Novel CLDN18.2 binding molecules

Abstract

The present invention relates to a novel CLDN18.2-binding molecule, a nucleic acid molecule encoding the CLDN18.2-binding molecule, an expression vector and a host cell for expressing the CLDN18.2-binding molecule, and a method and pathway for producing the CLDN18.2-binding molecule.

Description

[Technical field]

[0001] Claiming priority This application claims priority to Chinese Patent Applications Nos. 201910437800.2 and 201910437806.X, filed on May 24, 2019, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing, the contents of which are incorporated herein by reference in their entirety.

[0003] The present application generally relates to antibodies. More specifically, the present application relates to a single domain antibody that specifically recognizes CLDN18.2, its preparation method and its use. [Background technology]

[0004] Cell junctions are communication structures between cells and are an important basis for mutual communication and synergy between adjacent cells in multicellular organisms. Generally, there are four types of junctions in animal cells: tight junctions, adherens junctions, gap junctions, and desmosomes / hemidesmosomes.

[0005] Tight junctions, also known as closed junctions, are structures formed between endothelial or epithelial cells that can prevent substances between tissues from diffusing through the intercellular space and can only enter cells by active transport. The structure of tight junctions is formed by protein interactions between dozens of Claudin proteins within and between cells, and the expression of these proteins has a certain degree of tissue specificity. CLDN18 is one of the important proteins of Claudin proteins present in tight junctions.

[0006] CLDN18 is a membrane protein with four transmembrane domains and contains two extracellular domains. There are two CLDN18 variants in the human body, CLDN18.1 and CLDN18.2. Both are distributed in different tissues, the former is mainly expressed in lung epithelial cells, and the latter is specifically expressed in gastric epithelial cells and not expressed in gastric stem cells. In terms of protein sequence, CLDN18.1 and CLDN18.2 have very high sequence similarity, and the main difference between them is in the N-terminus, with only 8 amino acids difference between them in the first extracellular domain. Except for the N-terminus, the C-terminus sequences are exactly the same.

[0007] Antibody therapy has become one of the most promising methods to treat cancer patients. Due to its expression specificity in tumor cells and normal tissues, CLDN18.2 is currently a highly potential target for antibody drugs. However, due to the high sequence similarity between CLDN18.1 and CLDN18.2, the development of antibodies against CLDN18.2 alone without those against CLDN18.1 has become very difficult and has been rarely reported worldwide. To date, IMAB362, developed by Ganymed, Germany, is the only clinically studied antibody targeting CLDN18.2. IMAB362 is a monoclonal antibody consisting of two protein heavy chains and two protein light chains. Currently, there is no single domain antibody targeting CLDN18.2.

[0008] Single domain antibodies, abbreviated as sdAb (single domain antibody), are antibodies consisting of a single monomeric variable antibody domain (e.g., antibody heavy chain variable domain). Like complete antibodies (e.g., IgG), they can selectively bind to a specific antigen. However, the molecular weight of single domain antibodies is much smaller than that of typical antibodies consisting of two heavy protein chains and two light protein chains. The first single domain antibodies were adapted from heavy chain antibodies found in camelids (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363 (6428): 446-448). These heavy chain antibodies found in camelids are also called VHH fragments. Currently, most research on single domain antibodies is based on the heavy chain variable domain.

[0009] Single domain antibodies have many advantages. For example, they usually exhibit high solubility, excellent thermal stability and tissue permeability, and because of the presence of a second pair of intramolecular disulfide bonds, they can further resist degradation by papain and the like. In addition, single domain antibodies can be expressed in various host cells, such as yeast, plant and mammalian cells, and have high expression levels, which provides a significant cost advantage. Due to their advantages, single domain antibodies are suitable for various biotechnological and therapeutic applications. For example, they can be used to treat diseases, including but not limited to cancer, infectious diseases, inflammatory diseases and neurodegenerative diseases.

[0010] Currently, there are already monoclonal antibody drugs that target CLDN18.2 that are being clinically investigated, but there is an urgent need to continue to develop antibodies that target CLDN18.2 as therapeutic agents. In this field, there is a need to develop new antibodies that target CLDN18.2, especially single domain antibodies that specifically recognize only CLDN18.2 but not CLDN18.1. Summary of the Invention

[0011] In a broad sense, the present disclosure provides novel compounds, preparation methods, compositions and products of antibodies. The advantages provided by the present disclosure are widely applicable to the fields of antibody therapy and diagnosis. More specifically, the present disclosure provides single domain antibodies targeting CLDN18.2, as well as preparation methods of the antibodies, expression vectors and host cells for expressing the antibodies, etc. The antibodies of the present disclosure provide methods and applications for treating or preventing diseases associated with Claudin proteins, particularly those associated with CLDN18.2.

[0012] The present inventors have discovered, for the first time, a CLDN18.2-binding molecule (eg, a single domain antibody targeting CLDN18.2) that can specifically bind to the extracellular domain 1 (ECD1) of human CLDN18.2.

[0013] The present disclosure includes at least the following embodiments, which are listed in order by "N" (where "N" represents a number). The following list is not exhaustive, and those skilled in the art may combine different technical solutions.

[0014] 1. A CLDN18.2 binding molecule comprising at least one immunoglobulin single variable domain, said immunoglobulin single variable domain comprising: (a) a CDR1 comprising an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:1, a CDR2 comprising an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:2, and a CDR3 comprising an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:3, and (b) a CLDN18.2 binding molecule comprising any one set of CDR1, CDR2 and CDR3 selected from CDR1 comprising an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:30, CDR2 comprising an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:31, and CDR3 comprising an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:32.

[0015] 2. (a) CDR1 has an amino acid sequence difference of 2 or less amino acids compared to SEQ ID NO:1, CDR2 has an amino acid sequence difference of 2 or less amino acids compared to SEQ ID NO:2, and / or CDR3 has an amino acid sequence difference of 2 or less amino acids compared to SEQ ID NO:3, or (b) The CLDN18.2 binding molecule of embodiment 1, wherein CDR1 has an amino acid sequence difference of no more than 2 amino acids with respect to SEQ ID NO:30 by amino acid addition, deletion or substitution, CDR2 has an amino acid sequence difference of no more than 2 amino acids with respect to SEQ ID NO:31 by amino acid addition, deletion or substitution, and / or CDR3 has an amino acid sequence difference of no more than 2 amino acids with respect to SEQ ID NO:32 by amino acid addition, deletion or substitution.

[0016] 3. (a) CDR1 has an amino acid sequence difference with respect to SEQ ID NO:1 by the addition, deletion or substitution of one amino acid, CDR2 has an amino acid sequence difference with respect to SEQ ID NO:2 by the addition, deletion or substitution of one amino acid, and / or CDR3 has an amino acid sequence difference with respect to SEQ ID NO:3 by the addition, deletion or substitution of one amino acid, or (b) The CLDN18.2 binding molecule of embodiment 1, wherein CDR1 has an amino acid sequence difference of one amino acid addition, deletion or substitution relative to SEQ ID NO:30, CDR2 has an amino acid sequence difference of one amino acid addition, deletion or substitution relative to SEQ ID NO:31, and / or CDR3 has an amino acid sequence difference of one amino acid addition, deletion or substitution relative to SEQ ID NO:32.

[0017] 4. The CLDN18.2 binding molecule of any one of embodiments 1 to 3, wherein said CLDN18.2 binding molecule is an antibody against CLDN18.2 or an antigen-binding fragment thereof.

[0018] 5. The CLDN18.2 binding molecule according to any one of embodiments 1 to 4, wherein said immunoglobulin single variable domain is a VHH, for example a VHH derived from a Camelidae (for example an alpaca).

[0019] 6. The immunoglobulin single variable domain comprises: (a) CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in the formula ISRGGX1T (X1 is T or S), and CDR3 having the amino acid sequence shown in the formula NAQAWDX2GTX3RYLEV (X2 is P or V, and X3 is F or I), and (b) A CLDN18.2 binding molecule according to any one of embodiments 1 to 5, comprising any one pair of CDR1, CDR2 and CDR3 selected from CDR1 having an amino acid sequence as shown in SEQ ID NO:30, 33, 34, 35, 36, 38, 39 or 40, CDR2 having an amino acid sequence as shown in the formula X4STGGTT (X4 is I or M), and CDR3 having an amino acid sequence as shown in the formula NVLVX5SGIGSX6LEV (X5 is I or V, and X6 is H or T).

[0020] 7. The immunoglobulin single variable domain comprises: (a) CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:2, and CDR3 having the amino acid sequence shown in SEQ ID NO:3; (b) CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:4, and CDR3 having the amino acid sequence shown in SEQ ID NO:5; or (c) CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:2, and CDR3 having the amino acid sequence shown in SEQ ID NO:6; (d) CDR1 having the amino acid sequence shown in SEQ ID NO: 30, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 32; (e) CDR1 having the amino acid sequence shown in SEQ ID NO: 30, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 37; (f) CDR1 having the amino acid sequence shown in SEQ ID NO: 33, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 62; (g) CDR1 having the amino acid sequence shown in SEQ ID NO: 33, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 37; (h) CDR1 having the amino acid sequence shown in SEQ ID NO: 34, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 37; (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 35, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 62; (j) CDR1 having the amino acid sequence shown in SEQ ID NO: 36, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 62; (k) CDR1 having the amino acid sequence shown in SEQ ID NO: 38, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 62; (l) CDR1 having the amino acid sequence shown in SEQ ID NO: 39, CDR2 having the amino acid sequence shown in SEQ ID NO: 41, and CDR3 having the amino acid sequence shown in SEQ ID NO: 37, or (m) A CLDN18.2 binding molecule according to any one of embodiments 1 to 6, comprising a CDR1 having the amino acid sequence shown in SEQ ID NO:40, a CDR2 having the amino acid sequence shown in SEQ ID NO:31, and a CDR3 having the amino acid sequence shown in SEQ ID NO:37.

[0021] 8. The immunoglobulin single variable domain comprises: (a) an amino acid sequence of SEQ ID NO:7, an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:7, or an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions relative to SEQ ID NO:7, and (b) A CLDN18.2 binding molecule according to any one of embodiments 1 to 7, comprising any one selected from the amino acid sequence set forth in SEQ ID NO:47, an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO:47, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions relative to SEQ ID NO:47.

[0022] 9. The immunoglobulin single variable domain comprises: (a) one or more of the amino acids at positions 1, 4, 5, 14, 16, 35, 47, 56, 58, 65, 92, 102, 105, or 121 of SEQ ID NO:7 have been modified (e.g., an amino acid substitution); or (b) A CLDN18.2 binding molecule according to any one of embodiments 1 to 8, in which one or more of the amino acids at positions 1, 4, 5, 11, 27, 28, 29, 30, 31, 32, 35, 51, 75, 76, 92, 100, 106 or 120 of SEQ ID NO:47 have been modified (e.g., an amino acid substitution and / or addition).

[0023] 10. The CLDN18.2 binding molecule according to any one of embodiments 1 to 9, wherein said immunoglobulin single variable domain comprises or consists of the sequence of any one of SEQ ID NOs: 7 to 14, SEQ ID NOs: 42 to 51 and 63.

[0024] 11. The CLDN18.2 binding molecule of any one of embodiments 1 to 10, wherein said CLDN18.2 binding molecule is a single domain antibody, such as a heavy chain single domain antibody, a chimeric antibody, or a humanized antibody.

[0025] 12. The CLDN18.2 binding molecule according to any one of embodiments 1 to 11, wherein said immunoglobulin single variable domain is fused to another molecule, said other molecule being, for example, the Fc domain of an immunoglobulin (for example IgG) or a fluorescent protein.

[0026] 13. The CLDN18.2 binding molecule of embodiment 12, wherein said CLDN18.2 binding molecule is a chimeric antibody comprising a VHH derived from a camelid and an Fc domain of human IgG (e.g., human IgG1 or IgG4).

[0027] 14. The CLDN18.2 binding molecule of embodiment 13, wherein said CLDN18.2 binding molecule is a chimeric antibody comprising a VHH derived from an alpaca and an Fc domain of human IgG1.

[0028] 15. The CLDN18.2 binding molecule of any one of embodiments 1 to 14, wherein said CLDN18.2 binding molecule binds to the extracellular domain 1 (ECD1) of human CLDN18.2.

[0029] 16. A CLDN18.2 binding molecule that competes with a CLDN18.2 binding molecule described in any one of embodiments 1-15 for the same epitope.

[0030] 17. A CLDN18.2 binding molecule according to any one of embodiments 1 to 16, which specifically binds to CLDN18.2 but does not bind to CLDN18.1.

[0031] 18. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a CLDN18.2 binding molecule as defined in any one of embodiments 1 to 17.

[0032] 19. An isolated nucleic acid molecule according to embodiment 18, comprising or consisting of any one of the sequences of SEQ ID NOs: 22-29, SEQ ID NOs: 52-61 and 64.

[0033] 20. An expression vector comprising an isolated nucleic acid molecule as described in embodiment 18 or 19.

[0034] 21. A host cell comprising an expression vector according to embodiment 20.

[0035] 22. The host cell of embodiment 21, wherein the host cell is a bacterial cell (e.g., E. coli), a fungal cell (e.g., yeast) or a mammalian cell.

[0036] 23. A pharmaceutical composition comprising at least one CLDN18.2 binding molecule as defined in any one of embodiments 1 to 17 and a pharma- ceutically acceptable vector.

[0037] twenty four. Expressing a CLDN18.2 binding molecule as defined in any one of embodiments 1 to 17 in a host cell of embodiment 21 or 22; A method for preparing a CLDN18.2 binding molecule as defined in any one of embodiments 1 to 17, comprising the step of isolating the CLDN18.2 binding molecule from the host cell.

