Anti-CLD18A2 nanoantibodies and their applications

Abstract

The present invention relates to the biological field, and in particular to an anti-CLD18A2 nanobody. The present invention provides an anti-CLD18A2 nanobody, wherein the complementarity determining regions (CDRs) of the anti-CLD18A2 nanobody include CDR1 to CDR3 with the following amino acid sequences: CDR1 with an amino acid sequence as shown in one of SEQ ID NOs. 4 to 11, CDR2 with an amino acid sequence as shown in one of SEQ ID NOs. 19 to 26, and CDR3 with an amino acid sequence as shown in one of SEQ ID NOs. 33 to 39. The anti-CLD18A2 nanobody provided by the present invention is a nanobody that can specifically bind to an epitope present on CLD18A2, and the corresponding fusion protein has good specificity and affinity for CLD18A2 and has a significant tumor inhibitory effect.

Description

[0001] This application is a divisional application of the Chinese invention patent application No. 2021114603413, entitled “Anti-CLD18A2 Nanobodies and Applications Thereof,” filed on January 15, 2019. This application is a divisional application of the Chinese invention patent application No. 2019100339954, entitled “Anti-CLD18A2 Nanobodies and Applications Thereof.” Technical Field

[0002] The present invention relates to the biological field, and in particular to an anti-CLD18A2 nanobody and applications thereof. Background Art

[0003] Claudin 18 (CLD18) is a transmembrane protein with a molecular weight of approximately 28kD, located in the tight junctions of the epithelium and endothelium, tightly connected between adjacent cells. In normal epithelial tissue, the tight intercellular spaces make it difficult for claudin on the cell surface to be accessed, while the spaces between tumor cells are relatively loose. Therefore, claudin on tumor cells becomes a potential target for extracellular antibodies and immunotherapy. CLD18 has four hydrophobic regions, which serve as transmembrane regions to form two extracellular domains, of which hydrophobic region 1 and hydrophobic region 2 surround to form extracellular domain 1, and hydrophobic region 3 and hydrophobic region 4 surround to form extracellular domain 2. Due to different gene splicing, CLD18 forms two splice bodies: CLD18A1 and CLD18A2. CLD18A1 is selectively expressed in normal lung and gastric epithelia, while CLD18A2 is only expressed in gastric cells. More importantly, CLD18A2 is localized in differentiated short-lived gastric epithelial cells but absent in gastric stem cells (Niimi T, et al. Biol. 2001; 21(21): 7380–7390.). These characteristics suggest that CLD18A2 is a clinically valuable therapeutic target for the treatment of gastric cancer and other CLD18A2-positive tumors.

[0004] The successful application of monoclonal antibodies in cancer detection and targeted biological therapy has revolutionized tumor treatment. However, traditional monoclonal antibodies (approximately 150 kD) have a large molecular weight and difficulty penetrating tissue, resulting in low effective concentrations in tumor areas and insufficient therapeutic effects. Traditional antibodies are highly immunogenic, while modified antibodies struggle to achieve the same affinity as before. Furthermore, the long development cycle, high production costs, and insufficient stability of fully humanized traditional antibodies limit their clinical application and widespread adoption. Subsequently, the emergence of nanobodies, the smallest functional antigen-binding fragment of heavy-chain antibodies derived from adult camels, possesses high stability and high affinity for antigen binding. Nanobodies have many unique properties compared to conventional antibodies: 1) The sequences encoded by nanobodies are highly homologous to human VH families 3 and 4, making them less immunogenic; 2) Nanobodies have a small molecular weight of only about 15kDa and a simple structure, making them easily expressed in large quantities in microorganisms and easy to purify; 3) Nanobodies can recognize a large number of antigenic epitopes, including some epitopes hidden in molecular crevices; 4) Due to their small molecular weight, nanobodies can easily penetrate tissues and reach areas that are difficult for conventional antibodies to reach; 5) Nanobodies are highly soluble and stable under denaturing or high-temperature conditions. The unique properties and low cost of nanobodies have greatly expanded their application range. Therefore, nanobodies are of great value in the treatment and diagnosis of diseases and also have great development prospects in the antibody-targeted diagnosis and treatment of tumors.

[0005] However, there is currently no specific nanoantibody targeting the CLD18A2 antigen epitope. Therefore, it is of great value to develop new and effective specific nanoantibodies targeting CLD18A2. Summary of the Invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an anti-CLD18A2 nanobody to solve the problems in the prior art.

[0007] To achieve the above-mentioned objectives and other related objectives, the present invention provides an anti-CLD18A2 nanobody, wherein the complementarity determining region CDR of the anti-CLD18A2 nanobody includes CDR1 to CDR3 with the amino acid sequences shown below: CDR1 with an amino acid sequence as shown in one of SEQ ID NOs. 4 to 11, CDR2 with an amino acid sequence as shown in one of SEQ ID NOs. 19 to 26, and CDR3 with an amino acid sequence as shown in one of SEQ ID NOs. 33 to 39.

[0008] In some embodiments of the present invention, the complementarity determining region (CDR) of the anti-CLD18A2 Nanobody includes CDR1 to CDR3 with the amino acid sequences shown below:

[0009] (1) the CDR1 with an amino acid sequence as shown in SEQ ID NO. 4, the CDR2 with an amino acid sequence as shown in SEQ ID NO. 19, and the CDR3 with an amino acid sequence as shown in SEQ ID NO. 33; or

[0010] (2) the CDR1 amino acid sequence is as shown in SEQ ID NO. 5, the CDR2 amino acid sequence is as shown in SEQ ID NO. 20, and the CDR3 amino acid sequence is as shown in SEQ ID NO. 34; or

[0011] (3) the CDR1 amino acid sequence is as shown in SEQ ID NO.6, the CDR2 amino acid sequence is as shown in SEQ ID NO.21, and the CDR3 amino acid sequence is as shown in SEQ ID NO.35; or

[0012] (4) the CDR1 amino acid sequence is as shown in SEQ ID NO. 7, the CDR2 amino acid sequence is as shown in SEQ ID NO. 22, and the CDR3 amino acid sequence is as shown in SEQ ID NO. 36; or

[0013] (5) a CDR1 with an amino acid sequence as shown in SEQ ID NO. 8, a CDR2 with an amino acid sequence as shown in SEQ ID NO. 23, and a CDR3 with an amino acid sequence as shown in SEQ ID NO. 37; or

[0014] (6) a CDR1 with an amino acid sequence as shown in SEQ ID NO. 9, a CDR2 with an amino acid sequence as shown in SEQ ID NO. 24, and a CDR3 with an amino acid sequence as shown in SEQ ID NO. 38; or

[0015] (7) a CDR1 with an amino acid sequence as shown in SEQ ID NO.10, a CDR2 with an amino acid sequence as shown in SEQ ID NO.25, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.36; or

[0016] (8) CDR1 with an amino acid sequence as shown in SEQ ID NO.11, CDR2 with an amino acid sequence as shown in SEQ ID NO.26, and CDR3 with an amino acid sequence as shown in SEQ ID NO.39.

[0017] In some embodiments of the present invention, the anti-CLD18A2 Nanobody comprises a framework region FR, wherein the framework region FR comprises FR1 to FR4 with the amino acid sequence shown below:

[0018] (1) the amino acid sequence of FR1 as shown in SEQ ID NO.1, the amino acid sequence of FR2 as shown in SEQ ID NO.12, the amino acid sequence of FR3 as shown in SEQ ID NO.27, and the amino acid sequence of FR4 as shown in SEQ ID NO.40; or

[0019] (2) an amino acid sequence of FR1 as shown in SEQ ID NO. 2, an amino acid sequence of FR2 as shown in SEQ ID NO. 13, an amino acid sequence of FR3 as shown in SEQ ID NO. 28, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0020] (3) an amino acid sequence of FR1 as shown in SEQ ID NO. 3, 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. 29, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0021] (4) an amino acid sequence of FR1 as shown in SEQ ID NO. 1, an amino acid sequence of FR2 as shown in SEQ ID NO. 15, an amino acid sequence of FR3 as shown in SEQ ID NO. 30, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0022] (5) an amino acid sequence of FR1 as shown in SEQ ID NO. 2, an amino acid sequence of FR2 as shown in SEQ ID NO. 16, an amino acid sequence of FR3 as shown in SEQ ID NO. 31, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0023] (6) an amino acid sequence of FR1 as shown in SEQ ID NO. 2, an amino acid sequence of FR2 as shown in SEQ ID NO. 13, an amino acid sequence of FR3 as shown in SEQ ID NO. 31, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0024] (7) an amino acid sequence of FR1 as shown in SEQ ID NO. 1, an amino acid sequence of FR2 as shown in SEQ ID NO. 17, an amino acid sequence of FR3 as shown in SEQ ID NO. 30, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0025] (8) FR1 with an amino acid sequence as shown in SEQ ID NO. 2, FR2 with an amino acid sequence as shown in SEQ ID NO. 18, FR3 with an amino acid sequence as shown in SEQ ID NO. 32, and FR4 with an amino acid sequence as shown in SEQ ID NO. 41.

[0026] In some embodiments of the present invention, the amino acid sequence of the anti-CLD18A2 Nanobody comprises:

[0027] a) an amino acid sequence as shown in any one of SEQ ID NOs. 42 to 49; or

[0028] b) an amino acid sequence having a sequence identity of 80% or more to any one of SEQ ID NOs. 42 to 49, and having the function of the amino acid sequence defined in a).

[0029] In some embodiments of the present invention, the anti-CLD18A2 nanobody is a humanized antibody. Preferably, the amino acid sequence of the CLD18A2 nanobody is as shown in SEQ ID NOs. 67 to 90.

[0030] On the other hand, the present invention provides a fusion protein of an anti-CLD18A2 nanobody, comprising the first domain of the nanobody and a second domain for prolonging the in vivo half-life and / or having a binding effect on effector cells.

[0031] In some embodiments of the present invention, the second domain comprises a combination of one or more of a serum albumin fragment, a polyethylene glycol fragment, and a nanobody that binds to HSA.

[0032] In some embodiments of the present invention, the second domain comprises an immunoglobulin Fc region, preferably selected from a human immunoglobulin Fc region.

[0033] In some embodiments of the present invention, the human immunoglobulin Fc region includes mutations for altering Fc-mediated effector functions, and the effector functions include one or more combinations of CDC activity, ADCC activity, and ADCP activity.

[0034] In some embodiments of the present invention, the immunoglobulin is selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is selected from a combination of one or more of IgG1, IgG2, IgG3, or IgG4 subtypes.

[0035] In some embodiments of the present invention, the amino acid sequence of the immunoglobulin Fc region is selected from one of SEQ ID NOs. 91 to 95.

[0036] In some embodiments of the present invention, the second domain comprises a molecule that has affinity for and / or is capable of binding to CD3 present on T cells.

[0037] In some embodiments of the present invention, a connecting peptide is provided between the first domain and the second domain.

[0038] In some embodiments of the present invention, the connecting peptide is selected from a flexible polypeptide chain consisting of alanine and / or serine and / or glycine, and the length of the connecting peptide is 3 to 40 amino acids.

[0039] Another aspect of the present invention provides an isolated polynucleotide encoding the nanobody or the fusion protein.

