure time.

[0068] It is well known in the art that the CDR3 domain, independent of the CDR1 and / or CDR2 domains, can alone determine the binding specificity of an antibody to a homologous antigen, and it can be predicted that multiple antibodies with the same binding specificity can be generated based on this CDR3 sequence. See, for example, Klimka et al., British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol. Biol. 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. US.A. 95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533 (1995); Ditzel et al., J. Immunol. 157:739-749 (1996); Berezov et al., BIA journal 8:Scientific Review 8(2001); Igarashi et al., J. Biochem(Tokyo) 117:452-7 (1995); Bourgeois et al., J. Virol 72:807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. U.S.A. 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000); U.S. Pat. Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. Each of these references is hereby incorporated by reference in its entirety.

[0069] In another embodiment, the antibody of the present application comprises a heavy chain variable region CDR2 and at least a heavy chain and / or a light chain variable region CDR3, or another heavy chain and / or light chain variable region CDR3, wherein the antibody is capable of specifically binding to a human metastatic solid tumor target. Preferably, these antibodies (a) competitively bind to the metastatic solid tumor target; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have a binding affinity similar to that of the metastatic solid tumor target of the present application. In another embodiment, the antibody may also comprise a light chain variable region CDR2, or another light chain variable region CDR2, wherein the antibody is capable of specifically binding to a human metastatic solid tumor target. In another embodiment, the antibody of the present application may comprise a heavy chain / light chain variable region CDR1, or another heavy chain and / or light chain variable region CDR1, wherein the antibody is capable of specifically binding to a human metastatic solid tumor target.

[0070] In another aspect, the present application provides nucleic acid molecules encoding the heavy chain and / or light chain variable regions or CDRs of the antibodies of the present application. The nucleic acid may be present in intact cells, in cell lysates, or in a partially purified or substantially pure form. The nucleic acid is "isolated" or "presented as substantially pure" when purified from other cellular components or other contaminants such as other cellular nucleic acids or proteins by standard techniques. The nucleic acid of the present application may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0071] The nucleic acid of the present application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, which will be further described below), the cDNAs encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be retrieved from the gene library.

[0072] Preferred nucleic acid molecules of the present application include those encoding the VH and VL sequences or CDRs of monoclonal antibodies against metastatic solid tumor targets. Once the DNA fragments encoding the VH and VL fragments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein means that the two DNA fragments are joined together such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0073] In some embodiments of the present invention, the metastatic solid tumor target includes any one of EpCAM, Claudin 18.2, and AXL.

[0074] The antibodies (compositions, bispecific molecules, and immunoconjugates) of the present application have various in vitro and in vivo uses, which are related to, for example, the diagnosis, treatment, and / or prognosis of cancer. The antibodies can be administered to human subjects to, for example, inhibit tumor growth in vivo. In the diagnosis and prognosis of cancer, a target tissue sample can be collected and contacted with the antibodies of the present application. If a certain amount of EpCAM, Claudin 18.2, or AXL is detected in certain regions or cell types, then the subject can be diagnosed with cancer, and an increase / decrease in the expression of EpCAM, Claudin 18.2, or AXL indicates the development / alleviation of cancer.

[0075] In some embodiments of the present invention, the metastatic solid tumors include lung cancer, lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, liver cancer, squamous cell carcinoma, peritoneal cancer, brain tumor, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, rectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, penile cancer, anal cancer, thyroid cancer, head and neck cancer, skin cancer, osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcoma, carcinoid, eye cancer, mesothelioma, lymphocytic / lymphoblastic leukemia.

[0076] In some specific embodiments of the present invention, the lung cancer includes small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma or lung squamous cell carcinoma; the gastric cancer includes gastrointestinal cancer; the liver cancer includes hepatoblastoma, hepatocellular carcinoma or hepatoma; the brain tumor includes glioblastoma / multiform glioblastoma (GBM), non-glioblastoma brain tumor or meningioma; the glioma includes ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma or mixed glioma, and the oligodendroglioma includes oligoastrocytoma; the bladder cancer includes urothelial carcinoma; the kidney cancer includes renal rhabdomyoma; the anal cancer includes anal squamous cell carcinoma; the head and neck cancer includes nasopharyngeal carcinoma; the skin cancer includes melanoma or squamous cell carcinoma; the soft tissue sarcoma includes rhabdomyosarcoma, fibrosarcoma, Kaposi's sarcoma; the eye cancer includes retinoblastoma; the lymphocytic / lymphoblastic leukemia includes acute lymphocytic / lymphoblastic leukemia (ALL) of T cell lineage and B cell precursor lineage, chronic lymphocytic / lymphocytic leukemia (CLL), acute myeloid / myeloblastic leukemia (AML), including mast cell leukemia, chronic myeloid / myelocytic / myeloblastic leukemia (CML), hairy cell leukemia (HCL), Hodgkin's disease, non-Hodgkin's lymphoma, chronic myelomonocytic leukemia (CMML), follicular lymphoma (FL), diffuse large B cell lymphoma (DLCL), mantle cell lymphoma (MCL), Burkitt's lymphoma (BL), mycosis fungoides, Sézary syndrome, cutaneous T cell lymphoma, mastocytoma, medulloblastoma, nephroblastoma, solitary plasmacytoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, central nervous system tumors, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocytosis, gallbladder cancer, idiopathic myelofibrosis, and pediatric cancers, and the pediatric cancers include pediatric sarcomas, and the pediatric sarcomas include neuroblastoma, rhabdomyosarcoma and osteosarcoma.

[0077] Considering the ability of the metastatic solid tumor target antibody of the present application to inhibit the proliferation and survival of cancer cells, the present application provides a method for inhibiting the growth of tumor cells in a subject, including administering the antibody of the present application to the subject, thereby inhibiting tumor growth in the subject. Non-limiting examples of tumors that can be treated with the antibody of the present application include, but are not limited to, pancreatic cancer, gastric cancer, intestinal cancer, esophageal cancer, liver cancer, ovarian cancer, lung cancer and bladder cancer, primary or metastatic. In addition, the growth of refractory or recurrent malignant tumors may be inhibited by the antibody of the present application.

[0078] In some embodiments of the present invention, the VHH of the antibody or its antigen-binding portion is obtained from sharks, and the nanobody is a shark-derived nanobody.

[0079] In some embodiments of the present invention, the antibody or its antigen-binding portion comprises an amino acid sequence containing HCDR1 as shown in SEQ ID NO.1, an amino acid sequence of HCDR2 as shown in SEQ ID NO.2, and an amino acid sequence of HCDR3 as shown in SEQ ID NO.3. In some embodiments of the present invention, the antibody or its antigen-binding portion comprises an amino acid sequence of FR1 as shown in SEQ ID NO.13, an amino acid sequence of FR2 as shown in SEQ ID NO.14, an amino acid sequence of FR3 as shown in SEQ ID NO.15, and an amino acid sequence of FR4 as shown in SEQ ID NO.16. In some embodiments of the present invention, the amino acid sequence of the antibody or its antigen-binding portion is as shown in SEQ ID NO.17. In some embodiments of the present invention, the nucleotide sequence of the antibody or its antigen-binding portion is as shown in SEQ ID NO.18. In some embodiments of the present invention, the antibody or its antigen-binding portion comprises an amino acid sequence containing the amino acid sequence of HCDR1 as shown in SEQ ID NO.31, the amino acid sequence of HCDR2 as shown in SEQ ID NO.32, and the amino acid sequence of HCDR3 as shown in SEQ ID NO.33. In some embodiments of the present invention, the antibody or its antigen-binding portion comprises the amino acid sequence of HCDR1 as shown in SEQ ID NO.34, the amino acid sequence of HCDR2 as shown in SEQ ID NO.35, and the amino acid sequence of HCDR3 as shown in SEQ ID NO.36. In some embodiments of the present invention, the antibody or its antigen-binding portion comprises the amino acid sequence of HCDR1 as shown in SEQ ID NO.37, the amino acid sequence of HCDR2 as shown in SEQ ID NO.38, and the amino acid sequence of HCDR3 as shown in SEQ ID NO.39.

[0080] In some embodiments of the present invention, the antibody or its antigen-binding portion comprises an amino acid sequence of HCDR1 as shown in SEQ ID NO.4, an amino acid sequence of HCDR2 as shown in SEQ ID NO.5, and an amino acid sequence of HCDR3 as shown in SEQ ID NO.6. In some embodiments of the present invention, the antibody or its antigen-binding portion has an amino acid sequence of FR1 as shown in SEQ ID NO.19, an amino acid sequence of FR2 as shown in SEQ ID NO.20, an amino acid sequence of FR3 as shown in SEQ ID NO.21, and an amino acid sequence of FR4 as shown in SEQ ID NO.22.

[0081] In some embodiments of the present invention, the antibody or its antigen-binding portion comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO.7, an HCDR2 having the amino acid sequence shown in SEQ ID NO.8, and an HCDR3 having the amino acid sequence shown in SEQ ID NO.9. In some embodiments of the present invention, the antibody or its antigen-binding portion comprises an FR1 having the amino acid sequence shown in SEQ ID NO.23, an FR2 having the amino acid sequence shown in SEQ ID NO.24, an FR3 having the amino acid sequence shown in SEQ ID NO.25, and an FR4 having the amino acid sequence shown in SEQ ID NO.26.