[0038] 25. A method of treating a CLDN18.2 associated condition in a subject, comprising administering to the subject a therapeutically effective amount of a CLDN18.2 binding molecule as defined in any one of embodiments 1-17.

[0039] 26. The method of embodiment 25, wherein the CLDN18.2-associated condition comprises a disease involving cells expressing CLDN18.2 or a disease associated with cells expressing CLDN18.2.

[0040] 27. The method of embodiment 25 or 26, wherein said CLDN18.2 associated disease condition comprises cancer.

[0041] 28. The method of embodiment 27, wherein the cancer comprises bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, cancer of the genitals and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.

[0042] 29. The method of embodiment 28, wherein the cancer is gastric cancer.

[0043] 30. Use of a CLDN18.2 binding molecule as defined in any one of embodiments 1 to 17 in the preparation of a medicament for treating or preventing a disease associated with CLDN18.2.

[0044] 31. A kit for treating or preventing a disease associated with CLDN18.2, comprising a container containing a CLDN18.2 binding molecule as defined in any one of embodiments 1 to 17.

[0045] The above is a general description and may include simplifications, generalizations, and omissions of details as necessary. Therefore, those skilled in the art will appreciate that this general description is merely illustrative and is not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and / or devices and / or other subject matter described herein will become apparent in the teachings set forth herein. The general description is provided to briefly introduce some selected concepts, and these general descriptions are further described in the detailed description below. The above general description is not intended to identify key features or essential characteristics of the subject matter sought to be protected, nor is it intended to be used as an aid in identifying the scope of the subject matter sought to be protected. It is to be understood that the contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference in their entirety. [Brief description of the drawings]

[0046] [Figure 1a] The results of identification by flow cytometry of overexpressing cell lines and tumor cell lines in Example 1 are shown. Here, Fig. 1a shows the results of identification by flow cytometry of overexpressing cell lines of human CLDN18.2-HEK293T, human CLDN18.1-HEK293, mouse CLDN18.2-HEK293, and mouse CLDN18.1-HEK293. [Figure 1b]Figure 1 shows the results of flow cytometry identification of overexpressing cell lines and tumor cell lines in Example 1. Figure 1b shows the results of flow cytometry identification of human CLDN18.2-KATOIII tumor cell line. [Figure 2a] 1 shows a sequence comparison between multiple candidate antibodies, in which the full-length variable region sequences of the candidate antibodies and the CDR sequences therein are shown, and the differences in amino acid sequences between the candidate antibodies are shown. [Figure 2b] 1 shows a sequence comparison between multiple candidate antibodies, in which the full-length variable region sequences of the candidate antibodies and the CDR sequences therein are shown, and the differences in amino acid sequences between the candidate antibodies are shown. [Figure 3a] 1 shows the results of cross-binding experiments of candidate antibody NA1-S in cell lines overexpressing human CLDN18.2-HEK293T, human CLDN18.1-HEK293, mouse CLDN18.2-HEK293, and mouse CLDN18.1-HEK293. [Figure 3b] The results of cross-binding experiments of candidate antibodies NA3-S, NA5-S and NA6-S in cell lines overexpressing human CLDN18.2-HEK293T, human CLDN18.1-HEK293, mouse CLDN18.2-HEK293 and mouse CLDN18.1-HEK293 are shown. [Figure 4a] The results of a comparative experiment on the binding of candidate antibody NA1-S and a control antibody at the cell level are shown in Fig. 4a, where Fig. 4a shows the results of a comparative experiment on the binding of antibody NA1-S to human CLDN18.2-HEK293T overexpressing cells at the flow level. [Figure 4b] Figure 4b shows the results of a comparative experiment on the binding of candidate antibody NA1-S and a control antibody at the cell level. Figure 4b shows the results of a comparative experiment on the binding of antibody NA1-S and a control antibody at the flow level in human CLDN18.2-KATOIII tumor cell line. [Figure 5a] The results of a comparative experiment on the binding of candidate antibodies NA3-S, NA5-S, NA6-S and a control antibody at the cell level are shown. Here, Fig. 5a shows the results of a comparative experiment on the binding of antibodies NA3-S and NA6-S at the flow level in human CLDN18.2-HEK293T overexpressing cells. [Figure 5b]Figure 5b shows the results of a comparative experiment on the binding of candidate antibodies NA3-S, NA5-S, NA6-S and a control antibody at the cell level. Figure 5b shows the results of a comparative experiment on the binding of antibody NA5-S to human CLDN18.2-HEK293T overexpressing cells at the flow level. [Figure 5c] Figure 5c shows the results of a comparative experiment on the binding of candidate antibodies NA3-S, NA5-S, NA6-S and a control antibody at the cell level. Figure 5c shows the results of a comparative experiment on the binding of antibodies NA3-S, NA5-S, NA6-S and a control antibody at the flow level in human CLDN18.2-KATOIII tumor cell line. [Figure 6a] Complement-dependent cytotoxicity (CDC) mediated by candidate antibody NA1-S on human CLDN18.2-HEK293T overexpressing cell line and human CLDN18.2-KATOIII tumor cell line is shown, where FIG. 6a shows CDC mediated by antibody NA1-S on human CLDN18.2-HEK293T. [Figure 6b] Figure 6b shows complement-dependent cytotoxicity (CDC) mediated by candidate antibody NA1-S in human CLDN18.2-HEK293T overexpressing cell line and human CLDN18.2-KATOIII tumor cell line. Figure 6b shows CDC mediated by antibody NA1-S in human CLDN18.2-KATOIII tumor cell line. [Figure 7a] Figure 7 shows complement-dependent cytotoxicity (CDC) mediated by candidate antibodies NA3-S, NA5-S and NA6-S on human CLDN18.2-HEK293T overexpressing cell line and human CLDN18.2-KATOIII tumor cell line. Here, Figure 7a shows the CDC cytotoxicity effect mediated by antibody NA3-S on human CLDN18.2-HEK293T. [Figure 7b] Figure 7 shows complement-dependent cytotoxicity (CDC) mediated by candidate antibodies NA3-S, NA5-S, and NA6-S in human CLDN18.2-HEK293T overexpressing cell line and human CLDN18.2-KATOIII tumor cell line. Figure 7b and Figure 7c show the CDC killing effect of NA5-S and NA6-S, respectively, in human CLDN18.2-HEK293T cells. [Figure 7c]Figure 7 shows complement-dependent cytotoxicity (CDC) mediated by candidate antibodies NA3-S, NA5-S, and NA6-S in human CLDN18.2-HEK293T overexpressing cell line and human CLDN18.2-KATOIII tumor cell line. Figure 7b and Figure 7c show the CDC killing effect of NA5-S and NA6-S, respectively, in human CLDN18.2-HEK293T cells. [Figure 7d] Figure 7 shows complement-dependent cytotoxicity (CDC) mediated by candidate antibodies NA3-S, NA5-S and NA6-S in human CLDN18.2-HEK293T overexpressing cell line and human CLDN18.2-KATOIII tumor cell line. Figure 7d shows CDC mediated by antibody NA3-S in human CLDN18.2-KATOIII tumor cell line. [Figure 8a] Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by candidate antibodies NA1-S and NA3-S in human CLDN18.2-HEK293T and human CLDN18.2-KATOIII tumor cell lines, where Figures 8a and 8c show ADCC mediated by antibodies NA1-S and NA3-S in human CLDN18.2-HEK293T cell line. [Figure 8b] Figure 8b and Figure 8d show antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by candidate antibodies NA1-S and NA3-S in human CLDN18.2-HEK293T and human CLDN18.2-KATOIII tumor cell lines. Figure 8b and Figure 8d show ADCC mediated by antibodies NA1-S and NA3-S in human CLDN18.2-KATOIII tumor cell lines. [Figure 8c] Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by candidate antibodies NA1-S and NA3-S in human CLDN18.2-HEK293T and human CLDN18.2-KATOIII tumor cell lines, where Figures 8a and 8c show ADCC mediated by antibodies NA1-S and NA3-S in human CLDN18.2-HEK293T cell line. [Figure 8d]Figure 8b and Figure 8d show antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by candidate antibodies NA1-S and NA3-S in human CLDN18.2-HEK293T and human CLDN18.2-KATOIII tumor cell lines. Figure 8b and Figure 8d show ADCC mediated by antibodies NA1-S and NA3-S in human CLDN18.2-KATOIII tumor cell lines. [Figure 9a] The tumor inhibitory effect of candidate antibodies NA1-S and NA3-S in human CLDN18.2-HEK293T xenograft model is shown. Here, the mice are immunodeficient SCID mice, the doses of NA1-S and IMAB362 are calculated according to mass concentration, and the administration method is cross-administration of tail vein and abdominal cavity. [Figure 9b] The tumor inhibitory effect of candidate antibodies NA1-S and NA3-S in human CLDN18.2-HEK293T xenograft model is shown. Here, the mice are immunodeficient SCID mice, the doses of NA1-S and IMAB362 are calculated according to mass concentration, and the administration method is cross-administration of tail vein and abdominal cavity. [Figure 10a] The epitopes and key amino acid sites for binding of candidate antibodies NA1-S, NA3-S, and IMAB362 to CLDN18.2 are shown. Here, Figures 10a-10b show competitive binding assays of candidate antibodies NA1-S (Figure 10a) or NA3-S (Figure 10b) with IMAB362 in human CLDN18.2-HEK293T. [Figure 10b] The epitopes and key amino acid sites for binding of candidate antibodies NA1-S, NA3-S, and IMAB362 to CLDN18.2 are shown. Here, Figures 10a-10b show competitive binding assays of candidate antibodies NA1-S (Figure 10a) or NA3-S (Figure 10b) with IMAB362 in human CLDN18.2-HEK293T. [Figure 10c]The epitopes and key amino acid sites for binding of the candidate antibodies NA1-S, NA3-S, and IMAB362 to CLDN18.2 are shown. Figures 10c-10d show competitive binding assays of antibody IMAB362 and NA1-S (Figure 10c) or NA3-S (Figure 10d) in human CLDN18.2-HEK293T. [Figure 10d] The epitopes and key amino acid sites for binding of the candidate antibodies NA1-S, NA3-S, and IMAB362 to CLDN18.2 are shown. Figures 10c-10d show competitive binding assays of antibody IMAB362 and NA1-S (Figure 10c) or NA3-S (Figure 10d) in human CLDN18.2-HEK293T. [Figure 10e] The epitopes and key amino acid sites of the candidate antibodies NA1-S, NA3-S, and IMAB362 binding to CLDN18.2 are shown in Fig. 10e. The expression levels of mutant or wild-type CLDN18.2 in human CLDN18.2 mutant cell lines were measured using anti-CLDN18 antibodies. [Figure 10f] The epitopes and key amino acid sites for binding of candidate antibodies NA1-S, NA3-S, and IMAB362 to CLDN18.2 are shown. Figures 10f to 10g show the binding levels of NA1-S (Figure 10f) or NA3-S (Figure 10g) and IMAB362 to CLDN18.2 mutants in human CLDN18.2 mutant cell lines. [Figure 10g] The epitopes and key amino acid sites for binding of candidate antibodies NA1-S, NA3-S, and IMAB362 to CLDN18.2 are shown. Figures 10f to 10g show the binding levels of NA1-S (Figure 10f) or NA3-S (Figure 10g) and IMAB362 to CLDN18.2 mutants in human CLDN18.2 mutant cell lines. [Figure 10h]The epitopes and key amino acid sites for binding of candidate antibodies NA1-S, NA3-S, and IMAB362 to CLDN18.2 are shown. Figures 10h to 10i show the relative binding percentages of NA1-S (Figure 10h) or NA3-S (Figure 10i) and IMAB362 in human CLDN18.2 mutant cell lines compared to the wild-type cell line. [Figure 10i] The epitopes and key amino acid sites for binding of candidate antibodies NA1-S, NA3-S, and IMAB362 to CLDN18.2 are shown. Figures 10h to 10i show the relative binding percentages of NA1-S (Figure 10h) or NA3-S (Figure 10i) and IMAB362 in human CLDN18.2 mutant cell lines compared to the wild-type cell line. [Figure 11a] Figure 11 shows the cell killing efficiency of NA1-S (Figure 11a) or NA3-S (Figure 11b) and IMAB362 against CLDN18.2-HEK293T in the presence of Fab-ZAP, where the test is controlled with hIgG and Fab-ZAP, and prolonged incubation of cells with Fab-ZAP also has some toxic effect on cells. [Figure 11b] Figure 11 shows the cell killing efficiency of NA1-S (Figure 11a) or NA3-S (Figure 11b) and IMAB362 against CLDN18.2-HEK293T in the presence of Fab-ZAP, where the test is controlled with hIgG and Fab-ZAP, and prolonged incubation of cells with Fab-ZAP also has some toxic effect on cells. [Figure 12] The binding activity of NA3-S in CLDN18.2-HEK293T cells before and after the sequence was humanized is shown, where NA3S-H1 is the humanized molecule. [Figure 13a] 13a shows the antibody binding intensity and antibody binding positive rate of NA3S-H1 and IMAB362 at high concentrations in CLDN18.1-HEK293T cells. Here, FIG. 13a shows the binding intensity of NA3S-H1 and IMAB362 at different concentrations in CLDN18.1-HEK293T cells. [Figure 13b]Figure 13b shows the antibody binding strength and antibody binding positive rate of NA3S-H1 and IMAB362 in CLDN18.1-HEK293T cells at high concentrations. Figure 13b shows the binding positive rate of NA3S-H1 and IMAB362 in CLDN18.1-HEK293T cells at high concentrations (100 μg / ml), and the tests are all controlled by isotype antibodies. [Figure 14] Figure 2 shows the CDC cell killing efficiency of the humanized molecule NA3S-H1 in human CLDN18.2-KATOIII gastric cancer cells, where the control antibody is IMAB362. [Figure 15a] 15 shows the ADCC cell killing effect of NK cells mediated by NA3S-H1 and IMAB362 in different target cells. Here, Fig. 15a shows the ADCC cell killing effect mediated by NA3S-H1 and IMAB362 in human CLDN18.2-KATOIII target cells. [Figure 15b] Figure 15b shows the ADCC cell killing effect of NK cells mediated by NA3S-H1 and IMAB362 in different target cells. Figure 15b shows the ADCC cell killing effect mediated by NA3S-H1 and IMAB362 in human CLDN18.2-HEK293T target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] While the present invention may be embodied in many different forms, disclosed herein are specific and exemplary embodiments that demonstrate the principles of the present invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. In addition, any section headings used herein are for syntactic purposes only and should not be construed as limiting the subject matter described.