[0040] Another aspect of the present invention provides an expression vector containing the isolated polynucleotide.

[0041] Another aspect of the present invention provides an antibody expression system, which contains the expression vector or the exogenous polynucleotide integrated into the genome.

[0042] On the other hand, the present invention provides a method for preparing the nanobody or the fusion protein, comprising the steps of: culturing the antibody expression system under conditions suitable for expressing the antibody, thereby expressing the antibody, and purifying and isolating the antibody.

[0043] Another aspect of the present invention provides an immunoconjugate, which comprises the nanobody or the fusion protein.

[0044] In some embodiments of the present invention, the immunoconjugate further comprises a coupling moiety, wherein the coupling moiety comprises a detectable label, a cytotoxin, a radioactive isotope, or a combination of one or more biologically active proteins.

[0045] Another aspect of the present invention provides a detection kit, which comprises the nanobody, the fusion protein, or the immunoconjugate.

[0046] Another aspect of the present invention provides a pharmaceutical composition comprising the nanobody, the fusion protein, or the immunoconjugate.

[0047] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0048] Another aspect of the present invention provides a cell containing the membrane-bound polypeptide, wherein the cell is a T lymphocyte, a macrophage, or a NK cell. The polypeptide comprises an antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signaling domain, wherein the antigen recognition domain comprises the nanobody.

[0049] Another aspect of the present invention provides the use of the nanobody, or the fusion protein, or the immunoconjugate, or the pharmaceutical composition, or the cell in the preparation of a medicament for diagnosing, treating or preventing a disease associated with cells expressing CLD18A2.

[0050] In some embodiments of the present invention, the disease associated with cells expressing CLD18A2 is selected from tumors, and the tumor is selected from one or more combinations of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Shown is the binding curve of the Anti-C18.2-Fc fusion protein of the present invention to the cell surface antigen CLD18A2 (ELISA).

[0052] Figure 2 This shows the CDC activity of the Anti-C18.2-Fc fusion protein of the present invention.

[0053] Figure 3 This shows the CDC activity of the Anti-C18.2-Fc fusion protein of the present invention.

[0054] Figure 4 This shows the ADCC activity of the Anti-C18.2-Fc fusion protein of the present invention.

[0055] Figure 5 This shows the inhibitory effect of the Anti-C18.2-Fc fusion protein of the present invention on tumor growth in mice.

[0056] Figure 6 Shown is the binding curve of the Anti-CLDN18xCD3 fusion protein of the present invention to CHO-S-CLD18A2.

[0057] Figure 7 Shown is the binding curve of the Anti-CLDN18xCD3 fusion protein of the present invention to Jurkat cells.

[0058] Figure 8 It shows the killing effect of the Anti-CLDN18xCD3 fusion protein of the present invention on NUGC-4-CLD18A2 in vitro.

[0059] Figure 9 This shows the killing effect of the Anti-CLDN18xCD3 fusion protein of the present invention on NUGC-4-CLD18A1 in vitro.

[0060] Figure 10 This shows the inhibitory effect of the Anti-CLDN18xCD3 fusion protein of the present invention on tumor growth in mice.

[0061] Figure 11 Shown are the in vitro cytokine release detection results after the aC18.2-CAR-T cells of the present invention acted on NUGC-4-CLD18A2 and NUGC-4-CLD18A1.

[0062] Figure 12 This shows the inhibitory effect of the aC18.2-CAR-T cells of the present invention on tumor growth in mice. DETAILED DESCRIPTION

[0063] After in-depth research, the inventors have provided a nanobody that specifically binds to an epitope present on CLD18A2, and further provided fusion proteins, immunoconjugates and cells expressing a chimeric antigen receptor targeting CLD18A2 comprising the nanobody. The protein or cells have specificity and good affinity for CLD18A2 and have obvious tumor inhibitory effects. The present invention was completed on this basis.

[0064] The term "antibody" or "immunoglobulin" herein, whether referring to a heavy chain antibody or a conventional four-chain antibody, is used as a general term to include full-length antibodies, their individual chains, and all parts, domains, or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). In addition, the term "sequence" used herein (e.g., in terms such as "immunoglobulin sequence," "antibody sequence," "single variable domain sequence," "VHH sequence," or "protein sequence") is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0065] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0066] The term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and / or the domain.

[0067] The terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to cloning the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting solely of a single heavy chain variable region. This is the smallest fully functional antigen-binding fragment. Typically, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are obtained from alpaca immune serum, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0068] The term "single domain antibody (VHH)" refers to an immunoglobulin domain consisting essentially of four "framework regions," respectively referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," respectively referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3." Thus, the general structure or sequence of a single domain antibody (VHH) can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies (VHHs) confer specificity for an antigen by having an antigen-binding site.

[0069] The terms “heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH antibody fragment”, “VHH antibody” and “Nanobody” (“Nanobody” is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably.

[0070] The term "IMGT numbering system" is an integrated information system specifically for immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates, namely THE INTERNATIONAL IMMUNOGENETICS INFORMATION (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). Log in to IMGT (http: / / www.imgt.org / IMGT_vquest) and analyze the antibody light and heavy chain genes to determine the framework regions (FR) and complementarity determining regions (CDR) of the variable region. The "position" of CDR within the structure of the immunoglobulin variable domain is conserved between species and exists in structures called loops, so it is easy to identify CDR and framework residues by using a numbering system that aligns the variable domain sequence according to structural features. This information can be used to transplant and replace CDR residues from an immunoglobulin from one species into an acceptor framework, usually from a human antibody. Unless otherwise indicated, in this specification, claims and drawings, anti-CLD18A2 nanobodies are numbered according to the IMGT numbering method to identify CDR regions and FR regions.

[0071] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and immunofluorescence techniques.

[0072] The term "humanized antibody" refers to a molecule with an antigen binding site that is substantially derived from a non-human species immunoglobulin, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only complementarity determining regions (CDRs) transplanted into appropriate framework regions in the variable domain. The antigen binding site may be wild type or modified by one or more amino acid substitutions, for example, to be more similar to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences (e.g., humanized nanobodies containing all three CDRs from alpaca). Other forms have one or more CDRs that have changed relative to the original antibody.

[0073] The term "effector function" when referring to an antibody refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0074] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are absolutely required for the killing. NK cells, as the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcRs are known to be expressed on hematopoietic cells (see, e.g., Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9:457-92). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed (see, e.g., U.S. Patent Nos. 5,500,362 and 5,821,337). Suitable effector cells for use in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.

[0075] The term "complement-dependent cytotoxicity" or "CDC" is another method of cell killing directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also very effective in directing CDC through the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex results in the exposure of multiple C1q binding sites (C1q is one of the three subcomponents of complement C1) in close proximity to the CH2 domain of the participating antibody molecules (e.g., IgG molecules). Preferably, the exposed C1q binding sites convert the previously low-affinity C1q-IgG interaction into a high-affinity interaction, which triggers a cascade involving a series of other complement proteins and causes the proteolytic release of effector cell chemoattractants / activators C3a and C5a. Preferably, the complement cascade ultimately forms a membrane attack complex, which creates pores in the cell membrane, facilitating the free passage of water and solutes into and out of the cell.

[0076] The term "CD3" refers to the multi-subunit complex of the human CD3 protein. The CD3 protein multi-subunit complex is composed of six different polypeptide chains. These polypeptide chains include the CD3 γ chain (SwissProt P09693), the CD3 δ chain (SwissProt P04234), two CD3 ε chains (SwissProt P07766), and a CD3 ζ chain homodimer (SwissProt 20963) associated with the T cell receptor α and β chains. The term "CD3" includes any CD3 variants, isoforms, and species homologs that are naturally expressed by cells (including T cells) or expressed on cells transfected with genes or cDNAs encoding these polypeptides, unless otherwise noted. Cluster of differentiation 3 (CD3) on the surface of T cells is a co-receptor for the T cell receptor, assisting in the activation of cytotoxic T cells.

[0077] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program from the NCBI database can be used to determine identity.

[0078] The terms "chimeric antigen receptor" and "CAR" are artificial receptors that simulate TCR functions. They are composed of an antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signaling domain connected in sequence. When the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal is transmitted to the cell through the hinge region and the transmembrane region. The intracellular signaling domain then converts the signal into an activation signal, activating effector cells. The effector cells kill tumor cells by secreting perforin or producing cytokines. At the same time, the effector cells themselves also proliferate, further expanding the immune killing effect.

[0079] An "effective amount" of an agent is that amount necessary to effect a physiological change in the cell or tissue to which it is administered.

[0080] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0081] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0082] The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredient contained therein is effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0083] "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0084] The term "treatment / prevention" (and grammatical variations thereof) refers to an attempt to alter the natural course of a disease in a treated individual and can be a clinical intervention performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of the disease or slow the progression of the condition.

[0085] The term "antigen" refers to a predetermined antigen to which an antibody selectively binds. The target antigen can be a polypeptide, protein, nucleic acid, cell, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments herein, the target antigen is a cell expressing CLD18A2, and more preferably, a portion of a molecule expressing CLD18A2.

[0086] The term "whole-cell subtractive screening" is a high-throughput screening technology developed in recent years based on phage display technology. It uses paired cells to perform subtractive screening on phage libraries, and can screen out short peptides that bind to target cells with high affinity in a short time.

[0087] An "epitope" is a site on the surface of an antigen molecule that is bound by a single antibody molecule, such as a localized region on the surface of an antigen that is capable of binding to one or more antigen-binding regions of an antibody and that has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human), that is capable of eliciting an immune response. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide that is bound by an antibody, as determined by any method well known in the art, including, for example, by immunoassay. An antigenic epitope need not necessarily be immunogenic. Epitopes often consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear or conformational. Linear epitopes are formed by contiguous sequences of amino acids in a protein. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence but are brought together after the protein folds into its three-dimensional structure. Induced epitopes are formed when the three-dimensional structure of a protein is in an altered conformation, such as after activation or binding to another protein or ligand. In certain embodiments, a CLD18A2 epitope is a three-dimensional surface feature of the CLD18A2 protein. In other embodiments, a CLD18A2 epitope is a linear feature of the CLD18A2 protein. Typically, an antigen has several or many different epitopes and can react with many different antibodies.