[0082] In some embodiments of the present invention, the antibody or its antigen-binding portion comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO.10, an HCDR2 having the amino acid sequence shown in SEQ ID NO.11, and an HCDR3 having the amino acid sequence shown in SEQ ID NO.12. In some embodiments of the present invention, the antibody or its antigen-binding portion has an FR1 having the amino acid sequence shown in SEQ ID NO.27, an FR2 having the amino acid sequence shown in SEQ ID NO.28, an FR3 having the amino acid sequence shown in SEQ ID NO.29, and an FR4 having the amino acid sequence shown in SEQ ID NO.30.

[0083] In some embodiments of the present invention, the sequences involved are shown in Table 1.

[0084] Preparation and Screening of Nanobodies

[0085] Currently, the research on domestic nanobodies mainly focuses on camels and alpacas, which have problems such as high preparation cost, long cycle, and low efficiency. Chiloscyllium plagiosum is a new source for preparing nanobodies, with advantages such as small size, low price, and convenient experimental operation. Sanya has unique breeding conditions for Chiloscyllium plagiosum. The Deep Sea Compound Resource Center and Sanya Yazhou Bay Agriculture and Fishery Company are both located in Yazhou Bay Science and Technology City, where it is very convenient to breed Chiloscyllium plagiosum and perform antigen immunization. The Deep Sea Compound Resource Center has complete technologies, talents, and facilities for molecular biology, cell biology, antigen protein expression and purification, and antibody function research. By integrating upstream and downstream talents and technologies, it is expected to quickly establish a screening platform for shark-derived nanobodies. This project uses the above platform to prepare and screen Claudin18.2 nanobodies and conduct product development.

[0086] Previously, by optimizing the immunization method of Chiloscyllium plagiosum and screening anti-has nanobodies from Chiloscyllium plagiosum, a full-chain technology platform for antigen immunization, phage antibody library construction, antibody screening, and function identification based on Chiloscyllium plagiosum was established. Claudin18.2 nanobodies were prepared and screened through this platform, and the cell products of this project were developed.

[0087] The key technical problems to be solved in this project are as follows. The first one is the efficient screening and preparation of nanobodies. After immunizing Chiloscyllium plagiosum, IgNAR antibodies are produced. The possibility of using nanobodies derived from Chiloscyllium plagiosum as a superior and efficient source of nanobodies is studied innovatively. Given the great advantages of nanobodies, it has become an important trend in antibody drug research. In this project, Chiloscyllium plagiosum, which is easy to obtain, small in size, easy to breed, has stronger immune ability and is more suitable as an experimental animal, is used as the research object, which is more conducive to the large-scale application of shark nanobodies. This project can achieve the preparation and screening of Claudin18.2 nanobodies, conduct product research and development, and then realize the value transformation in clinical practice.

[0088] Harvest a certain number of wild Chiloscyllium plagiosum, put them into the circulating system of the industrial park for artificial breeding after isolation and artificial domestication, study the most suitable breeding conditions and the breeding model conducive to the extraction of nanobodies, and conduct research on reproductive biology, feed nutrition, etc., to form a feasibility report on the scientific breeding of Chiloscyllium plagiosum, providing guidance for the breeding of Chiloscyllium plagiosum. At the same time, a certain number of Chiloscyllium plagiosum will be randomly selected for the research of nanobodies.

[0089] Nano - antibody technology route of Chiloscyllium plagiosum 1. Breeding technology route of Chiloscyllium plagiosum: First: Isolate and domesticate the collected wild Chiloscyllium plagiosum in the breeding system, then conduct relevant breeding experiments and optimize the breeding conditions to form a report, providing guidance for the popularization of Chiloscyllium plagiosum breeding. Second: While conducting breeding research, randomly select some Chiloscyllium plagiosum to provide stable and healthy blood samples for the research of nano - antibodies. 2. Establishment of the screening technology platform for nano - antibodies, and the specific scheme design is as follows: (1) Design of the immunization scheme for Chiloscyllium plagiosum 1) According to the characteristics of different antigens, use different adjuvants to immunize Chiloscyllium plagiosum. 2) After immunization, draw the blood of Chiloscyllium plagiosum to detect the titer of specific IgNAR of Chiloscyllium plagiosum. When the IgNAR titer reaches the index, collect and isolate PBMC cells of Chiloscyllium plagiosum for subsequent experiments. (2) Construction of the Chiloscyllium plagiosum IgNAR phage library 1) Use the peripheral blood and spleen of antigen - immunized Chiloscyllium plagiosum as starting materials, extract RNA and reverse - transcribe it into cDNA. 2) Design primers according to the conserved sequence of the variable region of Chiloscyllium plagiosum IgNAR, and amplify the full set of nano - antibody - encoding genes by PCR. 3) Sub - clone the cloned full set of nano - antibody - encoding gene fragments into an appropriate vector to fuse and express the nano - antibody with the phage coat protein pIII. (3) Library panning and screening Utilize the re - amplifiable property of phages, immobilize the antigen molecule, and through several rounds of affinity adsorption - elution - amplification, pan and screen out the Chiloscyllium plagiosum nano - antibodies that can specifically bind to the antigen. (4) Expression preparation and functional verification of nano - antibodies from Chiloscyllium plagiosum 1) Clone the antibodies and their genes that can specifically bind to the antigen obtained by the previous phage antibody library technology into an Escherichia coli expression vector for expression and purification. 2) Study the characteristics such as the affinity and stability of nano - antibodies from Chiloscyllium plagiosum. 3. Preparation and application development of nano - antibodies against drug targets (1) For drug targets, prepare 10 related nano - antibodies, and clarify the activity characteristics of nano - antibodies through methods such as receptor - binding kinetics testing and downstream signal analysis. (2) Deeply evaluate the biological activity of shark - derived nano - antibodies, including pharmacodynamic and pharmacological evaluations at the cellular and animal levels, and explore their application research as molecular probes and therapeutic drugs.

[0090] Optimize the immunization method of Chiloscyllium plagiosum and screen out anti - Claudin18.2 nano - antibodies from Chiloscyllium plagiosum; prepare nano - antibodies against important drug target antigens, and study and evaluate their biological activity and application value.

[0091] (1) Optimize the immunization dose, cycle and adjuvant of Chiloscyllium plagiosum, and establish an antigen immunization, phage library construction and screening platform. 1) Prepare the secondary antibody of Chiloscyllium plagiosum heavy chain antibody IgNAR to prepare for the subsequent ELISA detection; 2) Immunize Chiloscyllium plagiosum with Claudin18.2 protein. At the same time, use the prepared secondary antibody of Chiloscyllium plagiosum IgNAR to detect the titer of specific IgNAR by indirect ELISA method, determine the peak time of IgNAR antibody after immunization of Chiloscyllium plagiosum, and determine the time of booster immunization. Detect the titer of specific IgNAR after immunization 2 - 5 times respectively to determine the optimal immunization time and number of immunizations; 3) Immunize with different antigen doses, detect the titer of specific IgNAR by indirect ELISA method, and determine the optimal immunization dose; 4) Immunize with different adjuvants such as Freund's adjuvant, aluminum hydroxide gel or microsilica powder, detect the titer of specific IgNAR by indirect ELISA method, and determine the optimal immunization adjuvant. 5) Construction of a phage antibody library from Chiloscyllium plagiosum and panning of nanobodies against HSA. Phage display technology is an in vitro cloning and recombinant expression technology for single - gene manipulation, and has developed into a phage display technology system that can operate in vitro cloning and in vivo cloning and recombinant expression of multiple genes (gene clusters, genomes). Phage antibody library technology combines PCR technology and phage display technology. Its principle is to clone the gene encoding the antibody molecule or peptide segment into a specific site of the genomic DNA of filamentous phage or phagemid vector, and fuse it with the coat protein of the phage for expression, so as to be displayed on the surface of the phage. Its main feature is to unify the genotype and phenotype of a specific molecule in the same phage, that is, the coding gene of the specific protein displayed on the surface of the phage exists in this phage, so that the coding gene of the required specific protein can be easily obtained. (2) Preparation and application research of nanobodies. 1) Prepare 10 related nanobodies for drug targets with which the research team has a deep research foundation; 2) Analyze the binding kinetics between shark - derived nanobodies and related antigens; 3) Explore the binding site between nanobodies and antigens and the molecular mechanism of their activity by means of structural biology; 4) Research on the biological activity of shark - derived nanobodies and their application as molecular probes and therapeutic drugs. (3) Library panning. Utilize the re - amplifiable property of phage, immobilize the antigen molecule, and through several rounds of affinity adsorption - elution - amplification, pan out the single - domain antibody of Chiloscyllium plagiosum that can specifically bind to the antigen. (4) Expression preparation and functional verification of single - domain antibodies from Chiloscyllium plagiosum: 1) Clone the antibody and its gene that can specifically bind to the antigen obtained by panning through the previous phage antibody library technology into an Escherichia coli expression vector for expression and purification; 2) Study the characteristics such as affinity and stability of single - domain antibodies from Chiloscyllium plagiosum.