[0048] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Also, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, the singular forms "one," "one," and "the" as used in this specification and the appended claims include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a type of protein" includes a plurality of proteins, reference to "a cell" includes a mixture of cells, and so forth. In this application, when "or" is used, unless otherwise indicated, it means "and / or." Also, the use of the term "consisting of" and other forms (e.g., "comprising" and "containing") is not limiting. Also, the ranges provided in the specification and the appended claims include the endpoints and all values ​​between the endpoints.

[0049] Generally, the terms and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics and proteins, and nucleic acid chemistry and hybridization described herein are those known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present invention are generally performed by conventional methods known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6, 2001; thed., WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terms, laboratory procedures and techniques relating to analytical chemistry, synthetic organic chemistry and medicinal chemistry described herein are those terms known and commonly used in the art. In addition, any chapter headings used herein are for syntactic purposes only and should not be construed as limiting the subject matter described.

[0050] definition In order to better understand the present invention, definitions and interpretations of relevant terms are provided below.

[0051] The term "antibody" or "Ab" as used herein generally refers to any form of antibody that exhibits the desired biological activity or binding activity, including, but not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies. An antibody may comprise a heavy chain and a light chain. Heavy chains can be divided into μ, δ, γ, α, and ε, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into relatively conserved regions (called framework regions (FR)) and hypervariable regions (called complementarity determining regions (CDRs)) separated by the FRs. Each VH and VL consists of three CDRs and four FRs in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The distribution of amino acids in the various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883. The antibodies can have different antibody isotypes, such as IgG (including, for example, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0052] As used herein, the term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, within which further framework region modifications can be made.

[0053] The term "chimeric antibody" as used herein broadly refers to an engineered antibody derived from one or more regions of one antibody and one or more regions of one or more other antibodies. Specifically, a chimeric antibody comprises a variable region derived from a non-human animal antibody and a constant region of another antibody, for example, a variable region derived from a mouse and a constant region derived from a human. A chimeric antibody can also refer to a multispecific antibody having specificity for at least two different antigens.

[0054] As used herein, the term "CLDN18.2 binding molecule" refers to a molecule that specifically binds to CLDN18.2.

[0055] As used herein, the terms "CLDN18.2 antibody", "antibody against CLDN18.2", "antibody that specifically binds to CLDN18.2", "antibody that specifically targets CLDN18.2" and "antibody that specifically recognizes CLDN18.2" may be used interchangeably and refer to an antibody that can specifically bind to the Claudin protein CLDN18.2. In particular, in a specific embodiment, the term refers to an antibody that specifically binds to human CLDN18.2, and in particular an antibody that specifically binds to human CLDN18.2 but does not specifically bind to human CLDN18.1. The amino acid sequences of human CLDN18.2 and human CLDN18.1 are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively, and the amino acid sequences of mouse CLDN18.2 and mouse CLDN18.1 are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively.

[0056] The term "immunoglobulin single variable domain" or "ISV" as used herein is defined herein as an amino acid sequence that generally comprises an immunoglobulin fold or is capable of forming (i.e. by folding) an immunoglobulin fold under appropriate conditions (e.g. physiological conditions), i.e. is capable of forming an immunoglobulin variable domain (e.g. a VH, VL or VHH domain), and that forms (or is capable of forming under appropriate conditions) an immunoglobulin variable domain that comprises a functional antigen binding site (in the sense of forming a functional antigen binding site without the need for interaction with another immunoglobulin variable domain (e.g. a VH-VL interaction)).

[0057] As used herein, the term "Ka" is intended to represent the association rate of a particular antibody-antigen interaction, and the term "Kd" is intended to represent the dissociation rate of a particular antibody-antigen interaction. D " or "K D The term "dissociation constant" is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). D The K value can be determined using methods well established in the art. D A preferred method of determining the value is using surface plasmon resonance, preferably using a biosensor system such as a Biacore® system.

[0058] As used herein, the terms "specific binding" or "specifically binds to" refer to a non-random binding reaction between two molecules, for example, between an antibody and an antigen.

[0059] As used herein, the ability to "inhibit binding," "block binding," or "compete for the same epitope" refers to the ability of an antibody to inhibit the binding of two molecules to any detectable extent. In some embodiments, an antibody that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In some embodiments, the inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0060] As used herein, the term "high affinity" antibody refers to an antibody with an affinity of 1×10 -7 M or less, preferably 5×10 -8 M or less, more preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less, more preferably 1×10 -9 K is less than or equal to M D It refers to an antibody having a value.

[0061] As used herein, "EC 50 The term "median effective concentration," also referred to as "median effective concentration," refers to the concentration of a drug, antibody, or toxic agent that induces a response that is 50% between the baseline and maximum after a specific exposure time. In the context of this application, EC 50The unit of measurement is "nM". As used herein, the term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to as "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains, and usually have a specific three-dimensional structure and specific charge characteristics. For example, an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique conformation, and may be a "linear epitope" or a "conformational epitope". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In conformational epitopes, the interaction sites span amino acid residues that are isolated from each other in a protein. Antibodies can be screened according to their competitiveness for binding to the same epitope, detected by conventional techniques known to those skilled in the art. For example, antibodies that compete or cross-compete with each other for binding to an antigen (e.g., CLDN18.2) can be obtained by studying competition or cross-competition. International patent application WO 03 / 048731 describes a high-throughput method for obtaining antibodies that bind to the same epitope, based on cross-competition.

[0062] The term "isolated" as used herein refers to the state of a substance or component that has been manually obtained from a natural state. If a certain "isolated" substance or component exists in nature, it may be because the natural environment in which it is located has been altered, or the substance or component has been isolated from the natural environment, or both. For example, a non-isolated polynucleotide or polypeptide naturally exists in a living animal body, and the same high-purity polynucleotide or polypeptide isolated from the natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude artificial or synthetic substances mixed in, nor does it exclude other impurities that do not affect the activity of the isolated substance.

[0063] The term "isolated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody may also be substantially free of other cellular material and / or chemicals.

[0064] The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows for the expression of a protein encoded by a polynucleotide inserted therein, the vector is called an expression vector. The vector can transform, transduce or transfect a host cell to express the genetic material element carried in the host cell. Vectors are familiar to those skilled in the art and include, but are not limited to, plasmids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). A vector can contain multiple elements that control expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. Additionally, a vector can contain an origin of replication.

[0065] The term "host cell" as used herein refers to any type of cell system into which a vector can be introduced, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0066] Methods for producing an antibody of the invention using a host cell are conventional in the art and include expressing the antibody in a prokaryotic or eukaryotic cell, followed by isolating and purifying the antibody, usually to a pharmaceutically acceptable degree of purity. For example, in some embodiments, a nucleic acid encoding the antibody is inserted into an expression vector by standard techniques known in the art, and the expression vector is introduced into a suitable prokaryotic or eukaryotic host cell, the host cell, such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6® cells, yeast or E. coli cells, is cultured under conditions and for a time sufficient to produce the antibody of the invention or a functional fragment thereof, and the antibody is recovered from the cells (supernatant or cells after lysis) and purified. Conventional methods for producing antibodies are known in the prior art and are described, for example, in the review articles by Makrides, SC, Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ, Mol. Biotechnol. 16 (2000) 151-160; Werner, RG, Drug Res. 48 (1998) 870-880.

[0067] The term "identity" as used herein refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning or comparing the sequences. "Percentage of identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. In these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0068] The term "immunogenicity" as used herein refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the property of an antigen to stimulate the activation, proliferation and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the property of an antigen being able to form a specific immune response of antibodies or sensitized T lymphocytes in the immune system of an organism after stimulating the organism. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce the development of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the immune means.

[0069] The term "transfection" as used herein refers to the process of introducing nucleic acid into eukaryotic cells, particularly mammalian cells. Solutions and techniques used for transfection include, but are not limited to, transfection by chemical and physical methods such as lipofection, electroporation, etc. Many transfection techniques are known in the art, see for example Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0070] The term "fluorescence activated cell sorting" or "FACS" as used herein refers to a special type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments for performing FACS are known to those of skill in the art and are commonly available commercially. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, CA), Epics C from Coulter Epics Division (Hialeah, FL) and MoFlo from Cytomation (Colorado Springs, Colorado).

[0071] The term "subject" includes any human or non-human animal, preferably a human.

[0072] As used herein, the term "CLDN18.2-associated disease" refers to any disease caused or exacerbated by, or otherwise associated with, increased or decreased expression or activity of CLDN18.2 (e.g., human CLDN18.2).

[0073] As used herein, the term "cancer" refers to any solid tumor or non-solid tumor, such as leukemia, mediated by the growth, proliferation or metastasis of malignant cells that causes a medical disease.

[0074] The term "treatment" as used herein when referring to treating a condition generally relates to treatment and therapy of a human or animal to achieve a desired therapeutic effect, such as slowing down the rate of progression, inhibiting the progression of a condition, including halting the rate of progression, regressing the condition, ameliorating the condition, and curing the condition. Treatments that are preventative measures (i.e., prophylaxis) are also included. With respect to cancer, "treatment" may refer to inhibiting or alleviating the growth, proliferation, and / or metastasis of a tumor or malignant cells. With respect to a tumor, "treatment" includes removal of all or part of the tumor, inhibiting or alleviating tumor growth and metastasis, preventing or slowing tumor progression, or a combination thereof.

[0075] The term "therapeutically effective amount" as used herein relates to the amount of an active compound or a material, composition or dosage form containing an active compound that is effective to produce a desired therapeutic effect commensurate with a reasonable benefit / risk ratio when administered according to a desired treatment regimen. Specifically, "therapeutically effective amount" refers to an amount or concentration of an antibody or antigen-binding portion thereof effective to treat a disease state associated with CLDN18.2.

[0076] As used herein, the term "pharmaceutical acceptable" refers to a vector, diluent, excipient, and / or salt thereof that is chemically and / or physically compatible with other ingredients in the formulation and physiologically compatible with the recipient.

[0077] The term "pharmacologically acceptable vector and / or excipient" as used herein refers to a vector and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, and is known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, and ionic strength enhancing agents. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween® 80. Ionic strength enhancing agents include, but are not limited to, sodium chloride.

[0078] The term "adjuvant" as used herein refers to a non-specific immunostimulant that can enhance or change the type of immune response to an antigen in an organism when delivered to the organism together with an antigen or delivered to the organism in advance. In the prior art, there are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0079] CLDN18.2 binding molecule In some embodiments, the disclosure includes a CLDN18.2 binding molecule.

[0080] Generally, a CLDN18.2 binding molecule may include any molecule that specifically binds to CLDN18.2. In some cases, a "CLDN18.2 binding molecule" may include a "CLDN18.2 antagonist." A CLDN18.2 binding molecule or CLDN18.2 antagonist is a polypeptide or protein, such as an antibody, more specifically, an antibody that specifically binds to CLDN18.2 (e.g., human CLDN18.2).

[0081] The antibody includes, but is not limited to, a chimeric antibody, a humanized antibody, or a single domain antibody. In a specific embodiment, the CLDN18.2 binding molecule is a single domain antibody, which generally refers to an antibody that consists of a single monomeric variable antibody domain. Like a complete antibody, a single domain antibody can selectively bind to a specific antigen.

[0082] More specifically, the CLDN18.2 binding molecule is a heavy chain single domain antibody, and is a "VHH", "VHH antibody", "VHH domain", "VHH antibody fragment ... HH " or "nanobody". VHH molecules derived from camelid antibodies have one of the smallest complete antigen-binding domains known (approximately 15 KDa, or 1 / 10 of a normal IgG), making them highly suitable for delivery to dense tissues and for entering the confined spaces between macromolecules.

[0083] The single domain antibodies disclosed herein can be prepared by the skilled person by methods known in the art or any future methods. For example, VHHs can be obtained by methods known in the art, such as by immunizing camels to obtain VHHs that bind and neutralize the target antigen, or by cloning a library of VHHs of the invention using molecular biology techniques known in the art, followed by selection using phage display. In some embodiments, the single domain antibodies of the invention are naturally produced in camelids, i.e. are produced by immunizing camels with CLDN18.2 or a fragment thereof, using the techniques described herein for other antibodies.