[0088] Nanobodies

[0089] In a first aspect, the present invention provides an anti-CLD18A2 Nanobody, wherein the complementary determining region (CDR) of the anti-CLD18A2 Nanobody comprises CDR1 to CDR3 with the amino acid sequences shown below: CDR1 with an amino acid sequence as shown in one of SEQ ID NOs. 4 to 11, CDR2 with an amino acid sequence as shown in one of SEQ ID NOs. 19 to 26, and CDR3 with an amino acid sequence as shown in one of SEQ ID NOs. 33 to 39. In some specific embodiments of the present invention, the complementary determining region (CDR) of the anti-CLD18A2 Nanobody comprises CDR1 to CDR3 with the amino acid sequences shown below:

[0090] (1) the CDR1 with an amino acid sequence as shown in SEQ ID NO. 4, the CDR2 with an amino acid sequence as shown in SEQ ID NO. 19, and the CDR3 with an amino acid sequence as shown in SEQ ID NO. 33; or

[0091] (2) the CDR1 with an amino acid sequence as shown in SEQ ID NO. 5, the CDR2 with an amino acid sequence as shown in SEQ ID NO. 20, and the CDR3 with an amino acid sequence as shown in SEQ ID NO. 34; or

[0092] (3) the CDR1 amino acid sequence is as shown in SEQ ID NO. 6, the CDR2 amino acid sequence is as shown in SEQ ID NO. 21, and the CDR3 amino acid sequence is as shown in SEQ ID NO. 35; or

[0093] (4) a CDR1 with an amino acid sequence as shown in SEQ ID NO. 7, a CDR2 with an amino acid sequence as shown in SEQ ID NO. 22, and a CDR3 with an amino acid sequence as shown in SEQ ID NO. 36; or

[0094] (5) the CDR1 amino acid sequence is as shown in SEQ ID NO. 8, the CDR2 amino acid sequence is as shown in SEQ ID NO. 23, and the CDR3 amino acid sequence is as shown in SEQ ID NO. 37; or

[0095] (6) a CDR1 with an amino acid sequence as shown in SEQ ID NO. 9, a CDR2 with an amino acid sequence as shown in SEQ ID NO. 24, and a CDR3 with an amino acid sequence as shown in SEQ ID NO. 38; or

[0096] (7) a CDR1 with an amino acid sequence as shown in SEQ ID NO. 10, a CDR2 with an amino acid sequence as shown in SEQ ID NO. 25, and a CDR3 with an amino acid sequence as shown in SEQ ID NO. 36; or

[0097] (8) The amino acid sequence of CDR1 is shown in SEQ ID NO.11, the amino acid sequence of CDR2 is shown in SEQ ID NO.26, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39.

[0098] The anti-CLD18A2 antigen Nanobody provided by the present invention may include a framework region FR, wherein the framework region FR includes FR1 to FR4 with the amino acid sequence shown below:

[0099] (1) the amino acid sequence of FR1 as shown in SEQ ID NO.1, the amino acid sequence of FR2 as shown in SEQ ID NO.12, the amino acid sequence of FR3 as shown in SEQ ID NO.27, and the amino acid sequence of FR4 as shown in SEQ ID NO.40; or

[0100] (2) an amino acid sequence of FR1 as shown in SEQ ID NO. 2, an amino acid sequence of FR2 as shown in SEQ ID NO. 13, an amino acid sequence of FR3 as shown in SEQ ID NO. 28, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0101] (3) an amino acid sequence of FR1 as shown in SEQ ID NO. 3, 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. 29, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0102] (4) an amino acid sequence of FR1 as shown in SEQ ID NO. 1, an amino acid sequence of FR2 as shown in SEQ ID NO. 15, an amino acid sequence of FR3 as shown in SEQ ID NO. 30, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0103] (5) an amino acid sequence of FR1 as shown in SEQ ID NO. 2, an amino acid sequence of FR2 as shown in SEQ ID NO. 16, an amino acid sequence of FR3 as shown in SEQ ID NO. 31, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0104] (6) an amino acid sequence of FR1 as shown in SEQ ID NO. 2, an amino acid sequence of FR2 as shown in SEQ ID NO. 13, an amino acid sequence of FR3 as shown in SEQ ID NO. 31, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0105] (7) an amino acid sequence of FR1 as shown in SEQ ID NO. 1, an amino acid sequence of FR2 as shown in SEQ ID NO. 17, an amino acid sequence of FR3 as shown in SEQ ID NO. 30, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or

[0106] (8) FR1 with an amino acid sequence as shown in SEQ ID NO. 2, FR2 with an amino acid sequence as shown in SEQ ID NO. 18, FR3 with an amino acid sequence as shown in SEQ ID NO. 32, and FR4 with an amino acid sequence as shown in SEQ ID NO. 41.

[0107] The amino acid sequence of the anti-CLD18A2 antigen Nanobody provided by the present invention may include: a) an amino acid sequence as shown in one of SEQ ID NOs. 42 to 49; or, b) an amino acid sequence having a sequence identity of more than 80% with one of SEQ ID NOs. 42 to 49, and having the function of the amino acid sequence defined in a); specifically, the amino acid sequence in b) specifically refers to: an amino acid sequence as shown in one of SEQ ID NOs. 42 to 49 by substitution, deletion or addition of one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or a polypeptide fragment obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the function of a polypeptide fragment as shown in one of SEQ ID NOs. 42 to 49, for example, specific binding ability to CLD18A2. The amino acid sequence in b) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with one of SEQ ID Nos. 42 to 49. The anti-CLD18A2 Nanobodies provided by the present invention can specifically bind to cells expressing CLD18A2. For example, the anti-CLD18A2 Nanobodies can bind to cells expressing CLD18A2, but may not bind to cells that do not express CLD18A2 but express CLD18A1, that is, they only recognize CLD18A2 but not CLD18A1.

[0108] The anti-CLD18A2 Nanobodies provided herein can be humanized antibodies. Humanization can effectively reduce the immunogenicity of antibodies, and the humanized Nanobodies can retain at least one functional property of an antibody, for example, specific binding ability to CLD18A2. In one embodiment of the present invention, the amino acid sequences of the anti-CLD18A2 humanized Nanobodies are shown in SEQ ID NOs. 67 to 90.

[0109] Fusion protein

[0110] In a second aspect, the present invention provides an anti-CLD18A2 Nanobody fusion protein comprising the first domain of the Nanobody provided in the first aspect of the invention and a second domain for prolonging the in vivo half-life and / or binding to effector cells. The fusion protein may be a binding molecule that is capable of specifically binding to cells expressing CLD18A2.

[0111] In the second domain, the fragment for extending the half-life in vivo may include a serum albumin fragment, a polyethylene glycol fragment, a domain that binds to HSA (for example, a nanobody that binds to HSA), etc. In the second domain, the fragment that has a binding effect on effector cells may include an immunoglobulin Fc region, etc., preferably selected from the human immunoglobulin Fc region. The human immunoglobulin Fc region includes mutations for changing the effector function mediated by Fc, and the effector function includes a combination of one or more of CDC activity, ADCC activity, and ADCP activity. The immunoglobulin may be selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, IgM, etc., and the IgG may be specifically selected from a combination of one or more of IgG1, IgG2, IgG3 or IgG4 subtypes, etc. The immunoglobulin Fc region contained in the nanobody fusion protein can cause the fusion protein to form a dimer, while extending the in vivo half-life of the fusion protein and increasing Fc-mediated related activities. In one embodiment of the present invention, the immunoglobulin Fc region can be the Fc region of human IgG1, more specifically, it can be a wild-type IgG1 Fc sequence, and the sequence can be introduced with mutations for altering Fc-mediated effector functions, for example, a) mutations that alter Fc-mediated CDC activity; b) mutations that alter Fc-mediated ADCC activity; or c) mutations that alter Fc-mediated ADCP activity. In another embodiment of the present invention, the amino acid sequence of the immunoglobulin Fc region is selected from one of SEQ ID NOs. 91 to 95. In the second domain, the fragment that has a binding effect on effector cells can also include a molecule that has a high affinity for / binds to cluster of differentiation 3 (CD3) present on T cells, preferably an anti-CD3 nanobody with an amino acid sequence of SEQ ID NO. 131.

[0112] In the fusion protein of the anti-CLD18A2 nanobody provided by the present invention, a connecting peptide may be provided between the first domain and the second domain. The connecting peptide may be a flexible polypeptide chain composed of alanine (A) and / or serine (S) and / or glycine (G), and the length of the connecting peptide may be 3 to 40 amino acids, preferably 3-9, 9-12, 12-16, 16 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40. In another specific embodiment of the present invention, the length of the connecting peptide may be 8, 15, or 35.

[0113] In a preferred embodiment, by using serum from healthy individuals as a complement source, the Anti-C18.2-Fc has a killing effect on cells expressing the CLD18A2 antigen, and the cell lysis rate is higher than that of the positive control ch-175D10.

[0114] In another preferred embodiment, the Anti-CLDN18×CD3 fusion protein has a tumor recognition portion Anti-C18.2, while the other arm of the molecule is specific for T cell antigens (effector binding arm) (mainly CD3). By simultaneously binding both arms to their respective target antigens, T lymphocytes are directed to tumor cells and activated at the tumor cells, where they can exert their cytolytic function. Anti-CD3 nanoantibodies can bind to CD3 in the TCR receptor complex on the surface of T cells, providing the first signal for T cell activation (similar to the binding of the MHC-peptide complex on antigen-presenting cells to the TCR), which is beneficial to the activation of T cells. In addition, the Anti-CLDN18×CD3 fusion protein containing an antigen binding portion for CD3 can achieve the enrichment of T cells around tumor cells and improve the efficiency of T cells in killing tumor cells.

[0115] Isolated polynucleotides

[0116] The third aspect of the present invention provides an isolated polynucleotide encoding the Nanobody provided in the first aspect of the present invention, or encoding the fusion protein provided in the second aspect of the present invention, wherein the polynucleotide may be RNA, DNA, or cDNA, etc. Methods for providing the isolated polynucleotide should be known to those skilled in the art. For example, the isolated polynucleotide may be prepared by automated DNA synthesis and / or recombinant DNA technology, or may be isolated from a suitable natural source. In a specific embodiment of the present invention, the nucleic acid sequence of the isolated polynucleotide is shown in one of SEQ ID NOs: 119-130.

[0117] expression vector

[0118] A fourth aspect of the present invention provides an expression vector comprising the isolated polynucleotide provided in the third aspect of the present invention. Methods for constructing such expression vectors should be known to those skilled in the art. For example, such expression vectors can be constructed by in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. More specifically, such vectors can be constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. The expression vectors herein generally refer to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. The vectors may further comprise one or more regulatory sequences operably linked to the polynucleotide sequence, and such regulatory sequences may include suitable promoter sequences. The promoter sequence is typically operably linked to the coding sequence for the amino acid sequence to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. Regulatory sequences may also include suitable transcription terminator sequences, sequences recognized by the host cell to terminate transcription. The terminator sequence is linked to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice can be used in the present invention.

[0119] Generally speaking, suitable vectors can contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers. For example, these promoters can include, but are not limited to, the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or viruses. Marker genes can be used to provide phenotypic traits for selection of transformed host cells, and can include, for example, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli. When the polynucleotide is expressed, the expression vector can also include an enhancer sequence. Insertion of an enhancer sequence in the vector enhances transcription. Enhancers are cis-acting DNA elements, typically about 10 to 300 base pairs, that act on the promoter to enhance gene transcription.

[0120] Expression system

[0121] The fifth aspect of the present invention provides an antibody expression system, comprising the expression vector provided in the fourth aspect of the present invention or the polynucleotide provided in the third aspect of the present invention integrated into its genome. Any cell suitable for expression by the expression vector can be used as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, specifically including but not limited to Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, HEK293 cells, or animal cells such as Bowes melanoma cells. Methods for constructing the expression system should be known to those skilled in the art, and for example, can include but are not limited to a combination of one or more of microinjection, gene guns, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, etc.