[0092] Table 1 Sequences

[0093]

[0094]

[0095]

[0096]

[0097] Example

[0098] Materials and Methods

[0099] (1) Preparation of Claudin18.2 nanobody is as follows

[0100] 1. Immune protocol design

[0101] 1.1 Selection of antigen

[0102] First, select a specific sequence of Claudin18.2 (please provide the amino acid sequence of the specific sequence, SEQ ID NO: 45) as the antigen. The commonly used antigen sequence can be an exogenous polypeptide fragment of Claudin18.2. For example, select a specific region of its extracellular domain to ensure a specific immune response is generated.

[0103] 1.2 Selection of adjuvant

[0104] For the immune response of the bamboo shark, a suitable adjuvant can be selected to enhance the immune effect. Commonly used adjuvants include:

[0105] Complete Freund's Adjuvant (CFA): Used for primary immunization, which can enhance the immune response.

[0106] Incomplete Freund's Adjuvant (IFA): Used for subsequent booster immunizations.

[0107] 1.3 Immunization

[0108] The immunization process generally follows the following steps:

[0109] Prepare the antigen: Dissolve the selected antigen and mix it well with the adjuvant.

[0110] Immunization: By subcutaneous injection, inject the mixture into the dorsal fin or abdominal muscle of the bamboo shark. Usually, multiple immunizations are carried out (such as 2 - 3 booster immunizations 2 - 4 weeks after the primary immunization) to increase antibody production.

[0111] 2. Blood collection and IgNAR titer detection

[0112] 2.1 Blood collection

[0113] After immunization, the immune response of sharks is monitored regularly. When the IgNAR titer reaches a predetermined level, blood sampling is carried out.

[0114] Anesthetize the shark: Anesthetize the shark using an appropriate anesthetic (such as MS-222).

[0115] Collect blood: Use a sterile syringe to collect blood from the caudal vein or heart of the shark, usually controlling the amount of blood collected each time within a safe range.

[0116] 2.2 Detection of IgNAR titer

[0117] Use immunological detection methods such as ELISA to evaluate the IgNAR antibody titer in shark serum:

[0118] Prepare the plate: Coat the Claudin18.2 antigen on the ELISA plate.

[0119] Dilute the serum sample: Dilute the collected shark serum with an appropriate buffer.

[0120] Add the sample: Add the diluted serum sample to the ELISA plate, and detect it with a specific secondary antibody after incubation.

[0121] Read the result: Use an enzyme-linked immunosorbent assay reader to measure the optical density (OD value), and calculate the IgNAR titer according to the standard curve.

[0122] 3. Isolation of PBMC cells

[0123] When the IgNAR titer reaches the standard, collect peripheral blood mononuclear cells (PBMC) from sharks:

[0124] Centrifugal separation: Place the blood sample in a centrifuge tube and use density gradient centrifugation (such as Ficoll-Paque) to isolate PBMC.

[0125] Collect cells: After centrifugation, collect the upper PBMC layer and wash it to remove residual plasma and cell debris.

[0126] Preparation process for constructing the IgNAR phage library of Chiloscyllium plagiosum

[0127] 1. Extract RNA and reverse transcribe it into cDNA

[0128] 1.1 Material preparation

[0129] Sample source: Extract peripheral blood and spleen from immunized Chiloscyllium plagiosum.

[0130] Reagents: Use an RNA extraction kit (such as TRIzol or RNeasy Kit) and a reverse transcription kit (such as SuperScript IV).

[0131] 1.2 RNA Extraction

[0132] Peripheral blood treatment:

[0133] Mix peripheral blood with normal saline and centrifuge to remove blood cells, then collect the supernatant (plasma).

[0134] Use an RNA extraction kit to extract RNA according to the instructions.

[0135] Spleen tissue treatment:

[0136] Place the spleen in cold normal saline and chop it up with a sterile scalpel.

[0137] Put the tissue sample into a centrifuge tube containing RNA extraction reagent and homogenize it thoroughly.

[0138] Extract and purify RNA according to the kit instructions.

[0139] 1.3 Reverse Transcription into cDNA

[0140] Reverse transcribe the extracted RNA using a reverse transcription kit. The specific steps are as follows:

[0141] Prepare the reverse transcription reaction system:

[0142] RNA (1 - 2 μg)

[0143] Primer (oligo(dT) or random primer, appropriate amount)

[0144] Reverse transcriptase buffer

[0145] dNTPs (each 100 μM)

[0146] Reverse transcriptase (such as SuperScript IV, appropriate amount)

[0147] Reaction conditions:

[0148] React at 50 °C for 50 minutes, then heat at 70 °C for 15 minutes to inactivate the enzyme. Storage: Store the reverse transcription product (cDNA) at -20 °C.

[0149] 2. PCR Amplification of the IgNAR Variable Region Encoding Gene

[0150] 2.1 Primer Design

[0151] Design specific primers based on the known IgNAR variable region sequence of Chiloscyllium plagiosum: Forward primer: Designed for a specific conserved region of IgNAR.

[0152] Reverse primer: Designed for another conserved region.

[0153] 2.2 PCR Amplification

[0154] Prepare the PCR reaction system:

[0155] cDNA (appropriate amount)

[0156] Designed primers (usually at a concentration of 0.1 - 0.5 μM)

[0157] dNTPs (200 μM each)

[0158] Taq DNA polymerase (appropriate amount)

[0159] PCR buffer (according to the enzyme instruction manual)

[0160] Make up to the required volume with ddH2O

[0161] PCR reaction program:

[0162] Initial denaturation: 95°C for 2 minutes Denaturation: 95°C for 30 seconds Annealing: 55 - 60°C for 30 seconds (set according to the Tm of the primer)

[0163] Extension: 72°C for 1 minute (set according to the length of the target fragment)

[0164] Perform 30 - 35 cycles, and finally extend for 5 minutes.

[0165] Product analysis:

[0166] Analyze the PCR products by agarose gel electrophoresis to confirm the band size and specificity.

[0167] 3. Subclone into the vector and fuse with phage pIII for expression

[0168] 3.1 Vector selection

[0169] Select a suitable phage display vector (such as pGEM - 3Z or other phage display vectors).

[0170] 3.2 Subcloning process

[0171] Digestion: Digest the PCR products and the vector with restriction enzymes, and select appropriate enzymes to ensure correct insertion of the fragments.

[0172] Common restriction enzymes such as EcoRI, BamHI, etc.

[0173] Ligation reaction:

[0174] Mix the digested vector and PCR products in an appropriate ratio, and add DNA ligase (such as T4 DNA ligase).

[0175] Ligation is carried out under suitable conditions (such as overnight at 16°C).

[0176] Transformation:

[0177] The ligation product is transformed into competent Escherichia coli (such as DH5α).

[0178] Transformation is carried out by heat shock method or electroporation method.

[0179] The transformed bacteria are inoculated on a medium containing selective antibiotics and cultured overnight.

[0180] Screening and identification:

[0181] Positive clones are obtained by antibiotic screening, and colony PCR or restriction enzyme analysis is performed to verify whether the inserted fragment is correct.

[0182] 3.3 Phage display

[0183] The constructed vector is transduced into M13 phage by bacterial infection.

[0184] The phage displays the fused single-domain antibody, binds to the pIII protein, and is expressed in bacteria.

[0185] (II) CAR T preparation steps

[0186] Main reagents and consumables

[0187] Cell culture reagents and consumables

[0188] DMEM (Hyclone)

[0189] RPMI (Hyclone)

[0190] FBS (Gibco)

[0191] PBS, pH 7.4 (Gibco, 10010023)

[0192] 0.25% Trypsin containing EDTA (Gibco)

[0193] Penicillin-Streptomycin-Glutamine (100X), Liquid (Gibco, 10378016) Recombinant human IL-2 protein (R&D system)

[0194] Sodium Pyruvate (100 mM) (100X) (Gibco, 11360070)

[0195] Human T cell electroporation kit

[0196] Human T Cell Kit (Lonza)

[0197] SgRNA1 - 6 (Suzhou Hongxun)

[0198] Reagents for flow cytometry

[0199] PE Anti - human CD3 OKT3 (eBioscience)

[0200] APC Anti - human CD3 OKT3 (Biolegend)

[0201] Streptavidin PE (eBioscience)

[0202] Biotin - Protein L Molecule (GenScript Biotech Corporation)

[0203] IC Fixation Buffer (Invitrogen)

[0204] Reagents for isolation of human CD3+ T cells

[0205] Ficoll - Paque TM PLUS Media (GE Healthcare, 17144002)

[0206] Human T Cell Isolation Kit (Stemcell techonologies)

[0207] EasySepTM Buffer (Stemcell technologies)

[0208] Reagents for virus packaging and transfection

[0209] Opti - MEM Reduced Serum Meidum (Gibco, 31985070)

[0210] Lentiviral expression envelope plasmid psPAX2 (GE Healthcare)

[0211] Lentiviral expression envelope plasmid pMD.2G (Hanheng Biotech Co., Ltd.)