[0084] In some embodiments, single domain antibodies are obtained by immunizing a llama or alpaca with the desired antigen and then isolating the mRNA encoding the heavy chain antibody. Gene libraries containing millions of cloned single domain antibodies are produced by reverse transcription and polymerase chain reaction. Screening techniques such as phage display and ribosome display help to identify clones that bind to the antigen. Phage display synthesizes an antibody library in phage, screens the library with the antigen of interest or an antibody-binding portion thereof, and isolates phages that bind to the antigen from which immunoreactive fragments can be obtained. Methods for preparing and screening such libraries are known in the art, and kits for producing phage display libraries may be commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). There are other methods available for producing and screening antibody display libraries (see, for example, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

[0085] When the most effective clone is identified, its DNA sequence can be optimized, for example by affinity maturation or humanization, to prevent the body's immune response to the antibody.

[0086] Thus, the single domain antibodies of the invention can be obtained by (1) isolating a VHH domain of a naturally occurring heavy chain antibody, (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain, (3) "humanizing" a naturally occurring VHH domain or expressing a nucleic acid encoding such a humanized VHH domain, (4) "camelizing" a naturally occurring VH domain from any animal species, in particular a mammalian species, such as human, or expressing a nucleic acid encoding such a camelized VH domain, (5) "camelizing" a "domain antibody" or "dAb" (see, for example, Ward et al., 1989, Nature 341:544-546), or expressing a nucleic acid encoding such a camelized VH domain, (6) preparing a protein, polypeptide or other amino acid sequence by synthetic or semi-synthetic techniques, (7) preparing a nucleic acid encoding a VHH by techniques used for nucleic acid synthesis and expressing the nucleic acid thus obtained, and / or (8) any combination thereof. Based on the disclosure herein, suitable methods and techniques for carrying out the above will be apparent to those of skill in the art, and include, for example, the methods and techniques described in more detail below.

[0087] Single domain antibodies are usually produced by PCR cloning of variable domain libraries into phage display vectors from cDNA of blood, lymph node or splenic lymphocytes obtained from immunized animals. Antigen-specific single domain antibodies are usually selected by panning the corresponding library on immobilized antigen (e.g. antigen coated on the plastic surface of a test tube, biotinylated antigen immobilized on streptavidin beads or membrane proteins expressed on the surface of cells). The affinity of sdAbs can be improved by simulating this strategy in vitro, for example by site-directed mutagenesis of the CDR regions, followed by further panning on immobilized antigen under additional stringent conditions (higher temperature, high or low salt concentration, high or low pH and low antigen concentration) (Wesolowski et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol (2009) 198:157-174).

[0088] Methods for preparing VHHs that specifically bind to an antigen or epitope are described in the literature, see for example R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645, 17 June, 2009 and WO 94 / 04678.

[0089] In some embodiments, the VHH in the CLDN18.2-binding molecule is fused to an Fc domain of an antibody (e.g., the Fc domain of an IgG (e.g., IgG1 or IgG4)). By fusing the VHH to the Fc domain, effector functions such as ADCC and CDC can be more effectively recruited. In addition, the fusion of the VHH to the Fc domain can not only help the CLDN18.2-binding molecule to form a dimer, but also help extend the in vivo half-life of the CLDN18.2-binding molecule.

[0090] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" as used herein refers to a form of cytotoxicity in which secreted antibodies binding to Fc receptors (FcR) present on certain cytotoxic effector 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. "Arming" the cytotoxic cells with antibodies is essential for such killing. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as the assay methods described in US Pat. No. 5,500,362 or US Pat. No. 5,821,337. Effector cells that can be used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0091] The term "complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its homologous antigen. To assess complement activation, a CDC measurement can be performed, for example, by the method described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0092] For ease of explanation, hereinafter the CLDN18.2 binding molecule will be described as a CLDN18.2 antibody.

[0093] CLDN18.2 antibodies capable of specifically binding to a specific epitope of CLDN18.2 In one embodiment, the present invention relates to single domain antibodies that specifically bind to CLDN18.2 but do not or do not substantially bind to CLDN18.1.

[0094] The present inventors have discovered, for the first time, a CLDN18.2-binding molecule (eg, a single domain antibody targeting CLDN18.2) that can specifically bind to the extracellular domain 1 (ECD1) of human CLDN18.2.

[0095] As described above, CLDN18.2 has two extracellular domains (ECDs), and the full-length sequence of human CLDN18.2 is shown in SEQ ID NO: 15. ECD1 is the amino acids 28 to 80 of SEQ ID NO: 15, as shown in SEQ ID NO: 19.

[0096] SEQ ID NO:15: [ka]

[0097] SEQ ID NO:19: [ka]

[0098] Mouse CLDN18.2 is also shown in SEQ ID NO:17.

[0099] SEQ ID NO:17: [ka]

[0100] For human and mouse CLDN18.1, the sequences are shown in SEQ ID NO:16 and SEQ ID NO:18, respectively.

[0101] SEQ ID NO:16: [ka]

[0102] SEQ ID NO:18: [ka]

[0103] CLDN18.2 antibodies containing CDRs with sequence identity to a particular sequence In some embodiments, a CLDN18.2 antibody of the present disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), wherein the VHH comprises CDR1, CDR2 and CDR3, and wherein CDR1 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:1, CDR2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, and CDR3 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:3.

[0104] Unless otherwise noted, the sequences are based on those used in Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5 thEd.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3 rd Amino acids can be assigned to each CDR based on one of the numbering schemes provided by Ed., Wily-VCH Verlag GmbH and Co.

[0105] The variable regions and CDRs in an antibody sequence can be identified based on general rules already developed in the art (e.g., the Kabat numbering system, as mentioned above) or by alignment of the sequence with a database of known variable regions. Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000, describe methods for identifying these regions. Exemplary databases of antibody sequences are described and can be obtained from the "Abysis" website at www.bioinf.org.uk / abs (maintained by AC Martin, Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Preferably, the sequences are analyzed using the Abysis database, which combines sequence data from Kabat, IMGT and the Protein Database (PDB) with structure data from the PDB, and see Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual, by Dr. Andrew C. Martin (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available at the website bioinforg.uk / abs). The Abysis database website further includes general rules developed for recognizing CDRs that can be used according to the teachings herein.

[0106] The percentage identity between two amino acid sequences can be determined by the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), and which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percentage identity between two amino acid sequences can also be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com), and which uses a Blossum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0107] Additionally or alternatively, the protein sequences of the present invention can be further used as a "query sequence" to perform searches against public databases, e.g., to identify related sequences. Such searches can be performed with the XBLAST program (version 2.0) of Altschul et al. (1990) J. MoI. Biol. 215:403-10. To obtain amino acid sequences homologous to the antibody molecules of the present invention, BLAST protein searches can be performed with the XBLAST program with a score=50 and wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used, see www.ncbi.nlm.nih.gov.

[0108] In another embodiment, the amino acid sequences of the CDRs are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each of the sequences given above.

[0109] CLDN18.2 Antibodies Comprising CDRs with Amino Acid Additions, Deletions, or Substitutions In some embodiments, a CLDN18.2 antibody of the disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), said VHH comprising CDR1, CDR2 and CDR3, and wherein CDR1 has an amino acid sequence difference of no more than two amino acids from SEQ ID NO:1 by amino acid addition, deletion or substitution, CDR2 has an amino acid sequence difference of no more than two amino acid addition, deletion or substitution from SEQ ID NO:2, and / or CDR3 has an amino acid sequence difference of no more than two amino acid addition, deletion or substitution from SEQ ID NO:3. For example, CDR1, CDR2 and CDR3 have an amino acid sequence difference of only one amino acid addition, deletion or substitution from the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0110] Preferably, the CDR of the isolated antibody or antigen-binding portion thereof contains no more than two amino acids or no more than one amino acid conservative substitution. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a physically or functionally similar residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) is replaced with the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which are incorporated herein by reference).

[0111] In some embodiments, the immunoglobulin single variable domain of the CLDN18.2 antibody comprises i) CDR1 of the amino acid sequence shown in SEQ ID NO:1, ii) a CDR2 having the amino acid sequence of the formula ISRGGX1T (wherein X1 is T or S); and iii) comprising a CDR3 of the amino acid sequence shown in the formula NAQAWDX2GTX3RYLEV (X2 is P or V, and X3 is F or I).

[0112] In some embodiments, the immunoglobulin single variable domain of the CLDN18.2 antibody comprises i) CDR1 of the amino acid sequence shown in SEQ ID NO: 30, 33, 34, 35, 36, 38, 39 or 40, ii) a CDR2 having the amino acid sequence of the formula X4STGGTT (wherein X4 is I or M); and iii) comprising a CDR3 having the amino acid sequence of the formula NVLVX5SGIGSX6LEV (X5 is I or V, and X6 is H or T).

[0113] CLDN18.2 antibodies containing CDRs In some embodiments, a CLDN18.2 antibody of the disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), the VHH comprising: (a) CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:3; (b) CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:5; or (c) comprises CDR1, CDR2 and CDR3 selected from CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:2 and CDR3 comprising the amino acid sequence shown in SEQ ID NO:6.

[0114] In some embodiments, a CLDN18.2 antibody of the disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), the VHH comprising: (a) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 30, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (b) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 30, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37; (c) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 62; (d) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37; (e) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 34, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37; (f) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 35, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 62; (g) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 62; (h) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 38, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 62; (i) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 39, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 37; or (j) comprises CDR1, CDR2 and CDR3 selected from CDR1 comprising the amino acid sequence set forth in SEQ ID NO:40, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:31 and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:37.

[0115] In some embodiments, a CLDN18.2 antibody of the disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), the VHH comprising: (a) CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:2, and CDR3 having the amino acid sequence shown in SEQ ID NO:3; (b) CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:4, and CDR3 having the amino acid sequence shown in SEQ ID NO:5; or (c) comprises CDR1, CDR2 and CDR3 selected from CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:2 and CDR3 having the amino acid sequence shown in SEQ ID NO:6.

[0116] In some embodiments, a CLDN18.2 antibody of the disclosure comprises at least one immunoglobulin single variable domain (e.g., a VHH), the VHH comprising: (a) CDR1 having the amino acid sequence shown in SEQ ID NO: 30, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 32; (b) CDR1 having the amino acid sequence shown in SEQ ID NO: 30, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 37; (c) CDR1 having the amino acid sequence shown in SEQ ID NO: 33, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 62; (d) CDR1 having the amino acid sequence shown in SEQ ID NO: 33, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 37; (e) CDR1 having the amino acid sequence shown in SEQ ID NO: 34, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 37; (f) CDR1 having the amino acid sequence shown in SEQ ID NO: 35, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 62; (g) CDR1 having the amino acid sequence shown in SEQ ID NO: 36, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 62; (h) CDR1 having the amino acid sequence shown in SEQ ID NO: 38, CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR3 having the amino acid sequence shown in SEQ ID NO: 62; (i) CDR1 having the amino acid sequence shown in SEQ ID NO: 39, CDR2 having the amino acid sequence shown in SEQ ID NO: 41, and CDR3 having the amino acid sequence shown in SEQ ID NO: 37; and (j) comprises CDR1, CDR2 and CDR3 selected from CDR1 having the amino acid sequence shown in SEQ ID NO:40, CDR2 having the amino acid sequence shown in SEQ ID NO:31 and CDR3 having the amino acid sequence shown in SEQ ID NO:37.

[0117] CLDN18.2 antibodies defined by VHH sequences In some embodiments, a CLDN18.2 antibody of the disclosure comprises at least one (e.g., one) immunoglobulin single variable domain (e.g., a VHH), the VHH comprising: (A) the amino acid sequence shown in SEQ ID NO: 7, 47 or 63; (B) an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 7, 47 or 63; or (C) Includes amino acid sequences having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions relative to SEQ ID NO:7, 47 or 63.

[0118] In some embodiments, a CLDN18.2 antibody of the disclosure comprises at least one (e.g., one) immunoglobulin single variable domain (e.g., a VHH), the VHH comprising: (A) the amino acid sequence shown in SEQ ID NO: 7, 47 or 63; (B) an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 7, 47 or 63; or (C) consists of an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions relative to SEQ ID NO:7, 47 or 63.

[0119] In other embodiments, the amino acid sequence of the VHH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each of the above sequences. As an illustrative example, an antibody may comprise a VHH having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:7, 47 or 63.

[0120] In some further embodiments, the CLDN18.2 antibodies of the present disclosure may include conservative substitutions or modifications of amino acids in the heavy chain variable region. As is understood in the art, conservative sequence modifications that do not eliminate antigen-binding properties can be made. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O' Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0121] In some embodiments, single domain CLDN18.2 antibodies of the disclosure have one or more amino acid modifications (e.g., amino acid substitutions) at positions 1, 4, 5, 14, 16, 35, 47, 56, 58, 65, 92, 102, 105, or 121 of SEQ ID NO:7. In some embodiments, single domain CLDN18.2 antibodies of the disclosure have one or more amino acid modifications (e.g., amino acid substitutions) at positions 1, 4, 5, 11, 27, 28, 29, 30, 31, 32, 35, 51, 75, 76, 92, 100, 106, or 120 of SEQ ID NO:47.

[0122] In some specific embodiments, the variable region of a single domain CLDN18.2 antibody of the present disclosure comprises any one of SEQ ID NOs:7-14 and SEQ ID NOs:42-51, 63.

[0123] In some specific embodiments, the variable region of a single domain CLDN18.2 antibody of the present disclosure consists of any one of SEQ ID NOs:7-14 and SEQ ID NOs:42-51, 63.

[0124] Nucleic acid molecules encoding antibodies of the present disclosure In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a CLDN18.2 antibody of the present disclosure, or a variable region fragment thereof.