[0122] Immunoconjugates

[0123] The sixth aspect of the present invention provides an immunoconjugate, which includes the nano antibody provided by the first aspect of the present invention, or the fusion protein provided by the second aspect of the present invention. The immunoconjugate generally also includes a coupling portion, and the coupling portion can be a combination of one or more including but not limited to a detectable marker, a cytotoxin, a radioisotope, or a biologically active protein. The method for preparing the immunoconjugate should be known to those skilled in the art. For example, the nano antibody and / or fusion protein can be connected to the coupling portion directly or through a spacer of appropriate length. The connection method can be chemical cross-linking or genetic engineering fusion expression to obtain the immunoconjugate. For therapeutic purposes, it may be appropriate to combine with therapeutic effector groups such as radioactive groups, which are composed of radioisotopes or radionuclides (e.g. 3 H. 14 C. 15 N. 33 P. 35 S. 90 Y. 99 Tc, 111 ln、 123 1. 125 1. 131 1. 201 TI, 213In some embodiments, the polypeptide of the present invention can be coupled to a labeling group (labeled polypeptide) or a group containing a radioisotope, a radionuclide, a fluorescent group (e.g., fluorescent proteins such as GFP, RFP, etc., dyes, rhodamine, fluorescein and derivatives thereof such as FITC, cyanine dyes such as and), an enzyme group (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase), a chemiluminescent group, a biotin group, a metal particle (e.g., a gold particle), a magnetic particle (e.g., having a core containing magnetite (Fe3O4) and / or maghemite (Fe2O3)), a predetermined polypeptide group, etc.

[0124] Detection kit

[0125] In a seventh aspect, the present invention provides a detection kit comprising the Nanobody provided in the first aspect of the present invention, the fusion protein provided in the second aspect of the present invention, or the immunoconjugate provided in the sixth aspect of the present invention. The kit may further comprise, as required, a container, a control (negative or positive control), a buffer, an adjuvant, and the like, which can be selected by those skilled in the art according to the specific circumstances.

[0126] The present invention further provides a detection method that can be used to detect CLD18A2 protein. The detection method may include: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of CLD18A2 protein in the dissolved sample. In one embodiment of the present invention, the detection target may be a cell-containing sample present in a cell preservation solution. In another embodiment of the present invention, the Nanobody is further conjugated with a fluorescent dye, chemical substance, peptide, enzyme, isotope, label, etc. that can be used for detection or can be detected by other reagents.

[0127] Pharmaceutical composition

[0128] The eighth aspect of the present invention provides a pharmaceutical composition comprising the anti-CLD18A2 nanobody provided by the first aspect of the present invention, or the fusion protein of the anti-CLD18A2 nanobody provided by the second aspect of the present invention, or the immunoconjugate provided by the sixth aspect of the present invention.

[0129] The pharmaceutical composition may also include various pharmaceutically acceptable carriers in the art. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used, and may include, but are not limited to: buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parahydroxybenzoates such as methyl parahydroxybenzoate or propyl parahydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; tonicity modifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactants such as polysorbate; salt-forming counterions such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants such as Or polyethylene glycol (PEG). Pharmaceutical preparations for in vivo administration are generally sterile. Methods for achieving sterility of pharmaceutical preparations should be known to those skilled in the art. For example, it can be achieved by filtration through a sterile filtration membrane. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the desired dosage form of the pharmaceutical composition to prepare it into different dosage forms. For example, the pharmaceutical composition of the present invention can be in various dosage forms including but not limited to tablets, injections, lyophilized agents, etc.

[0130] In the pharmaceutical composition, the content of the fusion protein and the immunoconjugate is generally an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined according to the subject to be treated and the specific administration method. For example, based on the total mass of the pharmaceutical composition, the content of the fusion protein and the immunoconjugate can range from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99%.

[0131] The fusion protein, immunoconjugate and pharmaceutical composition of the present invention can be administered as a single active ingredient or in combination therapy, i.e., in combination with other agents. For example, the combination therapy can be the fusion protein, immunoconjugate, pharmaceutical composition combined with at least one other anti-tumor drug. For another example, the combination therapy can be the fusion protein, immunoconjugate, pharmaceutical composition combined with an immune checkpoint inhibitor, the immune checkpoint inhibitor including but not limited to a combination of one or more of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor, etc., and the inhibitor can preferably be a monoclonal antibody.

[0132] Cells expressing a chimeric antigen receptor targeting CLD18A2

[0133] The ninth aspect of the present invention provides a cell expressing a chimeric antigen receptor (CAR) targeting CLD18A2. The cell targeting CLD18A2 generally includes a polypeptide as a chimeric antigen receptor, and the polypeptide may include an antigen recognition domain, a hinge region, a transmembrane region and an intracellular signaling domain. The method for constructing the chimeric antigen receptor should be known to those skilled in the art. For example, the transmembrane region may be the following transmembrane region: CD proteins such as CD4, CD8, CD3 or CD28, subunits of T cell receptors such as α, β, γ or δ, subunits of IL-2 receptors (α chain), subunits of low affinity nerve growth factor receptors (LNGFR or p75) (β chain or γ chain), or subunit chains of Fc receptors. In a specific embodiment of the present invention, the transmembrane region comprises a transmembrane domain of CD4, CD8 or CD28. In another specific embodiment of the present invention, the transmembrane region comprises a transmembrane region of CD4 or CD8 (e.g., CD8α chain, as described in NCBI reference number: NP_001139345.1, or a fragment thereof). In another specific embodiment of the present invention, CAR further comprises a hinge region between the antigen recognition domain and the transmembrane region. In another specific embodiment of the present invention, the hinge region is selected from the CH2 and / or CH3 domains of CD8 (e.g., CD8α) or IgG1 or IgG4. Preferred examples of the intracellular signaling domains for CAR can be cytoplasmic sequences of natural T cell receptors and auxiliary receptors that act synergistically to initiate signal transduction after antigen binding, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same function. The intracellular signaling domain can be divided into two categories: those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or costimulatory signals. The primary activation effector domain may include a signaling motif, which is known as an immunoreceptor tyrosine-based activation motif (ITAM). ITAMs are well-defined signaling motifs that are typically found in the cytoplasmic tails of various receptors and serve as binding sites for syk / zap70-class tyrosine kinases. As non-limiting examples, examples of ITAMs used in the present invention may include those derived from CD3ζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment of the present invention, the intracellular signaling domain comprises a CD3ζ signaling domain (also known as CD247). Natural TCRs contain CD3ζ signaling molecules, so the use of this effector domain is closest to TCR constructs occurring in nature. In another embodiment of the present invention, the CD3ζ signaling domain comprises the sequence described in NCBI Reference Number: NP_932170, or a fragment thereof having activation or stimulatory activity. As described herein, the intracellular signaling domain may also provide a secondary or co-stimulatory signal.T cells additionally include costimulatory molecules, which are combined with the cognate costimulatory ligands on antigen presenting cells to enhance T cell responses, such as by increasing proliferation activation, differentiation, etc. Therefore, in a specific embodiment of the present invention, the intracellular signaling domain additionally includes a costimulatory domain. In another specific embodiment of the present invention, the costimulatory domain includes the intracellular domain of a costimulatory molecule, which is selected from CD28, CD27, 4-1BB (CD137), OX40 (CD134), ICOS (CD278), CD30, CD40, PD-1 (CD279), CD2, CD7, NKG2C (CD94), B7-H3 (CD276) or any combination thereof. In another embodiment, the costimulatory domain includes the intracellular domain of a costimulatory molecule, which is selected from CD28, CD27, 4-1BB, OX40, ICOS or any combination thereof. In another embodiment of the invention, the costimulatory domain comprises CD28, such as described in NCBI Reference No. NP_006130, or a fragment thereof having activation or stimulatory activity.

[0134] The method of further constructing the cell targeting CLD18A2 through the chimeric antigen receptor should also be known to those skilled in the art. For example, the cell can be a T lymphocyte, a macrophage and / or a NK cell. When the nanobody binds to the CLD18A2 antigen, the T lymphocyte, macrophage and / or NK cell can be activated and / or stimulated to kill the cell expressing CLD18A2.

[0135] In a preferred embodiment of the present invention, the antigen recognition domain comprises the nanobody provided by the first aspect of the present invention, the hinge region is selected from CD8, the transmembrane region is CD28 (labeled as CD28a in the embodiment), the costimulatory domain in the intracellular signaling domain is selected from CD28 (labeled as CD28b in the embodiment) or a combination of CD28 and CD137, and the intracellular signaling domain further comprises a CD3ζ signaling domain. In a preferred embodiment, the CAR-T cells targeting CLD18A2 have a significant killing effect in vitro and in vivo.

[0136] use

[0137] In the tenth aspect, the present invention provides the use of the nanobody provided in the first aspect of the present invention, or the fusion protein provided in the second aspect of the present invention, or the immunoconjugate provided in the sixth aspect of the present invention, or the pharmaceutical composition provided in the eighth aspect of the present invention, the polypeptide as a chimeric antigen receptor provided in the ninth aspect, or the cell expressing a chimeric antigen receptor targeting CLD18A2 provided in the ninth aspect of the present invention in the preparation of a medicament for diagnosing, treating or preventing a disease associated with a cell expressing CLD18A2.

[0138] A "therapeutically effective amount" of the Nanobodies, fusion proteins, immunoconjugates, and pharmaceutical compositions provided herein preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevents damage or disability caused by the suffering of the disease. For example, for the treatment of CLD18A2-associated tumors (including, for example, gastric cancer), a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that is predictive of efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which inhibition can be determined in vitro using assays known to those skilled in the art. A therapeutically effective amount of a Nanobody, fusion protein, immunoconjugate, or pharmaceutical composition is generally capable of reducing tumor size or otherwise alleviating symptoms in a subject. A person skilled in the art can select an appropriate therapeutically effective amount based on the specific circumstances, for example, the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected. The prescription of treatment (e.g., determination of dosage, etc.) can be determined by a physician, and factors generally considered include, but are not limited to, the disease being treated, the individual patient's condition, the delivery site, the method of administration, and other factors. A prophylactically effective amount refers to an amount that is effective to achieve the desired prophylactic effect at the necessary dose and time. Typically, but not necessarily, a prophylactic dose is administered to a subject before the onset of the disease or in the early stages of the disease, so a "prophylactically effective amount" is generally lower than a "therapeutically effective amount." Examples of diseases associated with cells expressing CLD18A2 that can be diagnosed, treated, and / or prevented by the present invention may include all cancers and tumor entities that express CLD18A2, and specifically may include, but are not limited to, gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, breast cancer, colorectal cancer, liver cancer, gallbladder cancer, and head and neck cancer, etc. These cancers may be in the early, middle, or late stages, such as metastatic cancer.