[0212] CAR plasmid

[0213] Polybrene (Sigma - Aldrich)

[0214] Lipofectamine TM Stem Transfection Reagent(Invitrogen)

[0215] Cell killing experiment consumables

[0216] Enhanced Cell Counting Kit-8 / Enhanced CCK-8 Kit (Beyotime)

[0217] Culture medium preparation

[0218] Complete medium for 293T cells and HCT116 cells (DMEM medium + 10% serum + 1% double antibody)

[0219] Complete medium for human PBMC and CD3+ T cells (RPMI medium + 10% serum + 1% double antibody + 10 ng / mL IL-2 + 1 mM Sodium Pyruvate)

[0220] 0.25% Trypsin containing 0.02% EDTA (Gbico)

[0221] hPBMC cell cryopreservation solution (90% calf serum + 10% DMSO)

[0222] Cell cryopreservation solution (50% serum + 40% medium + 10% DMSO)

[0223] Experimental reagent preparation

[0224] FAC Buffer (PBS + 2% FBS)

[0225] 75% ethanol

[0226] Main instruments and equipment

[0227] xCElligence RTCA DPlus (ACEA Biosciences)

[0228] Protein and nucleic acid concentration detector NanoDrop2000 (Thermo Fisher)

[0229] CP100NX ultracentrifuge (Hitachi)

[0230] CytoFlex S flow cytometer (Beckman)

[0231] 5% CO2 constant temperature cell culture (S71 Thermo Fisher)

[0232] 10 cm and 6 cm cell culture dishes (Corning)

[0233] 12-well plate, 24-well plate, 6-well plate (Corning)

[0234] 1.5 mL, 2 mL, 15 mL, 50 mL centrifuge tubes (KIRGEN)

[0235] Inverted microscope (Olympas)

[0236] Cell counting chamber (Thermo Fisher)

[0237] Low-temperature ultra-high-speed centrifuge D3024R (Scilogex)

[0238] Bench-top high-speed centrifuge D1008 (Scilogex)

[0239] Vortex mixer MX-s (Scilogex)

[0240] Electric thermostatic water bath DK-8D (Jinghong)

[0241] Water purifier (Milli-Q)

[0242] Biological safety cabinet (Heal force)

[0243] Microbalance (ME204 METTLER TOLEDO)

[0244] Pipette (Eppendorf & Pipetman)

[0245] Ultra-low temperature freezer (Thermo Fisher)

[0246] Vernier caliper (Shanghai Tool Works Co., Ltd.)

[0247] 2. Experimental procedures

[0248] 2.1 Clone the CDR sequence into the CAR vector

[0249] 1. Design primers: Design specific primers based on the CDR sequence of the VHH nanobody extracted from sharks. The 5'-end of the primers should contain restriction enzyme cleavage sites suitable for viral vectors to facilitate subsequent cloning.

[0250] 2. PCR amplification: Use the designed primers and high-fidelity DNA polymerase to perform PCR amplification on the CDR sequence of the VHH nanobody to obtain a sufficient amount of DNA.

[0251] 3. Restriction Enzyme Digestion: Perform restriction enzyme digestion on the PCR product and the CAR-T viral vector plasmid using the same enzyme as in primer design. Ensure that the restriction enzyme digestion reaction proceeds fully, usually by incubating at 37°C for 1 - 2 hours.

[0252] 4. Purify the PCR Product: Separate the PCR amplification product by gel electrophoresis, and extract and purify the target fragment by gel excision to remove primer and enzyme residues.

[0253] 5. Ligation Reaction: Mix the purified CDR sequence and the digested viral vector plasmid, add DNA ligase for the ligation reaction, and usually incubate at room temperature for 1 hour.

[0254] 6. Transformation: Transform the ligation product into competent cells (such as E. coli) and culture on selective medium to screen for successfully transformed recombinant strains.

[0255] 7. Screen for Positive Clones: Verify the screened strains by colony PCR or restriction enzyme digestion analysis to confirm successful cloning of the CDR sequence of the VHH nanobody.

[0256] 8. Amplification and Purification: Perform large-scale culture on the confirmed positive clones, extract plasmid DNA for subsequent functional verification and cell transduction experiments.

[0257] 2.2 Virus Packaging Method

[0258] Resuscitate 293T cells frozen in liquid nitrogen and passage them 2 - 3 times. Culture them in a 10 cm culture dish with DMEM complete medium. When the cell density reaches about 70% of the culture dish area, replace the culture medium with DMEM medium without serum and antibiotics. Transfect 293T cells according to the steps provided by Lipofectamine TM Stem Transfection Reagent. Add 5 μg of pMD.2G, 10 μg of psPAX2, and 10 μg of the CAR plasmid. After 24 hours, replace the culture medium with DMEM medium containing 20% serum and no antibiotics, and collect the viral supernatant at 48 hours and 72 hours (replace with fresh culture medium after collecting the viral supernatant at 48 hours). After collection, filter through a 0.45 μm filter, place in a 30 ml ultracentrifuge tube, centrifuge at 4°C, 72000g for 2 hours, remove the supernatant, concentrate by 40 times, resuspend the virus with an appropriate amount of serum-free RPMI medium, and store at -80°C in the refrigerator after collection.

[0259] 2.2 Cell Line Culture and Treatment Method

[0260] 2.2.1 Cell Culture Method

[0261] HCT116 cells: Cultured in the prepared DMEM complete medium in an incubator at 37°C, 5% CO2 and saturated humidity. These are adherent cells. When the cells need to be used or when the area occupied by the cells reaches more than 70% of the culture dish, the adherent HCT116 cells are digested with 0.25% trypsin containing EDTA at 37°C for 1 - 2 minutes, the digestion is terminated by adding the medium at a 1:1 ratio, centrifuged at 1000 rpm at 24°C for 5 minutes, the supernatant is discarded and the cells are collected. If subculture is needed, the cells are resuspended according to the required number of cells and then added to the fresh DEME complete medium.

[0262] Human peripheral blood mononuclear cells PBMC / human CD3+ T cells: Cultured in the prepared RPMI complete medium in an incubator at 37°C, 5% CO2 and saturated humidity. These are suspension cells. When the cells need to be used or when there are a large number of cells, the culture solution is pipetted and collected, centrifuged at 600g at 24°C for 5 minutes, the supernatant is discarded, and the cells are collected. If subculture is needed, the cells are resuspended and then added to the fresh RPMI complete medium.

[0263] 2.2.2 Isolation of T cells from human blood

[0264] Take the blood sample of healthy volunteers into a sterile 50 ml centrifuge tube, dilute it 2 times with PBS, and then slowly add the diluted blood along the tube wall to the Ficoll reagent (diluent: Ficoll = 3:1), centrifuge at 800g for 30 minutes, adjust the acceleration of the centrifuge to 2 and the deceleration to 1, after centrifugation, aspirate the middle white flocculent cell layer into a 50 ml centrifuge tube. This layer is the human peripheral blood mononuclear cell (PBMC) layer. Subsequently, dilute the cell solution with an appropriate amount of PBS, centrifuge at 400g for 15 minutes, discard the supernatant, resuspend the cells with EasySepTM Buffer, and isolate human T cells according to the steps provided by the Human T Cell Isolation Kit, and culture them in the RPMI complete medium.

[0265] 2.2.3 Construction of CD3-T cells

[0266] Take the isolated T cells, according to Human T Cell Kit provided steps, electrotransfect the GFP gene (as the control group) and the sgRNA and Cas9 protein targeting CD3 knockout (experimental group) into T cells respectively. Observe the expression of the GFP gene under a fluorescence microscope the next day, and detect the CD3 expression of T cells by flow cytometry.

[0267] 2.2.4 Construction of CAR-T cells

[0268] Virus transfection of CAR gene: Use the virus packaged by Hanheng Biotechnology for transfection: virus titer: 2.5×108 TU / ml. Take PBMC (CD3+ T cells) and place them in a 24-well plate for transfection. The transfection system is as follows:

[0269] GroupPolybreneVirusIL-2Total volume

[0270] Blank control000.5 μL250 μL

[0271] Experimental group0.5 μL20 μL

[0272] After adding the reagents according to the system, centrifuge at room temperature at 200g for 1 h, place it in an incubator for continued culture. After 4 h, make up the liquid volume to 500 μl. The next day, centrifuge, change the liquid, and take the cells for flow cytometry to detect the CAR expression.

[0273] Electroporation: According to Human T Cell Kit provided steps, electroporate GFP (as the control group) and CAR (experimental group) genes into T cells respectively. The next day, observe the GFP gene expression under a fluorescence microscope and detect the CAR expression by flow cytometry.

[0274] 2.3 CAR-T cell killing experiment

[0275] CCK8 assay for cell killing experiment: Spread 100 μL of HCT116 cell suspension with a density of 5×104 cells / mL onto a 96-well plate and culture it in an incubator. After the cells are completely adherent, discard the supernatant. Add the successfully transfected CAR-T cells to each well at different ratios and simultaneously add 10 μL of CCK8 solution. Mix gently. After incubation for a period of time, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value at 450 nm.