[0125] The nucleic acids of the invention can be obtained by standard molecular biology techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acids encoding such antibodies can be recovered from a gene library.

[0126] The sequences of exemplary nucleic acid molecules of the invention are set forth in any one of SEQ ID NOs:22-29, SEQ ID NOs:52-61 and 64.

[0127] In some embodiments, the nucleic acid has at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to a nucleic acid molecule set forth in any one of SEQ ID NOs: 22-29, 52-61, 64, respectively. In some embodiments, the percentage of identity results from the degeneracy of the genetic code and does not alter the encoded protein sequence.

[0128] Using recombinant techniques known in the art, the nucleic acid molecule encoding the CLDN18.2 antibody of the present disclosure can be inserted into a vector for further cloning (amplification of DNA) or for expression. Many vectors are available. A vector or vector components typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence. The selectable marker gene serves to select a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, the selectable marker gene typically confers resistance to a drug (e.g., G418, hygromycin, or methotrexate) on a host cell into which the vector has already been introduced. In one embodiment, selectable marker genes can include a dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and a neo gene (for use in G418 selection). In another embodiment, antibodies can be produced by homologous recombination, as known in the art. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can bind specifically to genes encoding the antibody heavy chain).

[0129] In some embodiments, vector systems include mammalian, bacterial, yeast systems, etc., and include plasmids, including but not limited to pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., and other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). In one embodiment of the invention, the vector may be pET, such as pETbac, which contains a hexahistidine tag and a c-Myc-tag gene.

[0130] A vector containing a nucleic acid sequence encoding the CLDN18.2 antibody of the present disclosure can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria such as gram-negative or gram-positive bacteria, such as Enterobacteriaceae such as Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescens and Shigella, and Bacillus such as Bacillus subtilis and Bacillus licheniformis, and Pseudomonas such as Pseudomonas aeruginosa and Streptomyces.

[0131] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable host cells for expressing the CLDN18.2 antibodies of the present disclosure. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 56,500), Kluyveromyces spp. ... 36,906, K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g. Schwanniomyces occidentalis. occidentalis); as well as many other genera, species and strains of filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are commonly available and can be used in the present invention.

[0132] Other suitable host cells for expressing the CLDN18.2 antibody of the present disclosure are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. Currently, many baculovirus strains and variants and corresponding permissive insect host cells have been identified from hosts such as Spodoptera Frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster and Bombyx mori. Various virus strains used for transfection, such as the L-1 mutant of Autographa californica NPV and the Bm-5 strain of silkworm NPV, are publicly available, and according to the present disclosure, these viruses can be used for the transfection process when the CLDN18.2 antibody is expressed in a suitable host cell, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0133] Host cells are transformed with the vectors used to produce the CLDN18.2 antibodies of the present disclosure and cultured in conventional nutrient media modified as necessary for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0134] The host cells for producing the CLDN18.2 antibody of the present disclosure can be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnesr et al., Anal. Biochem. 102:255 (1980), US Pat. No. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469; WO 90 / 03430, WO 87 / 00195, or US Pat. Re. 30,985 can be used as the host cell culture medium. If necessary, hormones and / or other growth factors (e.g., insulin, transferrin or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN drug), trace elements (defined as inorganic compounds, usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source can be added to any of these media. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those used with the host cell previously selected for expression and will be apparent to those skilled in the art.

[0135] When using recombinant techniques, antibodies can be produced intracellularly, into the periplasmic space, or can be secreted directly into the medium. If antibodies are produced intracellularly, the first step is to remove particulate debris (host cells or lysed fragments), for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a method for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cells are lysed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethanesulfonyl fluoride (PMSF) within about 30 minutes. Cell debris can be removed by centrifugation. If antibodies are secreted into the medium, the supernatant from this expression system is usually first concentrated through a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any of the steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0136] For example, the antibodies prepared from the cells can be purified using hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting precipitation, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0137] After any preliminary purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography in an elution buffer having a pH between about 2.5 and 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0138] Drug Compositions In some embodiments, the present invention relates to a pharmaceutical composition comprising at least one CLDN18.2 binding molecule disclosed herein (eg, a CLDN18.2 antibody of the present disclosure) and a pharma- ceutically acceptable vector.

[0139] Components of the Drug Composition The pharmaceutical composition may optionally include one or more additional active pharmaceutical ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention may further be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, so as to enhance the immune response to the vaccine of the anti-CLDN18.2 antibody. The pharmaceutically acceptable vector may include, for example, a pharmaceutically acceptable liquid, gel, or solid vector, an aqueous vehicle, a non-aqueous vehicle, an antimicrobial agent, an isotonicity agent, a buffer, an antioxidant, an anesthetic, a suspending / dispersing agent, a chelating agent, a diluent, an adjuvant, an excipient, or a non-toxic auxiliary substance, a combination of various components known in the art, or more.

[0140] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propyl arsenate. As disclosed herein, the antibody or antigen-binding fragment may be oxidized in a solvent containing the antibody or antigen-binding fragment of the composition disclosed herein, which includes one or more antioxidants, such as methionine, that reduce the antibody or antigen-binding fragment thereof. The oxidation reduction can prevent or reduce the loss of binding affinity, improving the stability of the antibody and extending the shelf life. Thus, in some embodiments, the present invention provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. The present invention further provides a number of methods of combining the antibody or antigen-binding fragment thereof with one or more antioxidants, such as methionine, so that the antibody or antigen-binding fragment thereof can be protected from oxidation, extending its shelf life, and / or increasing its activity.

[0141] To further illustrate, pharma- ceutically acceptable vectors may include, for example, aqueous vectors such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection or Dextrose and Lactated Ringer's Injection; non-aqueous vectors such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; bacteriostatic or fungistatic concentrations of antimicrobial agents; isotonicity agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (TWEEN® 80); chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents as vectors can be added to drug compositions in multi-dose containers including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerin or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0142] Administration, Formulation and Dosage The pharmaceutical composition of the present invention can be administered in vivo to a subject in need thereof via various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The pharmaceutical composition of the present invention may be prepared into a solid, semisolid, liquid or gas form formulation, including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and administration route can be selected according to the intended application and treatment regimen.

[0143] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and sustained release formulations thereof.

[0144] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended or otherwise provided (e.g., in liposomes or other microparticles). These liquids may further include other pharma- ceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerin, vegetable oils, and the like. Examples of isotonic vehicles suitable for such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Similarly, the particular dosing regimen (i.e., dose, time, and repetition) is determined by empirical considerations, such as the particular individual and their medical history, as well as pharmacokinetics (e.g., half-life, clearance rate, and the like).

[0145] The frequency of administration can be determined and adjusted during the course of treatment, and based on the reduction in proliferation or number of tumorigenic cells, maintaining such reduction in tumor cells, reducing tumor cell proliferation or slowing metastatic progression. In some embodiments, the dosage of administration can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, sustained continuous release formulations of the therapeutic drug compositions of the present invention may be appropriate.

[0146] Those skilled in the art will understand that the appropriate dose may vary from patient to patient. Usually, the optimal dose is determined by balancing the level of therapeutic effect with any risk or adverse side effects. The selected dose level depends on many factors, including, but not limited to, the activity of the particular compound, administration, administration time, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the disease, and the species, sex, age, weight, disease state, general health and previous medical history of the patient. The amount of the compound and the route of administration are ultimately determined by the physician, veterinarian or clinician, but usually the dose is selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0147] Typically, the CLDN18.2 binding molecule can be administered in a variety of dosage ranges. In some embodiments, the CLDN18.2 binding molecule provided herein can be administered in a therapeutically effective amount of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In some of these embodiments, the antibody is administered at a dose of about 50 mg / kg or less, and in some of these embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In some embodiments, the administered dose can be varied over the course of treatment. For example, in some embodiments, an initial administered dose can be greater than a subsequent administered dose. In some embodiments, the administered dose can be varied over the course of treatment depending on the subject's response.

[0148] Regardless, the antibodies, or antigen-binding portions thereof, of the invention are preferably administered to a subject in need thereof as needed, with the frequency of administration being determined by one of skill in the art and will be considered, for example, by the attending physician based on the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc.

[0149] In some preferred embodiments, the therapeutic process for the antibody or antigen-binding portion thereof of the present invention includes administering multiple doses of the selected drug product over a period of several weeks or months. More specifically, the antibody or antigen-binding portion thereof of the present invention can be administered daily, every 2 days, every 4 days, weekly, every 10 days, every 2 weeks, every 3 weeks, monthly, every 6 weeks, every 2 months, every 10 weeks, or every 3 months. In this regard, it is understood that the dose can be modified and the interval can be adjusted based on the patient's response and clinical practice.

[0150] Doses and regimens of the drug compositions used in the disclosed treatments can also be empirically determined for individuals who receive one or more doses. For example, an individual can be administered increasing amounts of the drug compositions described herein. In selected embodiments, doses can be gradually increased or decreased based on empirically determined or observed side effects or toxicity, respectively. To assess the effectiveness of the selected composition, markers of the particular disease, condition, or condition can be tracked. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques, direct tumor biopsy and improved evaluation of tumor samples by microscopic examination, indirect tumor markers (e.g., PSA used in prostate cancer) or tumorigenic antigens identified based on the methods described herein, measurement of decreased pain or numbness, improved speech, vision, breathing, or other functional impairments related to the tumor, increased appetite, or improved quality of life or increased survival as measured by tests taken. Those skilled in the art will appreciate that dosages will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and previous and concurrent treatments used.

[0151] Formulations used for parenteral administration (e.g., intravenous injection) may contain a concentration of the CLDN18.2 binding molecule provided herein of about 10 μg / ml to about 100 mg / ml. In some embodiments, the concentration of the CLDN18.2 binding molecule may include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In other preferred embodiments, the concentration of the CLDN18.2 binding molecule comprises 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml. Calculated on a molar basis, in some embodiments, the concentration of the CLDN18.2 binding molecule can include, for example, 133 nM, 266 nM, 400 nM, 533 nM, 667 nM, 1.3 μM, 2 μM, 2.67 μM, 3.33 μM, 4 μM, 4.67 μM, 5.33 μM, 6 μM, or 6.67 μM.

[0152] Applications of the invention The CLDN18.2 binding molecules of the present invention have many in vitro and in vivo uses.

[0153] Treatment of disease The diseases and disorders associated with CLDN18.2 may be immune-related diseases or conditions, including but not limited to "diseases involving cells expressing CLDN18.2" or "diseases associated with cells expressing CLDN18.2" or similar expressions, and refer to the expression of CLDN18.2 in cells of diseased tissues or organs. In one embodiment, the expression of CLDN18.2 in cells of diseased tissues or organs is increased compared to the situation in the corresponding healthy tissues or organs. Increase means an increase of at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or more. In one embodiment, expression is found only in diseased tissues and expression in the corresponding healthy tissues is inhibited. According to the present invention, diseases associated with cells expressing CLDN18.2 include cancer diseases. Also according to the present invention, it is preferred that the cancer disease is a disease in which cancer cells express CLDN18.2.

[0154] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is usually characterized by unregulated cell growth. Examples of cancer include, but are not limited to, epithelial cancer, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancer include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, genital and reproductive cancer, Hodgkin's Disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumor, neuroectodermal cancer, spinal axis tumor, glioma, meningioma, and pituitary adenoma. The term "cancer" of the present invention further includes cancer metastasis. Preferably, the "cancer disease" is characterized by cells expressing CLDN18.2 and the expression of CLDN18.2 by cancer cells. The cells expressing CLDN18.2 are preferably cancer cells, preferably cancer cells of a cancer described herein.

[0155] According to the present disclosure, the term "tumor" or "tumor disease" refers to the abnormal growth of cells (called neoplastic cells, tumorigenic cells or tumor cells), preferably the formation of a swelling or lesion. "Tumor cells" refer to abnormal cells that grow by rapid uncontrolled cell proliferation and continue to grow after stimuli inducing new growth are discontinued. Tumors show partial or complete loss of structural organization and functional coordination with normal tissues, and usually form a special tissue mass that may be benign, pre-malignant or malignant. According to the present invention, a "cancer disease" is preferably a "tumor disease". However, in general, the terms "cancer" and "tumor" can be used interchangeably herein.

[0156] In one embodiment, the cancer according to the present disclosure relates to cancer cells expressing CLDN18.2. In one embodiment, the cancer is CLDN18.2 positive. In one embodiment, CLDN18.2 is expressed on the surface of the cells. In one embodiment, at least 50%, preferably 60%, 70%, 80% or 90% of the cancer cells are CLDN18.2 positive and / or at least 40%, preferably at least 50% of the cancer cells are positive for surface expression of CLDN18.2. In one embodiment, at least 95% or at least 98% of the cancer cells are CLDN18.2 positive. In one embodiment, at least 60%, at least 70%, at least 80% or at least 90% of the cancer cells are positive for surface expression of CLDN18.2.

[0157] In one embodiment, the cancer expressing CLDN18.2, the cancer of the cancer cells expressing CLDN18.2 or the CLDN18.2 positive cancer is selected from gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (e.g. non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer, and metastases thereof, in particular gastric cancer metastasis (e.g. Krukenberg tumor), peritoneal metastasis and lymph node metastasis. In one embodiment, the cancer is adenocarcinoma, in particular advanced adenocarcinoma. Particularly preferred cancer diseases are adenocarcinoma of the stomach, esophagus, pancreatic duct, bile duct, lung and ovary. In one embodiment, the cancer is selected from gastric cancer, esophageal cancer (in particular lower esophageal cancer), esophageal-gastric junction cancer and gastroesophageal cancer. In one particularly preferred embodiment, the cancer is gastroesophageal cancer, for example metastatic, refractory or recurrent advanced gastroesophageal cancer.