[0139] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0140] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0141] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0142] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in the literature, for example, by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0143] Example 1: Construction and detection of cell lines expressing CLD18A2

[0144] Plasmids were extracted from the pCDNA3.1 vector (Life Technologies) containing the full-length genes for CLD18A1 (amino acid sequence, SEQ ID NO. 96) and CLD18A2 (amino acid sequence, SEQ ID NO. 97) using a plasmid extraction kit (Biomiga). The expression plasmids were sterile-filtered and electroporated into CHO-S cells. Stable CHO-S-CLD18A1 and CHO-S-CLD18A2 cell lines were constructed in 96-well plates supplemented with G418. Stable cell lines were selected from the 96-well plates and cultured in the presence of G418, then scaled up stepwise. Dot blot analysis was performed using the anti-Claudin18 antibody [34H14L15] (abcam) to identify positively expressing CLD18A1 and CLD18A2 cell lines. NUGC-4-CLD18A1 and NUGC-4-CLD18A2 were constructed using the same method. Gastric adenocarcinoma cell NUGC-4 was purchased from Wuhan Jinkairui Co., Ltd., and the gastric adenocarcinoma cell NUGC-4 did not show positive expression of CLD18A2.

[0145] Example 2: Construction of Anti-CLD18A2 Nanobody Library

[0146] The CHO-S-CLD18A2 stably transfected cells in Example 1 were cultured at a rate of 1.0×10 7A healthy alpaca (Vicugnapacos) was immunized with 1 ml of complete Freund's adjuvant (Sigma) and then immunized again 21 days later for a total of three immunizations to stimulate B cells to express antigen-specific nanobodies. One week after the three immunizations, 30 ml of alpaca blood was collected using a vacuum tube. Lymphocytes were isolated using lymphocyte separation medium (Tianjin Haoyang Huake Biotechnology Co., Ltd.), and total RNA was extracted using Trizol. 3 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (Invitrogen) according to the manufacturer's instructions, and VHH was amplified using nested PCR with the following primers: the first-round PCR used an upstream primer 5′-CTTGGTGGTCCTGGCTG C-3′ (SEQ ID NO. 110) and a downstream primer 5′-GGTACGTGCTGTTGAACTGTTCC-3′ (SEQ ID NO. 111); the second-round PCR used the first-round PCR as a template with an upstream primer 5′-CATGCCATGACTGTGGCCCAGGCG GCCCAGKTGCAGCTCGTGGAGTC-3′ (SEQ ID NO. 112) and a downstream primer 5′-CATGCCAT GACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG-3′ (SEQ ID NO. 113) or 5′-CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG-3′ (SEQ ID NO. NO.114) for amplification. The target VHH nucleic acid fragment was recovered, digested with the restriction endonuclease SfiI (NEB), and inserted into the phage display vector pcomb3xss (Addgene plasmid #63890; RRID:Addgene_63890) that had been digested with the same enzyme, and ligated with T4 ligase (Takara). The ligation product was transformed into electroporation competent cells ER2738 to construct the Anti-CLD18A2 nanobody library. The library size was determined to be 1.23×10 by gradient dilution plating. 8 At the same time, 24 clones were randomly picked for colony PCR detection, and the results showed that the insertion rate of the constructed library was 100%.

[0147] Example 3: Screening and identification of anti-CLD18A2 nanobodies

[0148] 3.1 Screening of Anti-CLD18A2 Nanobodies:

[0149] The constructed Anti-CLD18A2 nanobody library was packaged using helper phage M13KO7 (NEB), and the titer of the recombinant phage display library was measured to be 5.7×10 13 PFU / ml. Take 18ml, 7×10 5 CHO-S-CLD18A2 cells / ml and 15ml, 3×10 6 CHO-S cells were centrifuged at 300 g for 5 minutes at 4°C, the supernatant was removed, the cells were resuspended in PBS, centrifuged again, and blocked with 2% skim milk powder (diluted with PBS) at room temperature for 1 hour. 11 PFU was added into the blocked CHO-S cells (about 4.5×10 7 The supernatant was added with about 1.5×10 7 The cells were incubated at room temperature for 1 hour in the blocked CHO-S-CLD18A2 stably transfected cells for binding. After centrifugation, the cells were resuspended and washed with PBS and washed five times. The washed phage-bound cells were incubated for 10 minutes with 1 ml of 0.1 Mgly-HCl 1 mg / ml BSA (pH 2.2) buffer, centrifuged, and the supernatant was neutralized with 1 M Tris-Cl, pH 8.0. The phage titer was determined to be 3.6 × 10 5 PFU / ml. The above phage eluate was amplified and the titer was determined to be 1×10 13 PFU / ml.

[0150] Take about 2×10 recombinant phages from the amplified library in the first round of panning 11 PFU, after blocking, 3 × 10 7 The CHO-S cells were incubated at room temperature for 30 minutes for differential selection, and centrifuged at 300g for 5 minutes at 4°C. The supernatant was washed with 1×10 7 The CHO-S-CLD18A1 cells were incubated at room temperature for 30 minutes and then subjected to differential selection. 7 CHO-S-CLD18A2 cells were incubated at room temperature for 1 hour for binding, centrifuged at 300g for 5 minutes at 4°C, resuspended and washed with PBS, washed 5 times with PBS, and incubated with 500μl of 0.1M gly-HCl 1mg / ml BSA (pH2.2) for 10 minutes for elution. After centrifugation at 300g for 5 minutes at 4°C, the supernatant was collected and neutralized with 1M Tris-Cl, pH8.0. The phage titer of the second round of panning was 6×10 5 PFU / ml, the phage eluate from the second round of panning was amplified and stored in 50% glycerol.

[0151] 3.2 First round screening using phage enzyme-linked immunosorbent assay (ELISA):

[0152] 80 single clones were picked from the phage titer determination plate after the second round of panning and cultured in 96-well plates. They were then infected and packaged with M13KO7 helper phage to obtain the accumulation of recombinant phage in the supernatant. CHO-S, CHO-S-CLD18A1, and CHO-S-CLD18A2 were cultured at 5×10 5 Each well of the 96-well plate was plated with 3% BSA and blocked with 3% BSA for 1 hour at room temperature. The 96-well plates corresponding to the three types of cells were centrifuged at 2000 rpm for 10 minutes in a well plate centrifuge and the supernatant was carefully removed. The monoclonal recombinant phage supernatant was diluted three times with 3% BSA in the 96-well plate, and added to the 96-well plate containing the three types of cells at a volume of 50 μl per well and incubated at room temperature for 1 hour. After washing with PBS three times, 100 μl of diluted HRP-anti-M13 antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added to each well and incubated at room temperature for 1 hour. After washing with PBS three times, TMB substrate was added and incubated at 37°C. After incubation for 5 minutes to develop color, 1M sulfuric acid was added to terminate the reaction and the OD450nm reading was taken. According to the ELISA test results of three 96-well plates of CHO-S, CHO-S-CLD18A1, and CHO-S-CLD18A2, the OD value of the negative wells (the wells corresponding to CHO-S) was 1.5 times or more and was considered positive. The clones that were negative on the CHO-S-CLD18A1 plate and positive on the CHO-S-CLD18A2 plate were selected.

[0153] 3.3 Second round of screening using enzyme-linked immunosorbent assay (ELISA) of E. coli expression supernatant:

[0154] Based on the colorimetric analysis of three cell lines (CHO-S, CHO-S-CLD18A1, and CHO-S-CLD18A2) in 96-well plates, clones were cultured. The pCoB3xss plasmids containing a single nanobody sequence were extracted and transformed into the E. coli Rosetta DE3 expression host. Each expressing clone was cultured and induced overnight with 0.2 mM IPTG at 30°C to allow periplasmic protein to leak into the culture supernatant. Culture supernatants from 16 of these clones were assayed by ELISA: 100 μl / well of a 1:5000 dilution of mouse anti-his tag antibody (R&D Systems, Inc.) was added and incubated at room temperature for 1 hour. After washing, 100 μl / well of a 1:10,000 dilution of HRP-goat anti-mouse IgG antibody (Thermo Scientific) was added and incubated at room temperature for 1 hour. After washing, the cells were visualized with TMB. One negative clone plasmid was selected as a negative control. The cell ELISA results are shown in Table 1. Specifically binding clones were further selected. These clones were sequenced individually, and their amino acid sequences were aligned. Repeated sequences were removed to obtain eight specific binding clones with different sequences. Table 1 exemplifies the specific binding clones obtained.

[0155] Table 1

[0156]

[0157]

[0158] Example 4: Preliminary evaluation and identification of anti-CLD18A2 nanobodies

[0159] 4.1 Expression and purification of anti-CLD18A2 nanobodies in host Escherichia coli

[0160] The specific positive sequence plasmid obtained by screening was used as a template, the upstream primer 5'-gtttaactttaagaaggagatatacatatgcaggtgcagctcgtggagtct-3' (SEQ ID NO.115) and the downstream primer 5'-ggccgcaagcttgtcgacggagctcgaattcttactaatggtgatggtgatggtgctg-3' (SEQ ID NO.116) were used for PCR amplification using the high-fidelity enzyme GVP8 (General Biosystems (Anhui) Co., Ltd.), the signal peptide sequence was retained at the 5' end of the sequence, and the histidine tag coding sequence was retained at the 3' end. The PCR product was electrophoresed and the gel was cut to recover a band of about 500 bp. The recovered PCR product was cleaved with pET32a digested with endonucleases NdeI and EcoRI.+ The vector (Novagen) was recombined and ligated using a recombination kit (Jinan Protein Technology Co., Ltd.) to construct an E. coli expression plasmid. This plasmid was then transformed into competent E. coli Top10F' cells, plated on ampicillin-resistant plates, and cultured overnight at 37°C. Colonies isolated from the ampicillin-resistant plates were isolated and sequenced to confirm correct insertion of the sequence into the pET32a+ vector.

[0161] The E. coli expression plasmid identified by sequencing was transformed into the E. coli Rosetta (DE3) expression host to construct an E. coli expression strain. Recombinant clones were selected and cultured on ampicillin-resistant plates. Expression was induced overnight with 1 mM IPTG at 30°C. The culture solution was ultrasonically disrupted and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was then purified using a Ni column (Borglon Biotechnology Co., Ltd.), achieving a final protein purity exceeding 90%.

[0162] 4.2 Specific Binding of Anti-CLD18A2 Nanobody Protein

[0163] CHO-S, CHO-S-CLD18A1, and CHO-S-CLD18A2 were added at 5×10 5 Cells were plated per well in a 96-well plate and blocked with 3% BSA for 1 hour at room temperature. Purified histidine-tagged anti-CLD18A2 nanobody was diluted to 2 μg / ml and 100 μl was added to the blocked cells. The cells were incubated at room temperature for 1 hour. After washing, 100 μl / well of a 1:5000 dilution of mouse anti-his tag antibody (R&D Systems, Inc.) was added and incubated at room temperature for 1 hour. After washing, 100 μl / well of a 1:10000 dilution of HRP-goat anti-mouse IgG antibody (Thermo Scientific) was added and incubated at room temperature for 1 hour. After washing, TMB was added for color development, and OD values ​​were measured at 450 nm. The results are shown in Table 2.