[0276] RTCA (real-time cell analyzer without labels) assay for cell killing experiment: Spread 100 μL of HCT116 cell suspension with a density of 5×104 cells / mL onto a special well plate with an electronic chip and culture it in an incubator. Measure the cell proliferation and adhesion by RTCA instrument. After the cells are completely adherent, add the successfully transfected CAR-T cells to each well at different ratios and continuously measure the proliferation of HCT116 cells.

[0277] 2.4 Flow cytometry

[0278] Detection of CAR expression: Collect the transfected cells, centrifuge at 800 g for 5 min at 4°C, discard the supernatant, wash the cells with 200 μL of PBS, centrifuge under the same conditions, resuspend the cells with 100 μL of PBS, add 5 μL of Protein L, incubate at 4°C for 45 min, centrifuge under the same conditions again, resuspend the cells with 100 μL of PBS, add 4 μL of PE Streptavidin, incubate at 4°C for 45 min in the dark. After that, wash the cells with 100 μL of PBS to remove non-specific binding, centrifuge under the same conditions, resuspend the cells with 200 μL of PBS, and then detect the CAR expression level using a flow cytometer.

[0279] Detection of CD3 expression: Centrifuge at 800 g for 5 min at 4°C, discard the supernatant, wash the cells with 200 μL of PBS, centrifuge under the same conditions, resuspend the cells with 100 μL of PBS, add 2 μL of PE Anti-human CD3 OTK3, incubate at 4°C for 45 min in the dark. After that, wash the cells with 100 μL of PBS to remove non-specific binding, centrifuge under the same conditions, resuspend the cells with 200 μL of PBS, and then detect the CAR expression level using a flow cytometer.

[0280] Flow cytometry detection of cell infiltration in animal experiments: 10 days after injecting CAR-T cells into the tail vein, sacrifice the mice by cervical dislocation, remove the tumors formed by subcutaneous HCT116 cells in the mice with sterilized forceps and scissors, and immerse them in PBS containing 2% FBS. Subsequently, moisten with PBS containing 2% FBS, and then grind the tumors successively through 30 μm and 70 μm filters to obtain a single-cell suspension of the tumors in an EP tube. Centrifuge at 800 g at room temperature, wash the cells with an appropriate amount of PBS, centrifuge again, resuspend the cells, add the corresponding CD3, CD4, and CD8 antibody dyes, incubate at 4°C in the dark for 45 min, wash the cells with PBS, and then measure using a flow cytometer.

[0281] (III) Affinity determination experimental materials and methods

[0282] Key reagents and consumables

[0283] BSA (purchased from Jackson ImmunoResearch), Tween 20 (purchased from Sinopharm), 1×PBS (purchased from Yuanpei), Probes (purchased from Gator), ultrapure water (prepared in the laboratory), multi-color Max plate (purchased from Gator), sucrose (purchased from Sigma).

[0284] Instrument: Label-free biomolecular analyzer Gator (purchased from Gator).

[0285] Methods and procedures

[0286] Turn on the GATOR instrument and related software, and select the Kientics experimental mode.

[0287] The analysis procedure is as follows in the table

[0288] Table 2. GATOR operation method

[0289]

[0290] The amino acid and DNA sequences of the obtained VHH antibodies are as follows:

[0291] VHH antibody 1 DNA sequence

[0292] CAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATC

[0293] TCTGAGACTGTCTTGTGCCGCCAGCGGCAGCATCTTCAACATCCCTGTGAT

[0294] GGGCTGGTACAGACAGGCCCCTGGCAAACAGAGAGAGCTGGTTGCCGGA

[0295] ATCTCTACCGGCGGCACCACCAATTACGGCGACTCTGTGAAGGGCAGATTC

[0296] ACCATCAGCCGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAG

[0297] CCTGAAGCCTGAGGACACCGCCGTGTACTACTGCAACGTGCTGGTGGTGT

[0298] CTGGCATCGGCAGCACACTGGAAGTTTGGGGCCAGGGCACACTGGTCACAGTGTCATCT(SEQ IDNO.44)

[0299] VHH antibody 1 amino acid sequence

[0300]

[0301] The VHH antibodies shown in Table 3 and Table 4 were also obtained. The full-length sequences of the VHH antibodies in Table 3 and Table 4 are as follows:

[0302] VHH antibody 2 (Claudin 18.2 nanobody) amino acid sequence

[0303] EVQLVESGGGLVQPGGSLRLSCAASGSFFRIVAMGWYRQAPGKGRELVATITRGGSTYYADSMKGRST ISRDNAKNTVYLQMNSLKPEDTAVYYCNVRVEVPFMQPNDYWGQGTLVTVSS (SEQ ID NO.42)

[0304] Amino acid sequence of VHH antibody 3

[0305] QVQLVESGGGLVQPGGSLRLSCAASGSIFNIPVMGWYRQACLAVHPGKQ RELVAGISTGGTTNYGDSVKGRFTISRDNAKNTVYLCLAVHQMNSLKPEDTA VYYCNVLVVSGIGSTLEVWGQGTLVTVS_(SEQ IDNO.43)

[0306] DNA sequence of VHH antibody 3

[0307] CAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCAGCATCTTCAACATCCCTGTGATGGGCTGGTACAGACAGGCCCCTGGCAAACAGAGAGAGCTGGTTGCCGGAATCTCTACCGGCGGCACCACCAATTACGGCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCAACGTGCTGGTGGTGTCTGGCATCGGCAGCACACTGGAAGTTTGGGGCCAGGGCACACTGGTCACAGTGTCATCT(SEQ IDNO.44)

[0308] Example 1: Preparation of overexpression lentiviral vector

[0309] Figure 1 For the vector plasmid of the present invention, refer to Figure 1 , the restriction enzyme sites and the sequence of Claudin 18.2 nanobody were provided by Professor Zhu Di of Fudan University, and the synthesis process of the vector plasmid was provided by Hanheng Biotechnology.

[0310] Plasmid extraction

[0311] After successful sequencing, according to the project requirements, arrange for bacterial liquid amplification and perform plasmid extraction and purification. The plasmid extraction protocol shall be based on the instructions of the extraction kit. The extracted plasmid needs to pass QC verification before being used for cell transfection.

[0312] Note: The principle of plasmid QC is that the concentration is greater than 200 ng / μL, and the ratio of 260-280 is between 1.8-2.0 (the detailed data varies according to different kits).

[0313] Example 2 Lentivirus Packaging and Quality Detection

[0314] CAR Amino Acid Sequence

[0315] MLLLVTSLLLCELPHPAFLLIP EVQLVESGGGLVQPGGSLRLSCAASGSFFR IVAMG WYRQAPGKGRELVA TITRGGSTYYADSMKG RSTISRDNAKNTVYLQMNSLKPEDTAVYYCNV RVEVPFMQPNDY WGQGTLVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO.17, the underlined part is the signal peptide, and the underlined and bold part is the CDR sequence)

[0316] CAR DNA Sequence

[0317]

[0318] The three-plasmid lentiviral system includes the following plasmids: a vector plasmid carrying the target gene, a viral packaging auxiliary plasmid (psPAX2 vector), and a viral packaging auxiliary plasmid (pMD2G vector, purchased from Hanheng Biotech).

[0319] Packaging cell line: 293T, the packaging cell of lentivirus, is an adherent-dependent epithelial-like cell, and the growth medium is DMEM (containing 10% FBS). The adherent cells grow and proliferate to form a monolayer of cells after culturing.

[0320] Strain: Escherichia coli strain DH5-α, which is used to amplify the lentiviral vector and the auxiliary packaging vector plasmid.

[0321] Three-plasmid system, pSPAX2, pMD2G and shuttle plasmid (carrying the target gene).

[0322] Lentivirus packaging, concentration and purification

[0323] Passage 293T cells in advance for transfection (provided that the cells have been cultured to meet the requirements of subsequent transfection experiments). After the operation, place them in an incubator at 37 °C and 5% carbon dioxide;

[0324] Observe the cell density before transfection. When the confluence rate reaches 70-80%, transfection can be carried out;

[0325] Prepare the lipid transfection complex. The components of the complex for transfecting a 100 mm dish are as follows:

[0326] Plasmids and Reagents Dosage pSPAX2 10 μg pMD2G 5 μg Shuttle Plasmid 10 μg <![CDATA[Lipofiter TM Dosage]]> 75 μL

[0327] Note: Lipofiter TM The transfection reagent is a product of Hanheng Biotech. For the usage instructions, refer to the Lipofiter TM Instruction manual (Appendix 1).

[0328] After mixing the lipid transfection complex, incubate it at room temperature for 15 min and then slowly add it dropwise to the 293T cells, and culture them in a cell incubator at 37 °C and 5% CO2;

[0329] Replace the fresh complete medium containing 10% fetal bovine serum (FBS) 16 h after transfection;

[0330] Virus collection: Collect the virus supernatant twice at 48 h and 72 h after transfection (replace with fresh complete medium after collection at 48 h). When collecting the virus at 48 h, pour the medium in the 100 mm dish into a 50 mL centrifuge tube, taking care not to let the dish wall touch the tube mouth to prevent bacterial contamination. Then, add 10 mL of fresh complete medium containing 10% fetal bovine serum (FBS), and place it gently in a constant temperature incubator at 37 °C and 5% CO2 for continued culture. When collecting the virus at 72 h, directly pour the medium in the 100 mm dish into a 50 mL centrifuge tube, again taking care not to let the dish wall touch the tube mouth to prevent bacterial contamination;

[0331] Ultracentrifugation: Centrifuge the virus supernatant in the 50 mL centrifuge tube at 2000×g for 10 min at 4 °C to remove cell debris; then collect the virus stock supernatant and place it in an ultracentrifuge tube, centrifuge at 82700×g for 120 min at 4 °C, resuspend the virus pellet with complete medium, and finally aliquot the ultracentrifugation resuspension into sterilized virus tubes.