[0158] Additionally, the antibodies, or antigen-binding portions thereof, of the present disclosure can be used in combination with chemotherapy or radiation therapy.

[0159] Combination use with chemotherapy The antibodies, or antigen-binding portions thereof, may be used in combination with anti-cancer, cytotoxic, or chemotherapeutic agents.

[0160] The term "anti-cancer agent" or "anti-proliferative agent" refers to any agent that can be used to treat a cell proliferative disorder, such as cancer, including, but not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiation therapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapy and anti-metastatic agents, and immunotherapeutic agents. It should be understood that in selected embodiments above, such anti-cancer agents may comprise a conjugate and may be bound to the disclosed site-specific antibody prior to administration. More specifically, in some embodiments, a selected anti-cancer agent is linked to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate as described herein. Thus, such engineered conjugates are expressly contemplated as being within the scope of the present invention. In other embodiments, the disclosed anti-cancer agents are administered in combination with a site-specific conjugate comprising a different therapeutic agent as described above.

[0161] The term "cytotoxic agent" as used herein refers to a substance that is toxic to cells and that reduces or inhibits cell function and / or causes cell destruction. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcal enterotoxin A), fungi (e.g., alphasarcin and restrictocin), plants (abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii proteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, soapwort inhibitor, mitegellin, phenomycin, neomycin, and the trichothecenes), animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase, DNase I, including fragments and / or mutants thereof).

[0162] For the purposes of the present invention, a "chemotherapeutic agent" includes chemical compounds (e.g., cytotoxic or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation and / or survival of cancer cells. These chemical agents are usually targeted to intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancer cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules and inhibits cells from entering mitosis. In general, chemotherapeutic agents may include any chemical agent designed to inhibit or inhibit cancer cells or cells that have the potential to become cancerous or produce tumorigenic progeny (e.g., TICs). These agents are usually used in combination and are usually most effective in regimens such as CHOP or FOLFIRI.

[0163] Examples of anti-cancer agents that can be used in combination with the site-specific constructs of the invention (either as components of a site-specific conjugate or unconjugated) include alkylating agents, alkanesulfonates, aziridines, ethylenimines and methylmelamines, acetogenins, camptothecins, bryostatins, callystatins, CC-1065, cryptophycins, dolastatins, duocarmycins, eleutherobins, pancratistatins, sarcodictyins, spongistatins, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicins, bisphosphonates, esperamicins, chromoprotein enediyne antibiotic chromophores, amylins, cytosines ... alacinomycins, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, antiadrenergics, folinic acid folic acid supplements such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazine, procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, bacillosporin A, and diacetoxyscirpenol); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; metronidazole; Lecaptopurine; methotrexate; platinum analogs; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine, NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine; retinoids; capecitabine; combretastatins;Leucovorin; oxaliplatin; inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A (reducing cell proliferation), and pharmaceutically acceptable salts, acids or derivatives of any of the above. This definition further includes antihormonal agents to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates the production of estrogen in the adrenal gland, antiandrogens; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors, vaccines, PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors, ABARELIX® rmRH; vinorelbine and esperamicin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0164] Combined use with radiation therapy The present invention further provides a combination of an antibody or antigen-binding portion thereof with radiation therapy (i.e., any mechanism that locally induces DNA damage in tumor cells, such as, for example, gamma irradiation, X-rays, UV irradiation, microwaves, electronic emissions, etc.). Combination therapy using targeted delivery of radioisotopes to tumor cells is also contemplated, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 week to about 2 weeks. Radiation therapy can be administered to subjects suffering from head and neck cancer for about 6 to 7 weeks. Optionally, radiation therapy can be administered in a single dose or multiple consecutive doses.

[0165] diagnosis The present invention provides in vitro and in vivo methods for the detection, diagnosis and monitoring of proliferative disorders, and methods for screening cells from a patient to identify tumor cells, including tumorigenic cells. The methods include identifying individuals suffering from cancer or monitoring the progression of cancer in need of treatment, and include contacting the patient or a sample obtained from the patient (in vitro or in vivo) with an antibody described herein and detecting the presence or level of antibody that binds to a bound or free target molecule in the sample. In some embodiments, the antibody comprises a detectable marker or reporter molecule.

[0166] In some embodiments, binding of the antibody to particular cells in a sample can indicate that the sample is likely to contain tumorigenic cells, demonstrating that individuals with cancer can be effectively treated with the antibodies described herein.

[0167] Samples can be analyzed by a number of assays, including radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblot assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostic or diagnostic assays can include imaging or monitoring techniques known in the art, such as, for example, magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like, as known to those skilled in the art.

[0168] Medication Packs and Kits The present invention further provides drug packs and kits of one or more containers containing one or more doses of the antibody or antigen-binding portion thereof. In some embodiments, a unit dose is provided containing a predetermined amount of a composition, the composition comprising, for example, an antibody or antigen-binding portion thereof with or without one or more other reagents. For other embodiments, the unit dose is provided by a disposable pre-filled injectable syringe. In other embodiments, the composition contained in the unit dose may contain a buffer, such as saline, sucrose or analogues, phosphate, and / or be prepared in a stable and effective pH range. Or, in some embodiments, the composition is a conjugate composition, provided as a lyophilized powder, and can be reconstituted upon addition of an appropriate liquid, such as sterile water or salt solution. Any label on or associated with the container indicates that the enclosed conjugate composition is used to treat a selected tumor disease. In some preferred embodiments, the composition includes one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine.

[0169] The present invention further provides kits for producing single or multiple dose dosage units of the site-specific conjugate and, optionally, one or more anti-cancer agents. The kits include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers may be formed of a variety of materials, for example, glass or plastic, and contain a pharma- ceutical effective amount of the disclosed conjugates in conjugated or unconjugated form. In other preferred embodiments, the containers include a sterile access port (e.g., the containers may be intravenous solution bags or vials with stoppers pierceable by hypodermic needles). Such kits typically include a pharma- ceutical acceptable formulation of the conjugate manipulated in a suitable container, and, optionally, one or more anti-cancer agents in the same or different containers. The kits may further contain other pharma- ceutical acceptable formulations used for diagnosis or co-therapy. For example, in addition to an antibody or antigen-binding portion thereof of the invention, such kits may include any one or more anti-cancer agents, such as, for example, chemotherapeutic or radiotherapeutic agents, anti-angiogenic agents, anti-metastatic agents, targeted anti-cancer agents, cytotoxic agents, and / or other anti-cancer agents.

[0170] More specifically, the kit may have a single container containing the disclosed antibody or antigen-binding portion thereof, with or without other components, or may have different containers for each desired reagent. When providing combination therapeutic agents used in the conjugate, they can be combined in molar equivalents or premixed in a single solution with more of one component than the other. Alternatively, the conjugate and optional anticancer agent of the kit can be stored separately in different containers before being administered to a patient. The kit may further include a second / third container device for containing a sterile pharma- ceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and glucose solution.

[0171] When the components of the kit are provided as one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a physiological saline solution.However, the components of the kit can be provided as dry powder.When a reagent or component is provided as dry powder, the powder can be reconstituted by adding a suitable solvent.It is envisioned that the solvent may be provided in a separate container.

[0172] As briefly described above, the kits may further include equipment for administering the antibody or antigen-binding portion thereof and optional components to a patient, such as one or more needles, an IV bag or syringe, or even an eye dropper, pipette or other similar device by which the formulation can be injected or introduced into an animal's body or applied to a diseased area of ​​the body. The kits of the invention will typically further include equipment for containing vials or analogs, such as injection or blow molded plastic containers into which the desired vials and other equipment are placed and held, and other components sealed for commercial sale.

[0173] Advantages of this invention The development of antibodies against CLDN18.2, particularly against human CLDN18.2, has at least the following advantages:

[0174] First, human CLDN18.2 is highly expressed in cancer cells, but is specifically expressed only in normal cells, specifically in gastric epithelial cells, and therefore has low toxicity and side effects and is more promising for drug development.

[0175] Second, CLDN18.2 is expressed in the tight junctions of gastric epithelial cells, making it difficult for antibodies to act on loose cancer cells; even if epithelial cells are killed by antibodies, the stem cells underneath the epithelial cells do not express CLDN18.2 and can therefore differentiate to replenish the damaged gastric epithelial cells.

[0176] Third, antibodies against CLDN18.2 can not only kill cells through ADCC and CDC, but also mediate cell apoptosis by cross-linking the antibodies with CLDN18.2, and can inhibit cell proliferation to a certain extent.

[0177] Up to now, there has been no nanoantibody against CLND18.2, and the antibody caplacizumab has been approved for sale as a novel antibody, fully explaining the drug discovery potential of nanoantibodies. Such antibodies have significant advantages over other antibodies, such as their half-life length can be adjusted by chemical modification or modification by protein fusion, they have high permeability, can recognize cryptic epitopes that are inaccessible to general antibodies, are pepsin-resistant, acid-resistant and heat-resistant, are easy to manufacture, and are single-chain, so they can be easily combined with other types of antibodies to form diabodies and multivalent antibodies.

[0178] In some embodiments of the present invention, an antibody against CLDN18.2 is obtained by screening through an alpaca immune library, on the basis of which the C-terminus is fused with the Fc fragment of IgG1, and currently, the experimental results of CDC and ADCC at the cell level of some candidate antibodies show better activity than or at least equivalent activity to the control antibody. Combined with the characteristics of the CLDN18.2 target, this antibody can be used in cancer immunotherapy, with lower toxicity and side effects and better clinical efficacy, providing more drug options for patients.

[0179] Working Example The invention generally described herein will be more readily understood with reference to the following examples, which are provided by way of illustration and are not intended to limit the invention, and are not intended to represent that the following experiments are all or the only experiments performed.

[0180] Example 1 Construction and identification of overexpressing cell lines and tumor cell lines In this example, different types of overexpressing cell lines and tumor cell lines were constructed and identified by flow cytometry.

[0181] 1. Construction and Identification of Overexpressing Cell Lines The nucleic acid sequences of full-length human CLDN18.1 (SEQ ID NO:16), mouse CLDN18.2 (SEQ ID NO:17), and mouse CLDN18.1 (SEQ ID NO:18) are constructed in pLVX-puro plasmid (Clontech, Cat#632164). The resulting plasmids are then electrotransformed into HEK293 cells (ATCC® CRL-1573™). Through screening, overexpression cell lines expressing full-length human CLDN18.1 (human CLDN18.1-HEK293), overexpression cell lines expressing mouse CLDN18.2 (mouse CLDN18.2-HEK293), and overexpression cell lines expressing mouse CLDN18.1 (mouse CLDN18.1-HEK293) are obtained. Then, the IMAB362 antibody (obtained by expression and purification based on the sequence information disclosed in patent US 20180127489 A1) and the anti-CLDN18 antibody [34H14L15] (Abcam, ab203563) that recognizes the C-terminal CLDN18 intracellular segment (GFKASTGFGSNTKN, SEQ ID NO: 21) were identified by flow cytometry to obtain a successfully transformed overexpression cell line, the specific method of which is as follows:

[0182] 1.1 Electrotransformation First, resuscitate and culture HEK293 cells for 2–3 serial passages, and then soak the cells at 3 × 10 for 1 day before transfection. 5 The cells were seeded on a cell culture dish at a density of 10 cells / mL and were ready for use the next day when the cell confluence reached approximately 70%. The cells were digested with Trypsin (Gibco, 25200-072) containing 0.25% EDTA by volume for 2 min, then harvested and centrifuged at 100 g for 5 min at room temperature. The supernatant was discarded, and the cells were resuspended in 1x DPBS (source culture medium, B210) and counted. The cells were counted at 5x10 6The cells are collected by centrifugation, resuspended in 250 μL of Buffer R (Invitrogen, Neon™ Kit, PK10096) buffer, add 25 μg of the target plasmid, and mix gently and uniformly with a pipette.Then, place the suspension in an electroporator (Invitrogen, Neon™ Transfection System, MP922947) for electric transformation, and set the reaction conditions to 1100 V / 20 ms / 2 times for electric transformation.

[0183] 1.2 Cell culture After electrotransformation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume but without antibiotics, then the cells were inoculated into a 10cm x 10cm cell culture dish and cultured for 48h, then the cells were dispensed into a 96-well cell culture plate at an average density of 0.5 cells / well, and a final concentration of 2μg / mL puromycin (Gibco, A111138-03) was added as a screening pressure, and the growth status of the cell line clones was observed for about 2 weeks, and the cell line that formed the clones was selected and identified.

[0184] 1.3 Identification of overexpressing cell lines by flow cytometry The cell lines obtained in section 1.2 above were identified by flow cytometry, specifically as follows.

[0185] 1.3.1 Flow cytometric characterization of human CLDN18.2-HEK293T and mouse CLDN18.2-HEK293 cell lines The IMAB362 antibody was directly used to identify human CLDN18.2-HEK293T (Kangyuan Bochuang, KC-0986) and mouse CLDN18.2-HEK293 cell lines.