[0164] Table 2

[0165]

[0166]

[0167] 4.3 Affinity Identification of Anti-CLD18A2 Nanobody Protein

[0168] CHO-S-CLD18A2 was cultured at 5 × 10 5Cells were plated per well in a 96-well plate and blocked with 3% BSA for 1 hour at room temperature. Purified histidine-tagged CLD18A2 nanobody was serially diluted in 1% BSA and added to the blocked cells. The cells were incubated at room temperature for 1 hour. After washing, 100 μl / well of a 1:5000 dilution of mouse anti-his tag antibody (R&D Systems, Inc.) was added and incubated at room temperature for 1 hour. After washing, 100 μl / well of a 1:10000 dilution of HRP-goat anti-mouse IgG antibody (Thermo Scientific) was added and incubated at room temperature for 1 hour. After washing, TMB was added for color development, and the OD values ​​were measured at 450 nm. Data were processed and analyzed using GraphPad Prism v5.0. EC50 values ​​of the anti-CLD18A2 nanobody for CLD18A2 binding in cells were calculated to reflect the antibody's affinity for CLD18A2. The results are shown in Table 3.

[0169] Table 3

[0170] Sample name EC50 (nM) Anti-C18.2-6 1.53 Anti-C18.2-7 2.12 Anti-C18.2-15 2.89 Anti-C18.2-19 1.25 Anti-C18.2-20 1.87 Anti-C18.2-28 2.54 Anti-C18.2-32 2.21 Anti-C18.2-69 2.23

[0171] Example 5: Humanization of Anti-CLD18A2 Nanobody

[0172] The humanization method was completed using the universal framework transplantation method for VHH humanization established by Vincke C et al. (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath KJ Biol Chem. 2009; 284(5): 3273–3284). The universal humanized VHH framework h-NbBcII10FGLA (PDB ID: 3EAK) was designed based on sequence homology. The corresponding CDR region was replaced with the CDR region of the CLD18A2 nanobody, and individual amino acids in the FR2 region were further humanized according to the sequence of the humanized antibody DP-47. At least three humanized variants were obtained for each anti-CLD18A2 nanobody. The antibody sequences before and after humanization are shown in Table 4:

[0173] Table 4

[0174]

[0175]

[0176] Example 6: Preparation of Anti-CLD18A2 Related Antibodies Using Mammalian Cells

[0177] 6.1 Expression and Purification of Anti-CLD18A2 Nanobody and Fc Fusion Protein (Anti-C18.2-Fc)

[0178] The specific positive sequence and humanized sequence obtained by screening were used as templates, the upstream primer 5'-gtgctgctgctgtgggtgc caggatccaccgggcaggtgcagctcgtggagtc-3' (SEQ ID NO.117) and the downstream primer 5'-gcaggacttgggctcagaag acacggtgaccagggtcccctggcc-3' (SEQ ID NO.118) were used for PCR amplification using the high-fidelity enzyme GVP8 (Anhui General Biotechnology Co., Ltd.). The PCR product was electrophoresed and the gel was cut to recover a band of about 400 bp. The recovered PCR product was recombined with the pCDNA3.1 vector containing the signal peptide and human IgG1 Fc sequence (amino acid sequence SEQ NO.91) to construct the anti-CLD18A2 nanobody and human IgG1 The Fc-fused cell expression plasmid and the anti-CLD18A2 nanobody human IgG1 Fc-fused cell expression plasmid were extracted using an endotoxin-free plasmid extraction kit (Biomiga). The plasmid was mixed with the transfection reagent PEI (Polysciences, Inc.) at a ratio of 1:3 and allowed to stand for 30 minutes. The plasmid was then added to HEK293F cells and cultured in a shaking incubator at 37°C, 5% CO2 for 7 days. The supernatant was then centrifuged and collected. The supernatant was adjusted to pH 7.0 and loaded onto a Protein A affinity chromatography column (Borgron Biotechnology Co., Ltd.) and eluted with 100% 0.1M Gly-HCl (pH 3.0); the eluate was pre-added with 10% 1M Tris-HCl (pH 8.5). The 100% eluate was diluted to a conductivity of 4 ms / cm, adjusted to pH 5.5, and centrifuged (8000 rpm, 4°C, 10 min). The supernatant was adjusted to pH 5.0 and loaded onto a DSP chromatography column (Borglon Biotechnology Co., Ltd.) and linearly eluted with 0-60% eluate (20 mM NaAc, 0.5 M NaCl, pH 5.0) at a flow rate of 2 ml / min for 15 min.

[0179] 6.2 Expression and Purification of Positive Control Antibody ch-175D10

[0180] A chimeric antibody composed of the heavy chain of SEQ ID NO: 118 and the light chain of SEQ ID NO: 125 in US9751934B2 (named ch-175D10 in US9751934B2) was used as a control antibody. The polynucleotide sequence corresponding to its amino acid sequence was recombinantly ligated into the pCDNA3.1 vector and transiently expressed and purified in HEK293F cells using the same method as in Example 6.1.

[0181] 6.3 Comparative Analysis of Aggregate Content of Anti-C18.2-Fc Fusion Protein and Positive Control Antibody ch-175D10

[0182] Purity of Anti-C18.2-Fc and control antibody ch-175D10 was determined using SEC-HPLC-UV analysis. Detector: Agilent 1100LC; Detection wavelength: 214 nm; Mobile phase: 150 mM PB pH 7.0 + 5% isopropanol; Column: Superdex 200 Increase 5 / 150 GL; Run time: 15 minutes; Column temperature: 25°C.

[0183] Table 5

[0184] Sample name SEQ ID NO purity(%) Aggregates (%) Anti-C18.2-6-Fc 98 99％ <1％ Anti-C18.2-7-Fc 99 99％ <1％ Anti-C18.2-15-Fc 100 98％ <1％ Anti-C18.2-19-Fc 101 99％ <1％ Anti-C18.2-20-Fc 102 97％ <1％ Anti-C18.2-28-Fc 103 98％ <1％ Anti-C18.2-32-Fc 104 98％ <1％ Anti-C18.2-69-Fc 105 98％ <1％ Anti-C18.2-hu6V2-Fc 106 98％ <1％ Anti-C18.2-hu6V3-Fc 107 98％ <1％ Anti-C18.2-hu19V1-Fc 108 99％ <1％ Anti-C18.2-hu19V3-Fc 109 99％ <1％ ch-175D10 94％ >5％

[0185] From the results in Table 5, it can be seen that the multimers of the Anti-C18.2-Fc fusion protein of the present invention are significantly less than that of the control ch-175D10.

[0186] Example 7: Identification of the function of Anti-C18.2-Fc fusion protein

[0187] 7.1 Affinity determination of Anti-C18.2-Fc fusion protein for CLD18A2

[0188] The Anti-C18.2-Fc fusion protein was serially diluted with 1% BSA. HRP-Goat anti-Human IgG Fc (Novex) secondary antibody was used at a 1:20,000 dilution. The remaining cell ELISA procedures were the same as described in Example 3.2. GraphPad Prism v5.0 was used for data processing and graphical analysis. Binding curves and EC50 values ​​for Anti-C18.2-Fc binding to CLD18A2 in cells were generated to reflect the antibody's affinity for CLD18A2.

[0189] See the results Figure 1 The affinity of Anti-C18.2-hu19V1-Fc, Anti-C18.2-hu19V3-Fc, and Anti-C18.2-19-Fc was comparable, and superior to the positive control ch-175D10. The EC50 values ​​of Anti-C18.2-19-Fc, Anti-C18.2-hu19V1-Fc, Anti-C18.2-hu19V3-Fc, and ch-175D10 were 0.71 nM, 0.82 nM, 0.41 nM, and 2.59 nM, respectively. This indicates that humanization of the Anti-C18.2 nanobody did not significantly alter its affinity.

[0190] 7.2CDC testing

[0191] Serum from healthy individuals was used as complement source, and serum incubated at 65°C for 30 minutes was used as inactivated serum control. 4 Each well was plated in a 96-well plate, and the Anti-C18.2-Fc fusion protein to be tested, the negative control (IgG1 Fc fragment without the Fab region, amino acid sequence SEQ NO.91) and the positive control antibody ch-175D10 were diluted with culture medium in a gradient manner and added to the 96-well plate so that the final concentration was gradually decreased from 750nM to 0.05nM. After incubation at 37°C for 30 minutes, 5% serum from healthy people or inactivated serum control was added, and incubated at 37°C for 4 hours. LDH release detection was performed according to the instructions of the LDH detection kit (Dongren Chemical Technology (Shanghai) Co., Ltd.). The results are as follows Figure 2 and Figure 3 shown.

[0192] 7.3 ADCC Assay

[0193] Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were washed and resuspended in 1640 medium supplemented with 5% fetal bovine serum (FBS). Anti-C18.2-Fc fusion protein to be tested and positive control ch-175D10 were diluted to 500 nM in 5% FBS 1640 medium, and 50 μl of each was added to a 96-well plate. NUGC-4-CLD18A2 was washed and resuspended in 5% FBS 1640 medium to prepare approximately 2×10 5 / ml cell density, 50 μl was added to the corresponding 96-well plate. 100 μl / well was added to the resuspended PBMC cells to make the cell volume of PBMC 1×10 5 Each well was set to a target ratio of 10:1, and the cells were incubated at 37°C for 4 hours. The LDH release was then detected using the LDH detection kit (Dongren Chemical Technology (Shanghai) Co., Ltd.). Figure 4 shown.

[0194] 7.4 Comparison of the inhibitory activity of fusion protein on tumor growth in model mice with positive and negative controls

[0195] This study used a patient-derived xenograft (PDX) model of gastric cancer tissue to establish a tumor-bearing mouse model to determine the anti-tumor effect of the fusion protein. 3Tumor-bearing mice of different sizes were randomly divided into groups, with 4-6 mice per experimental group. Fifteen days after tumor implantation, different proteins and doses were administered. Changes in tumor volume and body weight were monitored during the administration period. Administration was twice a week, and monitoring was performed twice a week for 5 consecutive weeks. The dosage and administration method are shown in Table 6. Tumor volume measurement: The maximum longitudinal axis (L) and maximum width axis (W) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: V = L × W 2 / 2.

[0196] Table 6

[0197] sample SEQ ID NO. Dosage (mg / kg) Dosage Anti-C18.2-hu6V3-Fc 107 10 Tail vein injection Anti-C18.2-hu19V3-Fc 109 10 Tail vein injection IgG1 Fc 91 10 Tail vein injection

[0198] The experimental results are as follows Figure 5 As shown, over time, the tumor volume of mice vaccinated with Anti-C18.2-hu6V3-Fc and Anti-C18.2-hu19V3-Fc was well controlled relative to the control group and did not show a significant increase, indicating that Anti-C18.2-hu6V3-Fc and AntiC18.2-hu19V3-Fc had a significant tumor inhibitory effect.

[0199] Example 8: Vector Construction, Expression and Purification of Anti-CLDN18×CD3 Fusion Protein

[0200] 8.1 Anti-CLDN18×CD3 Fusion Protein Sequence Design

[0201] The Anti-CLDN18×CD3 fusion protein is a bispecific nanobody targeting CLD18A2 and CD3. It consists of two nanobodies: the Anti-CLD18A2 nanobody sequence is the same as that in the present invention, and the Anti-CD3 nanobody sequence is as reported in WO2016 / 180982. The Anti-CLD18A2 and Anti-CD3 nanobodies are linked by a GS sequence (SEQ ID NO. 132). To facilitate post-expression purification, six His amino acids are attached to the C-termini of the Anti-CLDN18×CD3 fusion protein and Anti-CD3 nanobody.