[0332] Example 3 Lentivirus Quality Detection

[0333] Sterility Test

[0334] Detection method: Take 10 μL of the virus and add it to Hela cells in a 96-well plate for verification. After culturing for 24 h, examine under a microscope:

[0335] QC standard: The medium should be clear and transparent, there should be no obvious particles in the cell gaps, and there should be no contamination by any bacteria or fungi.

[0336] Mycoplasma Detection

[0337] Detection method: Take 10 μL of the virus, incubate in a water bath at 96 °C for 15 min, and then prepare a PCR reaction system in a laminar flow hood. After PCR reaction, perform electrophoresis to determine whether there is mycoplasma contamination.

[0338] QC standard: There should be no obvious bands in the PCR gel

[0339] Reference Figure 2 : If there are bands at around 500 bp, such as 1, 2, 3, 6, it indicates that the sample is contaminated with mycoplasma. Bands are absent in 4 and 5, indicating no mycoplasma contamination.

[0340] 3. Titer Detection

[0341] The lentivirus titer is detected by the dilution counting method:

[0342] Titer unit: TU / mL, which refers to the number of biologically active virus particles contained in each milliliter. "TU" is the abbreviation of "transducing units", which is translated as "transduction units" in Chinese and represents the number of viral genomes that can infect and enter target cells. IU / mL refers to the number of virus particles with integrative activity contained in each milliliter. "IU" is the abbreviation of integration units, which is translated as integration units in Chinese.

[0343] I. Cell preparation

[0344] Digest and count the 293T cells in good growth state and then dilute them to 1x10 5 / mL, add them to a 96-well plate, 100 μL per well, and prepare 6 wells for each virus. Place them in an incubator at 37 °C and 5% CO2 for culture.

[0345] II. Adding virus

[0346] On the second day, prepare 6 1.5-mL EP tubes. Add 10 μL of the virus solution to the first EP tube, and then perform 3-fold serial dilutions for a total of 6 dilution factors.

[0347] III. Adding additional culture medium

[0348] On the third day, for the wells that need to be screened with puromycin, first aspirate 100 mL of the virus-containing medium and add 100 μL of 10% FBS complete medium containing 1.5 μg / mL puromycin.

[0349] IV. Observing results and calculating titer

[0350] On the fifth day, observe the results under a fluorescence microscope. 6 hours before observing the results, replace the fresh 10% FBS complete medium. Aspirate 80 μL of the medium from the wells, then add 80 μL of fresh 10% FBS complete medium, and place them in an incubator at 37 °C and 5% CO2 for culture. After 6 hours, observe the results under a fluorescence microscope. Calculate the virus titer for the wells with a fluorescence or viable cell percentage of 10 - 50%.

[0351] Titer (TU / mL) = number of cells × percentage of positive clones × MOI (1) × virus dilution factor × 10 3 TU / mL

[0352] The lentivirus titer was detected to be 2×10 8 TU / mL.

[0353] Example 3 Extraction and culture of CAR-T cells containing Claudin 18.2 nanobody

[0354] 1. Take 10 ml of blood samples from healthy volunteers and dilute them with 20 mL of PBS;

[0355] 2. Slowly add the diluted blood along the tube wall to 10 ml of Ficoll-PaqueTM PLUS reagent (GE healthcare), and centrifuge at 800 g for 30 min at room temperature (the acceleration and deceleration rates are set to 2 and 1 respectively).

[0356] 3. After centrifugation, aspirate the serum layer, and aspirate the middle white flocculent cell layer <= 10 mL into a 50 ml centrifuge tube. This layer is the human peripheral blood mononuclear cell layer.

[0357] 4. Add 30 mL of PBS to dilute the cell suspension, and centrifuge at 400 g for 15 min at room temperature (the acceleration and deceleration rates are set to 2 and 1 respectively).

[0358] 5. Discard the supernatant, resuspend with 1 mL of PBS, filter through a 70 μm filter membrane, and centrifuge at 1500 rmp for 5 min at room temperature.

[0359] 6. Discard the supernatant, resuspend the cells with RPMI complete medium [RPMI1640 medium (Hyclone) + 10% FBS (Gibco, LifetechnologiesTM) + 1% penicillin-streptomycin mixture (Hyclone) + 1% sodium pyruvate (100 mM, Gibco)] supplemented with 200 μg / mL rhIL-2 (Biolegend) to 1 * 10^6 cells / mL.

[0360] 7. Incubate in a CO2 incubator for 2 hours, then stimulate with a human T cell activation / proliferation kit (Miltenyi Biotec GmbH), and then culture for 4 - 5 days to prepare for transfection. Passage the cells every two days.

[0361] Construction of CAR-T cells containing nanobodies against Claudin 18.2 by lentiviral transfection

[0362] First, separate the T cells using a magnet again: Collect the cells and centrifuge at 1500 rmp for 5 min at room temperature. Discard the supernatant and resuspend the cells with EasySepTM Buffer. Transfer to a 5 mL round-bottom tube, place the magnet for 5 min. Tilt the magnet and the tube together, pour out and collect the cell suspension. Centrifuge at 1500 rmp for 5 min at room temperature. Discard the supernatant and resuspend the cells with RPMI medium to 1 * 10^6 cells / mL.

[0363] Transfect using the virus packaged by Hanheng Biotech (virus titer: 1 * 108 TU / ml): Take 250 ul of the above cell suspension and place it in a 24-well plate for transfection. The transfection system is as follows:

[0364]

[0365] After adding reagents according to the system, centrifuge at room temperature at 200g for 1 hour. Place it in an incubator for further incubation. After 6 hours, add RPMI complete medium supplemented with 200 μg / mL rhIL-2 to make up the volume to 500 μL. Incubate for 48 - 72 hours to obtain CAR-T cells containing Claudin18.2, and then detect the transfection efficiency of CAR-T cells containing Claudin 18.2 by flow cytometry.

[0366] Flow cytometry

[0367] Collect the required cells. After counting, adjust the concentration to 1*10^6 / mL and take about 100 - 200 μL of cells. Centrifuge at 800g for 5 minutes at 4°C (replace with 4°C PBS, no need to cool down for centrifugation), discard the supernatant. Wash the cells with 200 μL of PBS, centrifuge under the same conditions, and resuspend the cells with 100 μL of PBS. Add 2 μL of biotinylated Protein L and incubate at 4°C for 45 minutes. After centrifugation under the same conditions, resuspend the cells with 100 μL of PBS, add 2 μL of streptavidin-PE and 2 μL of anti-CD3-APC (invitrogen), protect from light, and incubate at 4°C for 45 minutes. After completion, wash the cells with 500 μL of PBS as thoroughly as possible, centrifuge under the same conditions, resuspend the cells with 200 μL of PBS, and then detect the CAR expression level using a flow cytometer.

[0368] Real-time fluorescence quantitative PCR

[0369] Total RNA extraction:

[0370] 1. Wash the cell samples in the cell culture dish twice with PBS, add 1 mL of Trizol (invitrogen) solution, aspirate it into an RNase-free EP tube, and let it stand at room temperature for 5 minutes to lyse the cells.

[0371] 2. Add 200 μL of chloroform, vortex vigorously for 30 seconds, and let it stand at room temperature for 3 - 5 minutes.

[0372] 3. Centrifuge at 2,000g for 5 minutes at 4°C. It can be seen that it is divided into three layers. The RNA is in the upper aqueous phase and is transferred to another new RNase-free EP tube.

[0373] 4. Add an equal volume of isopropanol, gently invert 6 - 8 times to mix well, and let it stand at 4°C for 10 minutes.

[0374] 5. Centrifuge at 14,000g at high speed for 10 minutes at 4°C to collect the RNA precipitate.

[0375] 6. Add 100 μl of 75% ethanol, gently invert the EP tube, centrifuge at 12,000 g for 5 min at 4°C, and let it stand at room temperature for 10 min to air-dry; dissolve the precipitate by adding 20 μl of DEPC water (at least 15 μl) according to the amount of precipitate. Flick gently and centrifuge slightly.

[0376] Detection of total RNA purity and concentration: Use the NanoDrop2000 software to detect the purity and concentration of total RNA and record its concentration.

[0377] Reverse transcription reaction: Use Takara Prepare the following reaction system with the RT Master Mix reagent:

[0378]

[0379] After briefly centrifuging to remove air bubbles, incubate in a water bath at 37°C for 15 min and in an iron bath at 85°C for 5 s. Dilute it to 10 ng / μl by adding 40 μl of ddH2O and keep it on ice for use.