[0186] 1×10 5The cells are centrifuged at low speed (300g) and the supernatant is removed. The cells at the bottom of the centrifuge tube are rinsed once with the formulated FACS buffer (1x PBS buffer containing 2% FBS by volume), and 12.5μg / mL of antibody IMAB362 is added to the rinsed cells and incubated at 4℃ for 1h. Then, the cells are rinsed three times with the above FACS buffer, and 0.5μg of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) is added and incubated at 4℃ for 1h. Then, the cells are rinsed three times with FACS buffer, and 200μL of FACS buffer is added to resuspend the cells, and finally, the cells are detected by a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0187] 1.3.2 Flow cytometric characterization of human CLDN18.1-HEK293 and mouse CLDN18.1-HEK293 cell lines Since the IMAB362 antibody only recognizes CLDN18.2 and not CLDN18.1, it is necessary to identify human CLDN18.1-HEK293 and mouse CLDN18.1-HEK293 cell lines in combination with other methods. Considering the sequence identity of human CLDN18.1 and mouse CLDN18.1, first fix and rupture the cells according to the method in the specifications of the cell rupture kit (eBioscience, 88-8824-00), and then identify them by flow cytometry based on the recognition results of the anti-CLDN18 antibody [34H14L15] against the C-terminal CLDN18 intracellular segment and the specific binding results with the IMAB362 antibody.

[0188] The specific method is as follows: 1×10 5The cells are centrifuged at low speed (300g) and the supernatant is removed. The cells at the bottom of the centrifuge tube are rinsed once with FACS buffer, and 200 μL of IC fixative (eBioscience, 00-8222) is added to the rinsed cells, and incubated at 4 ° C for 1 h. Then, the cells are rinsed twice with rupture buffer (eBioscience, 00-8333), and the anti-CLDN18 antibody is added and incubated at 4 ° C for 1 h. After rinsing three times again with the rupture buffer, 0.5 μg of Alexa Fluor (registered trademark) 488 fluorescently labeled donkey anti-rabbit IgG H & L (abcam, ab150073) is added, and incubated at 4 ° C for 1 h, and finally detected by a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0189] At the same time, the IMAB362 antibody is used to measure whether it binds to human CLDN18.1-HEK293 and mouse CLDN18.1-HEK293 cell lines using the method described in 1.3.1 above.

[0190] The results of flow cytometry identification are shown in Figure 1a. As can be seen from Figure 1a, anti-CLDN18 antibody can recognize human CLDN18.2-HEK293T, human CLDN18.1-HEK293, mouse CLDN18.2-HEK293, mouse CLDN18.1-HEK293, HEK293 and HEK293T cells, while antibody IMAB362 only recognizes mouse CLDN18.2-HEK293 and human CLDN18.2-HEK293T, but does not recognize mouse CLDN18.1-HEK293 and human CLDN18.1-HEK293. This indicates that all four cell lines, including human CLDN18.2-HEK293T, human CLDN18.1-HEK293, mouse CLDN18.2-HEK293, and mouse CLDN18.1-HEK293, successfully expressed the corresponding CLDN18 protein.

[0191] 2. Construction and identification of overexpressing tumor cell lines The gastric cancer cell line KATOIII overexpressing human CLDN18.2 (human CLDN18.2-KATOIII tumor cell line) was constructed by lentivirus transfection and identified by antibody IMAB362. The specific method is as follows:

[0192] 5×10 healthy human gastric cancer cells KATOIII (ATCC® HTB-103®) 4 Add 1000 cells / well to lentivirus containing human CLDN18.2 sequence (SEQ ID NO:15) packaged at an MOI ratio of 30:1, mix thoroughly and homogenously, then add IMDM complete medium (Gibco, 12440061) containing 8 μg / mL polybrene (Polybrene, Sigma, 107689), mix homogenously, incubate at 37 °C, 5% CO2 for 20 h, remove the medium, replace with fresh IMDM complete medium and continue to incubate for 24 h, inoculate the transfected KATOIII cells into a 96-well plate at an average density of 0.5 cells / well, add puromycin at a final concentration of 2 μg / mL to perform resistance pressure screening, culture at 37 °C, 5% CO2 for 2 to 3 weeks, and select and identify clones.

[0193] As in Section 1.3.1 above, cell lines screened for resistance with antibody IMAB362 are subjected to identification by flow cytometry.

[0194] The result of identification by flow cytometry is shown in Figure 1b. As can be seen from Figure 1b, KATOIII cells not transfected with CLDN18.2 are hardly recognized by antibody IMAB362, indicating that this KATOIII cell line hardly expresses CLDN18.2 or the expression amount is extremely low. The human CLDN18.2-KATOIII tumor cell line constructed by lentivirus transfection can be identified by antibody IMAB362, indicating that this cell line was successfully constructed.

[0195] Example 2 Animal immunization and serum immune titer detection 1.Immunization In this embodiment, alpaca immunization is adopted. The specific procedure is as follows: As the immunogen, human CLDN18.2-HEK293T cell line (Kangyuan Bochuang, KC-0986) and hCLDN18.2-pLVX-puro plasmid containing human CLDN18.2 ECD1 (SEQ ID NO: 19) are adopted. Each of them is 2 × 10 7 10 human CLDN18.2-HEK293T cells (multiple subcutaneous injections) and 2 mg of plasmid (multiple intramuscular injections) were used to immunize alpacas (Nanchang Dajia Bioreactor) alternately every week. The alpaca's symbol is NSY002, and the total number of immunizations was 8. Finally, 2 × 10 7 Boost with human CLDN18.2-HEK293T cells.

[0196] 2. Measurement of serum immune titers The detection of immune titer is performed by ELISA based on the signal of immune serum in the antigen recombinant protein CLDN18.2 (GenScript, CP0007). The specific method is as follows:

[0197] One day before the immunological titration, dilute the antigen recombinant protein CLDN18.2 to 1 μg / mL with PBS to obtain a dilution solution. Add 30 μL of the dilution solution to the Elisa plate and coat it at 4°C overnight. On the day of serum immunological titration, rinse the coated plate twice with PBS, block it with PBST containing 5% skim milk powder by volume for 2 hours at room temperature, and rinse it twice more with PBS. Dilute the non-immunized negative serum and immunized serum with PBS in another 96-well dilution plate, dilute the first well 1000-fold, and dilute the remaining 7 wells 2-fold gradient. The diluted serum was added to the first Elisa plate coated with the antigen recombinant protein CLDN18.2 correspondingly, incubated at 37℃ for 1h, rinsed twice with PBS, and then added the secondary antibody MonoRab™ rabbit anti-camelidae VHH antibody (Jinsirui, A01862-200) at 1:5000, and finally read the OD value at a wavelength of 450nm by a microplate reader (Molecular Devices, SpecterMax 190). The results are shown in Table 1, and the alpaca immune titer is approximately 1:8000.

[0198] [Table 1]

[0199] Example 3 Construction and screening of an alpaca immune library After the animal immunization, 80 mL of blood was collected from the alpaca, and PBMCs were separated using Ficoll-Paque density gradient separation solution (GE, 17144003S) to be used for constructing an alpaca immune library. The specific method is as follows.

[0200] 15 mL of Ficoll-Paque density gradient separation solution is gradually added to a 50 mL centrifuge tube, followed by 15 mL of collected alpaca blood, so that the two liquids maintain a clear separation interface. Centrifuge at about 15°C, 400g, 20 min, acceleration 3, deceleration 0. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is the plasma mixture, the lower layer is red blood cells and granulocytes, the middle layer is the Ficoll-Paque liquid, and at the boundary between the upper and middle layers, there is a narrow band-like opaque layer mainly composed of PBMCs, that is, the PBMC cell layer. First, carefully aspirate the upper layer of the plasma mixture with a sterile Pasteur pipette, then aspirate the PBMCs with a new sterile Pasteur pipette to obtain isolated PBMCs. Rinse twice with PBS, horizontally centrifuge at 4°C, 1500 rpm for 10 min, and finally resuspend in 1.5 mL of PBS and count with a cytometer (CountStar, CountStar Altair).

[0201] RNA was extracted from the isolated PBMCs and reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). Since the molecular form of alpaca antibodies is different from that of general antibodies, and does not contain a light chain, and the heavy chain does not contain CH1, primers were designed at the front end and CH2 of the VH germline gene to obtain two fragments of different sizes by PCR, and the smaller target fragment was recovered by tapping. Degenerate primers containing NcoI and NotI restriction enzyme cleavage sites were designed by aligning all the sequences of all the V genes and J genes of the VHH antibody, and then all the VHH genes were amplified using the recovered DNA fragment products as templates, and finally the gene fragment of the target antibody was inserted into a vector for phage display by double enzyme cleavage and ligation, and the VHH was fused to the GIII gene at the C-terminus. The ligation products are recovered by a recovery kit (Omega, D6492-02) and finally transformed into competent E. coli SS320 (Lucigen, MC1061 F) by an electroporator (Bio-Rad, MicroPulser), which is then spread onto 2-YT solid plates with ampicillin resistance (prepared according to mass volume g / mL with 1.5% tryptone, 1% yeast extract, 0.5% NaCl, and 1.5% agar). To calculate the library size, the total number of all clones formed by electrotransformation, i.e. the library size, is calculated from the clones formed on the plate after dilution with 1 μL of bacterial solution. The library size of this immune library is 1 × 10 9 It is cfu.

[0202] Based on library size, 50 ODs (one OD is 5 × 10 8cfu) of alpaca library bacteria are selected and added to fresh 2-YT liquid medium to make the initial OD value 0.05. Culture at 37°C and 220 rpm until logarithmic growth phase, at which time VSCM13 helper phage is added in an amount 5 times the number of bacteria, mixed thoroughly and uniformly, left to stand for 30 min, then cultured at 220 rpm for 1 h, centrifuged at 10000 rpm for 5 min, discarded the supernatant, replaced with C+ / K+2-YT medium, and cultured at 30°C and 220 rpm overnight. The next day, centrifuge at 13000g for 10 min, add 20% PEG / NaCl (prepared with a volume concentration of 20% PEG6000 and 2.5M NaCl) to the supernatant to precipitate and obtain phages corresponding to the alpaca library, rinse once with PBS, and then use for phage screening.

[0203] The cell screening method uses human CLDN18.2-HEK293T cell line as the screening antigen to screen antibodies against human CLDN18.2 from the phage display library, and the specific method is as follows: Culture human CLDN18.2-HEK293T or human CLDN18.1-HEK293 in a T25 culture flask. When the cells grow to about 90% density, the growth condition is optimal. Remove the culture supernatant, rinse once with PBS (Genbyou, B310KJ), then add 5mL of 4% paraformaldehyde (Seiko, E672002-0500) to fix for 1h, and finally rinse twice with PBS, which can be used as an antigen material for phage cell screening. During screening, the phages corresponding to the alpaca library were first incubated with immobilized human CLDN18.1-HEK293 cells in a culture flask at room temperature for 1 h, and then the supernatant phages after adsorption were aspirated and co-incubated with immobilized human CLDN18.2-HEK293T cells in a culture flask for 2 h. After rinsing twice with PBS, 3 mL of glycine-HCl (pH 2.0) was added and mixed gently and uniformly for 10 min to elute the phages that specifically bind to the target membrane protein CLDN18.2. The eluted supernatant was then used to infect logarithmic phase SS320 bacteria (Lucigen, 60512-1), which were then left to stand for 30 min and then cultured at 220 rpm for 1 h. VSCM13 helper phages were then added, left to stand for 30 min, and cultured at 220 rpm for 1 h. The phages were then centrifuged and collected for 1 h. + / K + The medium was replaced with 2-YT, and the finally obtained phage was used for the second round of screening. This was repeated, and 10 randomly selected clones were subjected to sequence analysis each time. As a result, after three rounds of screening, there was obvious enrichment of sequences after the third round of screening.

[0204] The clones screened for the third time are selected to prepare phage supernatants in 96-well plates and screen positive clones against CLDN18.2 recombinant protein by phage ELISA, then all positive clones are selected for sequencing analysis, and then the clones with unique sequences are prepared as phage supernatants and further verified at the flow level to screen for candidate antibodies that bind only to human CLDN18.2 but not to human CLDN18.1.

[0205] The specific flow-level verification method is as follows.

[0206] First, 1×10 5 Human CLDN18.2-HEK293T and human CLDN18.1-HEK293 cells were centrifuged at low speed at 500g to remove the supernatant, and the cells were rinsed with FACS buffer as described in 1.3.1, and then the supernatant was prepared with phages blocked with 10% FBS (Gibco, 15140-141) for 1 h, incubated at 4 ° C for 1 h, followed by rinsing twice with FACS buffer, adding anti-M13 mouse monoclonal antibody (Sino Biological, 11973-MM05T) at 1:50, and incubating at 4 ° C for 1 h. Then, rinsing twice with FACS buffer, adding APC-labeled anti-mouse Fc secondary antibody (Jackson, 115136071), incubating at 4 ° C for 1 h, rinsing twice with FACS buffer, and finally detecting by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0207] Specific results are shown in Table 2 below.

[0208] [Table 2]

[0209] As can be seen from the above results, all of the antibodies of the present disclosure can specifically bind to human CLDN18.2.

[0210] The corresponding candidate antibodies are named by their clone numbers, and the amino acid sequences of each candidate antibody are shown in Tables 3 and 4 below.

[0211] [Table 3]

[0212] [Table 4-1] [Table 4-2] [Table 4-3]

[0213] The amino acid sequences of the screened candidate antibodies were compared, and the results are shown in FIG.

[0214] A comparison of the amino acid sequence identities of the variable regions between each of the candidate antibodies is shown in Tables 5-1 and 5-2 below.