[0202] 8.2 Anti-CLDN18×CD3 fusion protein vector construction, expression, and purification

[0203] The sequences for the anti-CLDN18×CD3 fusion protein and anti-CD3 nanobody were optimized and synthesized by General Biosystems (Anhui) Co., Ltd. The products digested with XhoI and EcoRI were ligated into the expression vector pPIC9 using T4 ligase (Takara), transformed into competent Escherichia coli Top10F', plated on ampicillin-resistant plates, and incubated overnight at 37°C. Clones from the ampicillin-resistant plates were isolated, and plasmids were sequenced to confirm correct insertion of the sequences into the pPIC9 vector. The expression plasmids confirmed by sequencing were transformed into Pichia pastoris GS115. Recombinant clones were isolated and cultured on MD plates, and expression was induced with methanol. The culture medium after overnight expression was centrifuged at 12,000g for 10 minutes at 4°C. The supernatant was then purified using a Ni column (Borglon Biotechnology Co., Ltd.), achieving a final protein purity exceeding 90%.

[0204] Example 9: Identification of the function of Anti-CLDN18×CD3 fusion protein

[0205] 9.1 Cell Binding Specificity of Anti-CLDN18×CD3 Fusion Protein

[0206] Jurkat cells (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were used as CD3-positive cells, and CHO-S-CLD18A2 cells constructed in Example 1 were used as CLD18A2-positive cells to determine the cell binding activity of the anti-CLDN18×CD3 fusion protein constructed and expressed by the present invention.

[0207] CHO-S-CLD18A2 and Jurkat cells were grown at 5×10 5 The cells were plated in a 96-well plate at 400 μg / ml per well and blocked with 3% BSA at room temperature for 1 hour. The purified Anti-CLDN18×CD3 fusion protein, the Anti-C18.2-6 and Anti-C18.2-19 nanobodies prepared in Example 4, and the Anti-CD3 nanobodies were diluted with 1% BSA and added to the blocked cells. The cells were incubated at room temperature for 1 hour. The subsequent experimental procedures were the same as in Example 4.3. GraphPad Prism v5.0 was used for data processing and graphical analysis of the affinity values ​​of the Anti-CLDN18×CD3 fusion protein for CHO-S-CLD18A2 and Jurkat cells. The results are shown in Figure 5. Figure 6 and Figure 7The results showed that the Anti-CLDN18×CD3 fusion protein had good binding activity to CLD18A2-positive cells and CD3-positive cells. For binding to CLD18A2-positive cells, the EC50 values ​​for Anti-CLDN18×CD3-hu6V3 (SEQ ID NO. 133) were 6.50 nM, and for Anti-CLDN18×CD3-hu19V3 (SEQ ID NO. 134), the EC50 values ​​were 4.60 nM. For binding to CD3-positive Jurkat cells, the EC50 values ​​for Anti-CLDN18×CD3-hu6V3 were 9.39 nM, and for Anti-CLDN18×CD3-hu19V3, the EC50 values ​​were 10.36 nM.

[0208] 9.2 In vitro cell killing assay of Anti-CLDN18×CD3 fusion protein

[0209] In order to evaluate the cell-killing effect of the Anti-CLDN18×CD3 fusion protein, the present invention used T cells (Miaotong Bio) as effector cells to perform cytotoxicity tests.

[0210] Anti-CLDN18×CD3 fusion protein was diluted serially, and 50 μl was added to each well. CLD18A2 and CLD18A1 stably transfected cells were washed and resuspended in 5% FBS 1640 medium (Gibco) to prepare approximately 2×10 5 / ml cell density, add 50μl to each well of the corresponding 96-well plate. Resuspend human T lymphocytes from healthy donors in 5% FBS 1640 medium and add 1×10 5 Each well was filled with cells, with an effector-target ratio of 10:1. After incubation at 37°C for 4 hours, the LDH release was detected using an LDH detection kit (Dongren Chemical Technology (Shanghai) Co., Ltd.) to evaluate the cell killing effect of the Anti-CLDN18×CD3 fusion protein.

[0211] In the in vitro cytotoxicity experiment, Anti-CLDN18×CD3-hu6V3 and Anti-CLDN18×CD3-hu19V3 had a very significant killing effect on NUGC-4-CLD18A2 with high expression of CLD18A2 ( Figure 8 ), with EC50 values ​​of 26.15pM and 20.73pM respectively; for NUGC-4-CLD18A1 cells, Anti-CLDN18×CD3 fusion protein had no significant killing effect ( Figure 9). This indicates that in in vitro experiments, the Anti-CLDN18×CD3 fusion protein has a specific killing effect on NUGC-4-CLD18A2 cells with the participation of T lymphocytes, and is basically non-toxic to cells that do not express CLD18A2.

[0212] 9.3 Tumor Suppressor Activity of Anti-CLDN18×CD3 Fusion Protein

[0213] The present invention uses a patient-derived xenograft (PDX) model established with gastric cancer tissue in NSG mice to analyze the tumor inhibitory effect of the anti-CLDN18×CD3 fusion protein. 3 At about 14 days, the tumor-bearing mice were randomly divided into groups of 5 and intraperitoneally injected with 2 x 10 7 Healthy human PBMC cells. One day later, tumor-bearing mice were intraperitoneally injected with 5 μg (25 μg / ml, 200 μl PBS) of Anti-CLDN18×CD3 fusion protein once a day for 4 weeks, and the tumor volume was recorded twice a week.

[0214] According to the experimental results Figure 10 As can be seen, Anti-CLDN18×CD3-hu6V3 and Anti-CLDN18×CD3-hu19V3 had a significant growth inhibitory effect on transplanted tumors. Over time, the tumor volume in the experimental groups gradually decreased.

[0215] Example 10: VHH specifically targeting CLD18A2 for use in chimeric antigen receptors

[0216] The VHH specifically targeting CLD18A2 of the present invention was used to construct a chimeric antigen receptor. Table 7 lists the constructed chimeric antigen receptor and its structure (antigen recognition domain-hinge region-transmembrane region-intracellular signaling domain, and the co-expressed eGFP structure is not listed).

[0217] Table 7

[0218]

[0219] 10.1 Construction of Lentiviral Plasmid Vectors for Expressing Specific VHHs

[0220] As an example of construction, the present invention uses a third-generation self-inactivating lentiviral vector system, which has three plasmids: the envelope plasmid PMD2.G (purchased from Addgene) encoding the VSV-G protein; the packaging plasmid psPAX2 (purchased from Addgene) encoding the protein Gag / Pol and the Rev protein; and a recombinant expression vector encoding the target gene CAR constructed based on the empty vector pWPT-eGFP (purchased from Addgene). Based on pWPT-eGFP, the present invention constructs a universal lentiviral plasmid vector for expressing specific VHHs to facilitate the insertion of a variety of VHH sequences to construct a complete CAR structure, and co-expression of the target gene CAR and eGFP is achieved through T2A in the recombinant expression vector encoding the target gene CAR. T2A is a 2A peptide from Thoseaasigna virus with a "self-cleavage" function, which can achieve co-expression of upstream and downstream genes. The expression of CAR can be indirectly detected by detecting eGFP.

[0221] A sequence (SEQ ID NO. 141) containing the CD8 signal peptide and the CD8 hinge-CD28a-CD28b-CD3-T2A-egfp structure was synthesized. A multiple cloning site was inserted between the CD8 signal peptide and the CD8 hinge for insertion of VHH or other specific recognition sequences. The synthesized sequence was ligated to the pWPT-GFP vector (Addgene) using T4 ligase (Takara) via the Mlu1 and salI restriction sites at both ends. The ligation product was transformed into Top10F', plated on ampicillin-resistant plates, and clones were selected for culture and sequencing to construct the universal CART vector pWPT-x-CAR-28Z. Similarly, a protein containing CD8 signal peptide and CD8 hinge-CD28a-CD28b-CD137-CD3-T2A-egfp (SEQ ID NO. 142) was synthesized, wherein a multiple cloning site was inserted between the CD8 signal peptide and CD8 hinge for the insertion of VHH or other specific recognition sequences. The synthesized sequence was inserted into the pWPT-GFP vector that was similarly digested through the Mlu1 and SalI restriction sites at both ends to construct the CART universal vector pWPT-x-CAR-28-137Z.

[0222] 10.2 Construction of Lentiviral Plasmid Expressing Anti-CLD18A2 CAR

[0223] Using the anti-C18.2-hu19V3-Fc plasmid as a template, a forward primer (SEQ ID NO. 143) and a reverse primer (SEQ ID NO. 144) were used for PCR amplification using the high-fidelity enzyme GVP8 (General Biosystems (Anhui) Co., Ltd.). PCR products were recovered by gel electrophoresis and excision. The universal CART vector pWPT-x-CAR-28Z was double-digested with the endonucleases NdeI (Takara) and PstI (Takara) and recovered by gel electrophoresis. The recovered PCR product and vector were recombined and ligated using a recombination kit (Jinan Protein Technology Co., Ltd.). The ligated product was transformed into Top10F' and plated with ampicillin-resistant plates. Colonies were selected, cultured, and sequenced to construct the anti-CLD18A2 CAR lentiviral plasmid pWPT-aC18.2-hu19V3-28Z. Similarly, the anti-CLD18A2 CAR lentiviral plasmid pWPT-aC18.2-hu19V3-28-137Z was constructed by recombining the recovered PCR product with the vector pWPT-x-CAR-28-137Z digested with NdeI (Takara) and PstI (Takara). Following the above procedures, pWPT-aC18.2-hu6V3-28Z and pWPT-aC18.2-hu6V3-28-137Z were constructed.

[0224] 10.3 Plasmid transfection 293T packaging lentivirus

[0225] Lentiviral packaging follows conventional methods, which are roughly as follows: 5×10 6HEK-293T cells (ATCC) were seeded at a cell density in a 10 cm dish and cultured overnight in a 37°C, 5% CO2 incubator. The culture medium was DMEM (Gibco) containing 10% fetal bovine serum (Gibco). Approximately 2 hours before transfection, the culture medium was replaced with serum-free DMEM. When transfecting cells, in addition to using a lentiviral plasmid expressing CAR, plasmids (providing viral membrane proteins and structural proteins) psPAX2 and pMD2.0G were also required for co-transfection. 5 μg of the lentiviral plasmid expressing the target sequence CAR or the empty vector was used, 3.75 μg of psPAX2, and 1.25 μg of pMD2.0G were used. During transfection, add the mixture of the three plasmids mentioned above to 500 μl MEM medium. In another microcentrifuge tube, add 25 μl Lipofectamine 2000 transfection reagent (Thermo Fisher) to 500 μl MEM medium. Then, add the diluted transfection reagent on top of the diluted plasmid and mix well. After standing at room temperature for 20 minutes, add the mixture of plasmid and transfection reagent to a 10 cm culture dish, shake and mix well, and place it in a 37°C incubator. After 6 hours, replace it with DMEM medium containing 10% fetal bovine serum. Three days after cell transfection, the virus can be harvested. The culture supernatant containing the virus is transferred to a centrifuge tube and centrifuged at 4°C, 1500 rpm, for 5 minutes to remove the cells. Then, the virus-containing culture medium is filtered, aliquoted, and frozen at -80°C. In DMEM with 10% fetal bovine serum, 1×10 5 / mL cell density 100μL / well inoculated HEK-293T cells in 96-well culture plates, 37 ℃, 5% CO2 overnight culture. The next day, discard 50μL / well culture supernatant, add 50μL / well fresh culture medium and polybrene with a final concentration of 6μg / mL, incubate at 37 ℃, 5% CO2 for 30min. Add 10μL / well virus stock solution, 37 ℃, 5% CO2 culture. After 48h of infection, eGFP was detected by flow cytometry. The titer was calculated to be about 2×10 6 U / mL.