[0380] Configuration of qPCR reaction system: There are 5 groups in this experiment (blank control, negative control, experimental group (MOI = 2.5), experimental group (MOI = 5), experimental group (MOI = 10)), with 3 replicates in each group, and 2 genes (GAPDH, AXL-Ab) are measured.

[0381] Human T cell activation / proliferation kit

[0382] Configuration method: Mix 100 μl of CD3-biotin + 100 μl of CD8-biotin, add 500 μl of anti-biotin beads (vortex well before adding), add 300 μl of EasySepTM Buffer, mix well, and shake overnight at 4°C.

[0383] (Configuration should be stored and used in the dark)

[0384] Operation of adding to cells: After mixing the above-prepared solution (abbreviated as beads), take out the required amount, centrifuge at 1500 rmp for 10 min, discard the supernatant, resuspend with the medium, and add it to the cell suspension at a ratio of cell number: magnetic bead number = 2:1.

[0385] Example 4 CAR-T cells containing Claudin 18.2 nanobody have an inhibitory effect on tumor cells.

[0386] The CAR-T cells containing Claudin 18.2 nanobody prepared in Example 3 were co-cultured with SW480 (human colon cancer cells), HCT116 (human colon cancer cells), A549 (human non-small cell lung cancer cells) and RKO (colon cancer cells), and the expression level of Claudin 18.2 was detected. Refer to Figure 3 , the expression level of Claudin 18.2 nanobody was the highest when co-cultured with HCT116, followed by RKO, then A549, and finally SW480, indicating that the inhibitory ability of CAR-T cells containing Claudin 18.2 nanobody on tumor cells was different, and the killing effect on tumor cells secreting Claudin 18.2 was the greatest.

[0387] The CART cells in Example 5 had an inhibitory effect on HCT116 cells

[0388] Prepare a tumor cell suspension at 1×10 5 cells / ml (it is best to screen the cell density of the tumor cells used for the first time); add 50 μl of complete medium to the wells of E-plate 16 and gently pat it into a semi-circular shape. Place E-Plate 16 on the RTCA Station and start Step1 to detect the baseline, ensuring that the selected wells are in normal contact and the CI of all wells is below 0.063; take out E-Plate 16, add 50 μl of the well-mixed tumor cell suspension to the wells so that the number of cells in each well is 5,000 cells / 100 μl; place E-Plate 16 in the ultra-clean bench at room temperature for 30 min; place E-Plate 16 on the RTCA Station in the incubator. After the system automatically scans, start Step2; click pause after the tumor cells adhere to the wall, carefully aspirate the medium, and add control T cells and CAR-T cells with E:T ratios of 2:1, 4:1, and 8:1 respectively; place it on the RTCA Station, click start, and continue to monitor for more than 48 h.

[0389] Prepare a tumor cell suspension at a certain concentration with RPMI complete medium. Add 50 μl of RPMI complete medium to the wells of E-plate 16 and gently pat it into a semi-circular shape. Place the E-Plate 16 on the RTCA Station, detect the baseline, and ensure that the selected wells are in normal contact and the CI of all wells is below 0.063. Take out the E-Plate 16, add 50 μl of well-mixed tumor cell suspension to the wells, so that the number of cells in each well is 5,000 / 100 μl (5,000 for LCLC-103H, 10,000 for H460 and MDA231, and 20,000 for MDA453). Place the E-Plate 16 in a laminar flow hood at room temperature for 30 min, and then place it on the RTCA Station in the incubator. After the system automatically scans, start step two. Pause after the tumor cells adhere (CI continuously rises and is at least 0.15 higher than the initial CI). Carefully discard the supernatant medium, add the control T cells and CAR-T cells with E:T ratios of 2:1, 4:1, and 8:1 respectively, and supplement with a certain amount of complete medium to make the liquid volume in each well consistent, and continue to monitor for 48 h.

[0390] The detection results refer to Figure 4 、 Figure 5 and Figure 6 , the CAR T cells of the present invention (the CAR-T cells containing Claudin18.2 nanobody prepared in Example 3) have an inhibitory effect on HCT116 cells expressing Claudin 18.2.

[0391] Table 3 and Table 4 also show the antibody binding affinities of different nanobodies and the cell killing effects of the corresponding CAR T cells on HCT116 cells. The experimental methods are as described in "Experimental Materials and Methods for Affinity Determination" above and Example 5.

[0392] Table 3 and Table 4 show the VHH antibody binding affinity data and CAR T cell killing results

[0393] Table 3

[0394]

[0395] Table 4

[0396]

[0397] Example 5 Experiment on the combination of VHH CART cells and chemotherapeutic drugs

[0398] Materials

[0399] CAR-T cells containing the VHH (SEQ ID NO.17) sequence

[0400] Tumor cell lines (such as human breast cancer cell line MDA-MB-231)

[0401] Different compounds:

[0402] Pembrolizumab (A01846-40, Thermo Fisher Scientific Inc.)

[0403] Paclitaxel (HY-B0015, MedChemExpress)

[0404] Ramucirumab (947687-13-0, Meilun Bio)

[0405] Docetaxel (RP-56976, MedChemExpress)

[0406] Culture medium (such as RPMI-1640)

[0407] 96-well plates

[0408] Cell counting kits (such as CCK-8 or MTT kits)

[0409] Centrifuge

[0410] CO2 incubator

[0411] Experimental procedures

[0412] Tumor cell culture:

[0413] Culture MDA-MB-231 cells in RPMI-1640 medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator until the cells reach 70%-80% confluence.

[0414] CAR-T cell preparation:

[0415] According to the experimental design, culture CAR-T cells containing the VHH sequence to an appropriate density (about 1×10^6 cells / mL).

[0416] Combined treatment experimental design:

[0417] Seed tumor cells into 96-well plates at a density (such as 5×10^4 cells / well) and culture for 24 hours to allow them to adhere.

[0418] Compound treatment:

[0419] Treatment groups:

[0420] Add CAR-T cells (about 1×10^5 cells / well) and different compounds (determine the appropriate concentration according to the drug instruction manual) simultaneously, and set the following groups:

[0421] VHH1 + Pembrolizumab 1 mM

[0422] VHH1 + Paclitaxel 500 nM

[0423] VHH1 + Ramucirumab 1 mM

[0424] VHH1 + Docetaxel 500 nM

[0425] Set 3 replicates for each group.

[0426] Control group:

[0427] Add VHH1 (CAR-T cells) alone

[0428] Add each compound (Pembrolizumab, Paclitaxel, Ramucirumab, Docetaxel) alone

[0429] Blank control (no treatment)

[0430] Culture:

[0431] Continue to culture at 37°C and 5% CO2 for 48 - 72 hours to observe the cell proliferation and killing effects.

[0432] Cell viability detection:

[0433] After the treatment, add cell counting reagent (such as CCK-8 or MTT), and operate according to the kit instruction manual.

[0434] Measure the absorbance (OD value) of each well on an enzyme-linked immunosorbent assay (ELISA) reader to evaluate cell viability.

[0435] Data analysis:

[0436] Calculate the cell viability of each group, compare it with the control group, and analyze the significant differences between different treatment groups using statistical software.

[0437] Result recording:

[0438] Record the killing rate of cells in each group, display the results in a chart, and analyze the effects of the combination of CAR-T cells and different drugs.

[0439] Table 5

[0440]

[0441]

[0442] Example 5: Single-cell Sequencing

[0443] The single-cell sequencing of Example 5 was completed by Shanghai OE Biotech Co., Ltd. First, cell separation and counting: Immunocytes in tumor tissues were separated using magnetic beads, and the cells were counted with II Automated CellCounter, and the cell concentration was adjusted to the ideal concentration of 1×10 6 / mL. Secondly, 10x-labeled cDNA fragments: Gel beads containing barcode information were combined with the mixture of cells and enzymes, and then were encapsulated by surfactant droplets on the oil surface in the microfluidic "double cross" connection. Thirdly, library construction: The cDNA was fragmented into fragments about 200 - 300 bp using a Biorupter ultrasonic disruptor, and the library construction process of traditional second-generation sequencing such as adding sequencing adapter P5 and sequencing primer R1 was carried out. Finally, PCR amplification was performed to obtain a DNA library. Cluster generation and sequencing: The library was quantified using a Qubit instrument, and the qualified library was placed in a cBot for bridge amplification. After generating clusters, sequencing was performed using a Hiseq or Miseq sequencer. The principle is that in each cycle, dNTPs labeled with 4 fluorescent groups of A, T, G, and C are added. According to the AT and GC pairing, the corresponding dNTPs are bound to the template DNA strand through DNA polymerase, and other unbound dNTPs are washed away. The fluorescence signal is released at the binding position and is captured by a computer and converted accordingly, so as to obtain the base information at this position.

[0444] Experimental Procedures for Single-cell TCR Sequencing Analysis

[0445] Cell suspension preparation:

[0446] Adjust the freshly prepared single-cell suspension to 700 - 1200 cells / μl.

[0447] Water-in-oil preparation and cDNA library amplification:

[0448] Use 10×Genomics Chromium Next GEM Single Cell 5′Reagent Kits v2.0 for water-in-oil reaction and cDNA library amplification.