[0226] Example 11: CAR-T cells specifically targeting CLD18A2

[0227] 11.1aC18.2-CAR-T Preparation

[0228] Human peripheral blood mononuclear cells (Miaotong, Shanghai) were obtained from healthy human peripheral blood by density gradient centrifugation and sorted using CD3 MicroBeads (Miltenyi Biotec GmbH) according to the manufacturer's instructions. 6The cells were cultured with Quantum007 lymphocyte culture medium (purchased from PAA Laboratories GmbH) at a density of 1 / mL and Dynabeads were added at a cell:magnetic bead ratio of 1:1. TM Human T-Activator CD3 / CD28 (thermofisher) and recombinant human IL-2 (Shanghai Jinan) at a final concentration of 100 U / mL were stimulated and cultured for 24 h. T cells were then infected with the recombinant lentivirus (Example 10.3) at an MOI of ≈ 5. After infection, 5×10 cells were plated every other day. 5 The cells were passaged at a density of 1 / mL, and recombinant human IL-2 was supplemented to the lymphocyte culture medium at a final concentration of 100 U / mL. On day 8 of culture, flow cytometry revealed that eGFP-positive cells were considered to be CAR-positive due to the co-expression of eGFP and CAR. Uninfected T cells served as negative controls, and the positive rate of virus-infected T cells expressing different CARs was approximately 66.4%.

[0229] 11.2aC18.2-CAR-T killing experiment

[0230] We investigated the cytotoxicity of different aC18.2-CAR-T cells against NUGC-4-CLD18A2 cells and the CLD18A2-negative cell line NUGC-4-CLD18A1 in vitro. The effector-target ratio was set at 3:1, 1:1, and 1:3, respectively, with 10,000 target cells per well. Five replicate wells were prepared for each group, and the average of the five replicates was taken. After 16 hours of co-culture, LDH levels in the supernatant were measured using an LDH assay kit (Shanghai Dongren). Table 8 shows that at an effector-target ratio of 3:1, specific aC18.2-CAR-T cells were able to effectively kill CLD18A2-positive cells, while showing little to no killing of CLD18A2-negative cells. These results demonstrate that aC18.2-CAR-T cells can specifically kill CLD18A2-positive cells, and the killing effect is positively correlated with the effector-target ratio.

[0231] Table 8

[0232]

[0233] 11.3 In vitro cytokine release

[0234] CLD18A2-positive NUGC-4-CLD18A2 cells were co-cultured with aC18.2-CAR-T cells at a 1:1 ratio. After 24 hours of incubation, the culture supernatant was collected and cytokines were detected using IL-2 (R&D Systems, Inc.), TNF-α (R&D Systems, Inc.), and IFN-γ (R&D Systems, Inc.) according to the kit instructions. Figure 11 The results showed that the secretion of cytokines such as IL-2, TNF-α and IFN-γ in NUGC-4-CLD18A2 was significantly higher than that in negative cells NUGC-4-CLD18A1 when co-incubated with aC18.2-CAR-T.

[0235] 11.4 In vivo pharmacodynamic study of aC18.2-CAR-T

[0236] A subcutaneous transplant tumor model was established using NUGC-4-CLD18A2. 6 NUGC-4-CLD18A2 was subcutaneously inoculated into NOD / SCID mice until the average tumor volume of the mice reached 100-150 mm 3 At the same time, 100 mg / kg of cyclophosphamide was injected intraperitoneally to eliminate the immune cells of NOD / SCID mice, so that the adoptively transferred transgenic T lymphocytes could better exert their anti-tumor function. On the second day, 1.0×10 7 The growth of subcutaneous transplanted tumors was observed and measured using aC18.2-CAR-T cell aC18.2-hu19V3-28-137Z and a 28-137Z expressing Mock group as a control. Figure 12 It showed that aC18.2-CAR-T cells could significantly inhibit the growth of NUGC-4-CLD18A2 transplanted tumors.

[0237] In summary, the present invention effectively overcomes various shortcomings of the prior art and has high industrial utilization value.

[0238] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. An anti-CLD18A2 nanobody, wherein the complementarity determining region (CDR) of the anti-CLD18A2 nanobody comprises CDR1 to CDR3 with the amino acid sequences shown below: (1) the CDR1 with an amino acid sequence as shown in SEQ ID NO. 6, the CDR2 with an amino acid sequence as shown in SEQ ID NO. 21, and the CDR3 with an amino acid sequence as shown in SEQ ID NO. 35; or (2) the CDR1 amino acid sequence is as shown in SEQ ID NO. 8, the CDR2 amino acid sequence is as shown in SEQ ID NO. 23, and the CDR3 amino acid sequence is as shown in SEQ ID NO. 37; or (3) the CDR1 amino acid sequence is as shown in SEQ ID NO. 9, the CDR2 amino acid sequence is as shown in SEQ ID NO. 24, and the CDR3 amino acid sequence is as shown in SEQ ID NO. 38; or (4) a CDR1 with an amino acid sequence as shown in SEQ ID NO. 10, a CDR2 with an amino acid sequence as shown in SEQ ID NO. 25, and a CDR3 with an amino acid sequence as shown in SEQ ID NO. 36; or (5) The amino acid sequence of CDR1 is shown in SEQ ID NO.11, the amino acid sequence of CDR2 is shown in SEQ ID NO.26, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

39.

2. The Nanobody according to claim 1, wherein The anti-CLD18A2 nanobody includes a framework region FR, and the framework region FR includes FR1 to FR4 with the amino acid sequence shown below: (1) an amino acid sequence of FR1 as shown in SEQ ID NO. 3, 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. 29, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or (2) an amino acid sequence of FR1 as shown in SEQ ID NO. 2, an amino acid sequence of FR2 as shown in SEQ ID NO. 16, an amino acid sequence of FR3 as shown in SEQ ID NO. 31, and an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or (3) an amino acid sequence of FR1 as shown in SEQ ID NO. 2, an amino acid sequence of FR2 as shown in SEQ ID NO. 13, an amino acid sequence of FR3 as shown in SEQ ID NO. 31, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or (4) an amino acid sequence of FR1 as shown in SEQ ID NO. 1, an amino acid sequence of FR2 as shown in SEQ ID NO. 17, an amino acid sequence of FR3 as shown in SEQ ID NO. 30, or an amino acid sequence of FR4 as shown in SEQ ID NO. 41; or (5) FR1 with an amino acid sequence as shown in SEQ ID NO. 2, FR2 with an amino acid sequence as shown in SEQ ID NO. 18, FR3 with an amino acid sequence as shown in SEQ ID NO. 32, and FR4 with an amino acid sequence as shown in SEQ ID NO.

41.

3. The Nanobody according to claim 1, wherein The amino acid sequence of the anti-CLD18A2 nanobody includes: a) the amino acid sequence shown in any one of SEQ ID NO. 44 and SEQ ID NO. 46 to 49; or b) an amino acid sequence having a sequence identity of 80% or more to one of SEQ ID NO. 44 and SEQ ID NOs. 46 to 49, and having the function of the amino acid sequence defined in a).

4. The Nanobody according to claim 1, wherein The anti-CLD18A2 nanobody is a humanized antibody, and the amino acid sequence of the CLD18A2 nanobody is shown in SEQ ID NOs. 69, 77, 85, 71-74, 79-82, and 87-90.

5. A fusion protein of an anti-CLD18A2 nanobody, comprising the first domain of the nanobody according to any one of claims 1 to 4, and further comprising a second domain for prolonging the in vivo half-life and / or having a binding effect on effector cells.

6. The fusion protein according to claim 5, characterized in that The second domain comprises a combination of one or more of a serum albumin fragment, a polyethylene glycol fragment, and a nanobody that binds to HSA; and / or, the second domain comprises an immunoglobulin Fc region; And / or, the second domain comprises a molecule having affinity for and / or capable of binding to CD3 present on T cells.

7. The fusion protein according to claim 6, wherein The second domain includes a human immunoglobulin Fc region.

8. The fusion protein according to claim 6, wherein The human immunoglobulin Fc region includes mutations for changing Fc-mediated effector functions, wherein the effector functions include one or more combinations of CDC activity, ADCC activity, and ADCP activity; And / or, the immunoglobulin is selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is selected from a combination of one or more of IgG1, IgG2, IgG3, or IgG4 subtypes; and / or, the amino acid sequence of the immunoglobulin Fc region is selected from one of SEQ ID NOs. 91 to 95; And / or, a connecting peptide is provided between the first domain and the second domain.

9. The fusion protein according to claim 8, wherein The connecting peptide is selected from a flexible polypeptide chain consisting of alanine and / or serine and / or glycine, and / or the length of the connecting peptide is 3 to 40 amino acids.

10. An isolated polynucleotide encoding the Nanobody according to any one of claims 1 to 4, or encoding the fusion protein according to any one of claims 5 to 9.

11. An expression vector comprising the isolated polynucleotide according to claim 10.

12. An expression system comprising the expression vector according to claim 11 or the exogenous polynucleotide according to claim 10 integrated into its genome.

13. A method for preparing the Nanobody according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 9, comprising the steps of culturing the expression system according to claim 12 under conditions suitable for expressing the antibody or fusion protein, thereby expressing the antibody or fusion protein, and purifying and isolating the antibody or fusion protein.

14. An immunoconjugate, comprising the Nanobody according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 7.

15. The immunoconjugate of claim 14, further comprising a coupling moiety comprising a detectable label, a cytotoxin, a radioisotope, or a combination of one or more of a biologically active protein.

16. A detection kit comprising the Nanobody according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 9, or the immunoconjugate according to any one of claims 14 to 15.

17. A pharmaceutical composition comprising the Nanobody according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 9, or the immunoconjugate according to any one of claims 14 to 15.

18. The pharmaceutical composition according to claim 17, wherein Also included are pharmaceutically acceptable carriers.

19. An isolated polypeptide comprising an antigen recognition domain, a hinge region, a transmembrane region and an intracellular signaling domain, wherein the antigen recognition domain comprises the nanobody according to any one of claims 1 to 4.

20. A cell comprising the membrane-bound polypeptide of claim 19, wherein the cell is a T lymphocyte, a macrophage and / or a NK cell.

21. Use of the Nanobody according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 9, or the immunoconjugate according to any one of claims 14 to 15, or the pharmaceutical composition according to any one of claims 17 to 18, the polypeptide according to claim 19, or the cell according to claim 20 in the preparation of a medicament for treating a disease associated with cells expressing CLD18A2, wherein the disease associated with cells expressing CLD18A2 is selected from a combination of one or more of gastric cancer, esophageal cancer, pancreatic cancer, and lung cancer.

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