[0449] Library construction:

[0450] Use the Chromium single-cell 3' / 5' library construction kit for library construction.

[0451] Sequencing:

[0452] The constructed library is sequenced, and the specific operation is completed by a professional sequencing company (such as Shanghai OE Biotech Co., Ltd.).

[0453] Data analysis:

[0454] Use the official 10×Genomics software Cell Ranger (version 8.0.1) for data analysis:

[0455] File preparation: Specify the FASTQ file path through the config CSV file, including 5' gene expression, feature barcodes, and V(D)J libraries.

[0456] Data processing:

[0457] Perform alignment, filtering, barcode counting, and UMI counting.

[0458] Perform sequence alignment on the gene expression and feature barcode libraries.

[0459] Perform sequence assembly and clonotype calling on the V(D)J library.

[0460] The cell calling results of gene expression data are used to improve the cell recognition of V(D)J data.

[0461] Reference genome:

[0462] Use an appropriate reference genome for analysis:

[0463] Transcriptome reference: refdata-gex-GRCh38-2024-A (human) or refdata-gex-GRCm39-2024-A (mouse).

[0464] Immune group reference: refdata-cellranger-vdj-GRCh38-alts-ensembl-7.0.0 (human) or refdata-cellranger-vdj-GRCm38-alts-ensembl-7.0.0 (mouse).

[0465] Clonotype information analysis:

[0466] Use the Seurat (version 4.0.0) package to integrate the clonotype information of each cell into single-cell gene expression analysis.

[0467] Use VDJtools (version 1.1.4) for downstream diversity analysis.

[0468] Specific steps of the TCR library

[0469] T cell V(D)J enrichment:

[0470] According to the operating instructions of the 10×Genomics Chromium TM Single Cell V(D)J Enrichment Kit, perform V(D)J enrichment of T cells.

[0471] Library amplification:

[0472] Use the Chromium Single Cell Human TCR Amplification Kit for library amplification, and the experimental operations are carried out in accordance with the product instructions.

[0473] The CDR sequences and HCDR sequences of the VHH antibodies obtained by screening in the examples were compared (and Figures 7 - 19 ) were obtained. Figures 7 - 19 The TCR VDJ sequencing results are shown, and the results show that there is partial overlap between the CDR sequences and HCDR sequences.

[0474] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. An antibody or an antigen-binding portion thereof, characterized in that It comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO.1 or an amino acid sequence in which one, two, three or four or more amino acid substitutions, additions, insertions, deletions or a combination thereof are carried out in SEQ ID NO.1, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO.2 or an amino acid sequence in which one, two, three or four or more amino acid substitutions, additions, insertions, deletions or a combination thereof are carried out in SEQ ID NO.2, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO.3 or an amino acid sequence in which one, two, three or four or more amino acid substitutions, additions, insertions, deletions or a combination thereof are carried out in SEQ ID NO.

3.

2. The antibody or antigen-binding portion thereof according to claim 1, characterized in that The amino acid sequence of the HCDR1 is shown in SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.3; The amino acid sequence of the HCDR1 is shown in SEQ ID NO.4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.6; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO.7, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.8, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.9; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO.10, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.11, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.12; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO.31, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.32, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.33; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO.34, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.35, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.36; or; The amino acid sequence of the HCDR1 is shown in SEQ ID NO.37, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.38, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.39; or; The amino acid sequence of the HCDR1 is shown in SEQ ID NO.40, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.41, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.

3.

3. The antibody or antigen-binding portion thereof according to claim 1, characterized in that The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.42 or 43, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid shown in SEQ ID NO.42 or 43.

4. The antibody or antigen-binding portion thereof according to any one of claims 1 to 3, characterized in that The antibody or its antigen-binding portion comprises FR1 as shown in SEQ ID NO.13, FR2 as shown in SEQ ID NO.14, FR3 as shown in SEQ ID NO.15, and FR4 as shown in SEQ ID NO.16; and / or the antibody or its antigen-binding portion is in the form of scFv.

5. A nanobody, characterized in that It comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO.1 or an amino acid sequence in which one, two, three or four or more amino acid substitutions, additions, insertions, deletions or a combination thereof are carried out in SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.2 or an amino acid sequence in which one, two, three or four or more amino acid substitutions, additions, insertions, deletions or a combination thereof are carried out in SEQ ID NO.2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.3 or an amino acid sequence in which one, two, three or four or more amino acid substitutions, additions, insertions, deletions or a combination thereof are carried out in SEQ ID NO.

3.

6. The Nanobody according to claim 5, characterized in that The amino acid sequence of the HCDR1 is shown in SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.3; The amino acid sequence of the HCDR1 is shown in SEQ ID NO.4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.6; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO.7, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.8, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.9; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO.10, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.11, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.12; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO.31, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.32, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.33; or The amino acid sequence of the HCDR1 is shown in SEQ ID NO.34, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.35, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.36; or; The amino acid sequence of the HCDR1 is shown in SEQ ID NO.37, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.38, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.39; or; The amino acid sequence of the HCDR1 is shown in SEQ ID NO.40, the amino acid sequence of the HCDR2 is shown in SEQ ID NO.41, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.

3.

7. The Nanobody according to claim 5, characterized in that The nanobody comprises an amino acid sequence such as FR1 as shown in SEQ ID NO.13, an amino acid sequence such as FR2 as shown in SEQ ID NO.14, an amino acid sequence such as FR3 as shown in SEQ ID NO.15, and an amino acid sequence such as FR4 as shown in SEQ ID NO.16; Preferably, the Nanobody comprises the amino acid sequence shown in SEQ ID NO. 42 or 43, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acids shown in SEQ ID NO. 42 or 43.

8. A chimeric antigen receptor, characterized in that Comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 4 or the Nanobody of any one of claims 5 to 7; The chimeric antigen receptor further comprises a signal peptide; The chimeric antigen receptor further comprises a hinge region; The chimeric antigen receptor further comprises a transmembrane domain; The chimeric antigen receptor further comprises a co-stimulatory signaling domain; The chimeric antigen receptor further comprises an intracellular signaling domain; The signal peptide is selected from the signal peptides of the following molecules: α chain and β chain of T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, IgG6; The hinge region is selected from the hinge regions of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, IL-11 receptor; The transmembrane domain is selected from the transmembrane domains of the following molecules: CD8, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, CD3ε, CD8α, IL-2 receptor, IL-7 receptor, IL-11 receptor; The costimulatory signaling domain is selected from the costimulatory signaling domains of the following molecules: 4-1BB, CD28, ICOS, CD27, CD19, CD4, CD8α, CD8β, HVEM, LIGHT, CD40, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, CD278; The intracellular signaling domain is selected from the intracellular signaling domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d, and FYN.

9. The chimeric antigen receptor according to claim 8, characterized in that An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO.17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acids shown in SEQ ID NO.17, and / or an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO.43, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acids shown in SEQ ID NO.

43.

10. A nucleic acid, characterized in that Encoding the antibody or antigen-binding portion thereof as described in any one of claims 1 to 4, or the Nanobody as described in any one of claims 5 to 7, or the chimeric antigen receptor as described in claim 9 or 10.

11. An expression vector, characterized in that: Comprising the nucleic acid of claim 10.

12. A host cell, characterized in that Comprising the nucleic acid of claim 10 or the expression vector of claim 11, the host cell comprises a T cell, a B cell, a NK cell, an iNKT cell, a CTL cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage or any combination thereof.

13. A use of the host cell according to claim 12, characterized in that: The host cells include CAR T cells or SNR CART cells, wherein the CART cells are co-cultured with cells of metastatic solid tumors to promote antigen presentation and thereby kill T cells, or the SNR CART cells are co-cultured with T cells to promote the formation of memory T cells and reduce exhausted T cells.

14. A pharmaceutical composition, characterized in that Comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 4, or the Nanobody of any one of claims 5 to 7, or the chimeric antigen receptor of claim 8 or 9, or the nucleic acid of claim 10, or the expression vector of claim 11, or the host cell of claim 12, and a pharmaceutically acceptable carrier; Optionally, the pharmaceutical composition further comprises one or more of Pembrolizumab, Paclitaxel, Ramucirumab, Docetaxel.

15. Use of an antibody or antigen-binding portion thereof according to any one of claims 1 to 4, or a Nanobody according to any one of claims 5 to 7, or a chimeric antigen receptor according to claim 8 or 9, or a nucleic acid according to claim 10, or an expression vector according to claim 11, or a host cell according to claim 12, or a pharmaceutical composition according to claim 14 in the preparation of a medicament for treating metastatic solid tumors.

16. The use according to claim 15, characterized in that The metastatic solid tumors include lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, liver cancer, squamous cell carcinoma, peritoneal cancer, brain tumor, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, rectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, penile cancer, anal cancer, thyroid cancer, head and neck cancer, skin cancer, osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcoma, carcinoid, eye cancer, mesothelioma, lymphocytic / lymphoblastic leukemia.

17. The VHH of the antibody or antigen-binding portion thereof according to any one of claims 1 to 4 is obtained from shark, and the Nanobody according to any one of claims 5 to 7 is a shark-derived Nanobody.

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