[0215] [Table 5-1]

[0216] [Table 5-2]

[0217] Example 4 Production and expression of chimeric VHH-Fc(hIgG1) antibodies CLDN18.2 is highly expressed in cancer cells such as gastric cancer, and antibody drugs targeting such tumor-related targets can kill tumors by complement-dependent cytotoxicity (CDC) and antibody-dependent, cell-mediated cytotoxicity (ADCC). In this example, chimeric antibodies are designed based on the candidate nanobodies screened in Example 3, expressed, and used in subsequent CDC and ADCC experiments. Considering the specificity of the target, when the candidate nanobody genes are constructed into the transient expression plasmid pcDNA3.4 (Thermofisher, A14697), a human IgG1 Fc fragment is inserted into the C-terminus of the candidate nanobody. [ka] and this fragment contains the joining region and the constant region of IgG1 and is used to mediate effects such as ADCC and CDC. In this example, candidate nanobody A-2-4 was selected, and a human IgG1 Fc fragment was fused to it to obtain chimeric antibody NA1-S (also referred to as "candidate antibody NA1-S", "antibody NA1-S" or "NA1-S antibody" in this disclosure); candidate nanobodies A18-1-63, A18-1-42 and A293-34 were selected, and a human IgG1 Fc fragment was fused to it to obtain chimeric antibodies NA3-S (also referred to as "candidate antibody NA3-S", "antibody NA3-S" or "NA3-S antibody" in this disclosure, which is a chimeric antibody obtained by fusing candidate nanobody A18-1-63 with human IgG1 Fc), NA5-S (also referred to as "candidate antibody NA5-S", "antibody NA5-S" or "NA5-S antibody" in this disclosure, which is a chimeric antibody obtained by fusing candidate nanobody A18-1-42 with human IgG1 Fc), The present invention provides a chimeric antibody obtained by fusing candidate nanobody A293-34 with human IgG1 Fc) and NA6-S (also referred to in the present disclosure as "candidate antibody NA6-S", "antibody NA6-S" or "NA6-S antibody", which is a chimeric antibody obtained by fusing candidate nanobody A293-34 with human IgG1 Fc).

[0218] The antibodies NA1-S, NA3-S, NA5-S and NA6-S were expressed using the ExpiCHO transient expression system (Gibco, A29133), and the specific method is as follows. On the day of transfection, the cell density was 7 × 10 6 ~1×10 7 The cells were cultured at a final concentration of 6 × 10 viable cells / mL, indicating that the cell viability was >98%. In this case, cells were cultured at a final concentration of 6 × 10 viable cells / mL using 25 mL of fresh ExpiCHO expression medium pre-warmed to 37 °C. 6 Adjust the concentration to cells / mL and dilute the target plasmid (25 μg in total) with 1 mL of OptiPRO™ SFM pre-cooled to 4°C. At the same time, dilute 80 μL of ExpiFectamine™ CHO with 920 μL of OptiPRO™ SFM. Mix the two together and gently pipette to mix evenly to prepare an ExpiFectamine™ CHO / plasmid DNA mixture. Incubate at room temperature for 1 to 5 minutes and then transfer to the prepared cell suspension. Gently shake the cell suspension while gradually adding the mixture. Finally, place on a cell culture shaker and culture at 37°C and 8% CO2.

[0219] Add ExpiCHO™ Enhancer and ExpiCHO™ Feed within 18-22 hours after transfection, and continue to culture the shake flask under the conditions of 32°C shaker and 5% CO2. On the 5th day after transfection, add the same volume of ExpiCHO™ Feed, gradually adding it while gently and uniformly mixing the cell suspension. On the 12th to 15th day after transfection, the cell expression supernatant is centrifuged at high speed (15000g, 10min), and the obtained supernatant is affinity purified with Protein A (Millipore, P2545). Then, the target protein is eluted with 100mM sodium acetate (pH3.0), followed by neutralization with 1M Tris-HCl, and finally the obtained protein (i.e., antibody NA1-S, NA3-S, NA5-S or NA6-S) is replaced with PBS buffer by the method of concentration tube (Millipore, UFC901096).

[0220] Example 5 Determining specific binding and species cross-specific binding of candidate antibodies Taking the candidate antibody NA1-S as an example, human CLDN18.2-HEK293T, HEK293T, HEK293, human CLDN18.1-HEK293, mouse CLDN18.2-HEK293 and mouse CLDN18.1-HEK293 cells in good growth condition were digested with Trypsin (Gibco, 25200-072) containing 0.25% EDTA, and 1 × 10 5 The cells were incubated with 10 μg / mL of candidate antibody NA1-S for 1 h, and hIgG1 isotype antibody was used as a control. The cells were rinsed twice with FACS buffer, and then incubated with 0.5 μg of PE-labeled goat anti-human IgG-Fc secondary antibody (Abcam, ab98596) at 4°C for 1 h. The cells were then rinsed three times with FACS buffer, and the binding of the candidate antibody to the cells was detected by a flow cytometer (Beckman, CytoFLEX AOO-1-1102). The specific binding and species cross-specific binding of the candidate antibodies NA3-S, NA5-S, and NA6-S were measured in the same manner as NA1-S.

[0221] As shown by the flow cytometry results (shown in FIG. 3), similar to the control antibody IMAB362, the candidate antibodies NA1-S (FIG. 3a), NA3-S, NA5-S and NA6-S (FIG. 3b) all specifically bind to human CLDN18.2-HEK293T and mouse CLDN18.2-HEK293, but do not bind to HEK293T, HEK293, human CLDN18.1-HEK293 and mouse CLDN18.1-HEK293 cells. This indicates that the candidate antibodies NA1-S, NA3-S, NA5-S and NA6-S can specifically bind to CLDN18.2, but not to CLDN18.1, and can cross-recognize human and mouse CLDN18.2.

[0222] Example 6 Comparison of the binding ability of candidate antibodies to human CLDN18.2-HEK293T cell line and human CLDN18.2-KATOIII tumor cell line Human CLDN18.2-HEK293T cells in good culture conditions were incubated with 3-fold gradient dilutions of candidate antibodies NA1-S, NA3-S, NA5-S, and NA6-S, respectively, for 1 h at 4°C. After rinsing twice with FACS buffer, 0.5 μg of PE-labeled goat anti-human IgG-Fc antibody (Abcam, ab98596) was added, incubated for 1 h at 4°C, and further rinsed twice with FACS buffer before detection by flow cytometer.

[0223] The flow cytometry results are shown in Figures 4a, 5a and 5b, and as can be seen from the results, all of the candidate antibodies show cell binding activity comparable to that of the control antibody in human CLDN18.2-HEK293T cells.

[0224] The binding ability of candidate antibodies NA1-S, NA3-S, NA5-S and NA6-S to human CLDN18.2-KATOIII tumor cell line was measured in a similar manner. The flow cytometry results are shown in Figures 4b and 5c. As can be seen from the results, in human CLDN18.2-KATOIII tumor cell line, all candidate antibodies show cell binding activity equivalent to that of the control antibody.

[0225] Example 7 Complement-dependent cytotoxicity (CDC) of candidate antibodies The MTS method is used to measure the CDC cell killing effect of the candidate antibodies. The candidate antibodies NA1-S, NA3-S, NA5-S and NA6-S contain IgG1 Fc fragment, so they can kill cells by CDC, and the MTS reagent can be reduced to a colored compound by NADPH or NADH produced by living cells, so the color shade represents the killing effect of CDC mediated by the antibody. The specific operation method is as follows:

[0226] Take the candidate antibody NA1-S as an example. First, human CLDN18.2-HEK293T cells in good culture conditions were digested with trypsin, and then 5 × 10 4The cells are mixed with rabbit serum diluted 5-fold, and 50 μL of gradient-diluted candidate antibody NA1-S or control antibody IMAB362 are added, and incubated at 37 ° C for 3 h. Then, 30 μL of MTS reagent (Promega, G3580) is added to each of the candidate antibody NA1-S or control antibody IMAB362, mixed thoroughly and homogenously, and cultured in a constant temperature incubator at 37 ° C and 5% CO2 for 4 h. During this period, the color change of the medium is observed, and the OD value at 492 nm wavelength is measured by a microplate reader. 10% Triton X-100 and target cells are used as the complete lysis control, only target cells are used as the blank negative control, and rabbit complement and target cells are used as the background negative control.

[0227] The cell killing rate was calculated by the following formula: Cell killing rate (%)=(OD value of candidate antibody well−OD value of background well) / (OD value of completely lysed well−OD value of blank well)×100%.

[0228] The results of the CDC killing effect of the candidate antibody NA1-S on human CLDN18.2-HEK293T tumor cells are shown in Figure 6a. As can be seen from Figure 6a, the candidate antibody NA1-S shows a higher CDC cell killing effect than the control antibody IMAB362 at equimolar concentrations, with the EC50 of NA1-S being 0.5289nM and the EC50 of IMAB362 being 0.936nM. The CDC killing effect of the candidate antibodies NA3-S, NA5-S and NA6-S on human CLDN18.2-HEK293T cells was measured in a similar manner, and the results are shown in Figures 7a to 7c. As can be seen from Figures 7a to 7c, the candidate antibodies NA3-S, NA5-S and NA6-S also show the same CDC cell killing effect against the control antibody IMAB362 at equimolar concentrations.

[0229] The CDC killing effect of the candidate antibody NA1-S on human CLDN18.2-KATOIII tumor cells was measured in a similar manner, and the results are shown in Figure 6b. As can be seen from Figure 6b, NA1-S shows a higher CDC cell killing effect than the control antibody IMAB362 at equimolar concentrations, with the EC50 of NA1-S being 1.91 nM and the EC50 of IMAB362 being 50.86 nM. The CDC effects of the candidate antibodies NA3-S, NA5-S and NA6-S were measured in the same manner as NA1-S, and were similar to NA1-S, and they also have a higher CDC cell killing effect than the control antibody IMAB362. For example, NA3-S shows a higher CDC cell killing effect than the control antibody IMAB362 at equimolar concentrations, with the EC50 of the nanobody NA3-S being 4.831 nM and the EC50 of IMAB362 being 85.83 nM (Figure 7d).

[0230] Example 8 Antibody-dependent cell-mediated cytotoxicity (ADCC) The ADCC effect is detected by the lactate dehydrogenase (LDH) release method. The principle is as follows: the variable region of the antibody binds to the target antigen on the target cell, and the Fc segment of the antibody binds to FcRIIIa (also known as CD16a) on the NK effector cells in PBMC, after which the NK cells release perforin, granzyme, etc. to lyse the target cells, and then the release of lactate dehydrogenase in the cell supernatant can be detected by the LDH lactate dehydrogenase kit (Takara, MK401), thereby measuring the degree of killing of the target cells by the NK cells. The specific operation is as follows:

[0231] Add 50 µL of cells at a density of 2 x 10 to each well of a 96-well cell culture plate. 5 Add 5 × 10 cells / mL of human CLDN18.2-HEK293T cells and incubate overnight (16 to 20 h) in a 37 °C incubator. Add 50 μL of gradient-diluted candidate antibody NA1-S or NA3-S, mix evenly, and incubate in a 37 °C incubator for 20 min. Then add 5 × 10 cells / mL of resuscitated antibody. 5Human PBMC cells (effector cell / target cell ratio 50:1) were added per well, incubated in a 37°C incubator for 4 hours, centrifuged at 300g to obtain the supernatant, and then LDH detection reagent was added and reacted for 60 minutes. Finally, the OD value at a wavelength of 492 nm was measured using a microplate reader (Molecular Devices, SpectraMax190) to analyze the detection results. 10% Triton X-100 and target cells were used as the complete lysis control, only target cells were used as the blank negative control, and PBMC and target cells were used as the background negative control.

[0232] The cell killing rate was calculated by the following formula: Killing rate (%)=(candidate antibody well OD value-background well OD value) / (completely lysed well OD value-background well OD value)×100%. As can be seen from the results (FIGS. 8a and 8c), both candidate antibodies NA1-S and NA3-S have cell killing activity comparable to that of the control antibody IMAB362 at equimolar concentrations.

[0233] The ADCC killing effect of the candidate antibodies NA1-S or NA3-S on human CLDN18.2-KATOIII tumor cells was measured in a similar manner, and the results are shown in Figures 8b and 8d. As can be seen from the results in Figure 8b, NA1-S exhibits a higher ADCC cell killing effect than the control antibody IMAB362 at equimolar concentrations, with the killing rate of the candidate antibody being as high as 45%, while the killing rate of the IMAB362 control antibody is only 17%. As can be seen from the results in Figure 8d, NA3-S also exhibits a higher ADCC cell killing effect than the control antibody IMAB362 at equimolar concentrations, with the killing rate of the candidate antibody being approximately 50%, while the killing rate of the IMAB362 control antibody is only 20-25%.

[0234] Example 9 In vivo tumor inhibition experiments (human CLDN18.2-HEK293T is used as the tumor-forming cell line) For the experiment, 6-8 week old female SCID mice (body weight 24-26g) were used. The experimental mice were housed in an independent ventilated box with constant temperature and humidity, with the temperature of the breeding room being 21-24°C and the humidity being 30-53%.

[0235] 1×10 7 Human CLDN18.2-HEK293T cells are injected subcutaneously into the right axilla. The volume of the subcutaneous tumor mass is 80–100 mm. 3 When the tumor volume reached 100%, mouse samples with large tumor volume differences were removed, and randomly divided into groups (8 mice per group) based on tumor volume into PBS treatment group, IMAB362 antibody treatment group, and candidate antibody NA1-S treatment group. Antibody treatment was started 6 days after cell inoculation (candidate antibody NA1-S was administered at a dose equimolar to IMAB362 antibody based on molecular weight, which is 2.5 mg / kg and 5 mg / kg in mass concentration), and was administered twice a week, alternating between intravenous and intracavitary injections. The length (mm) and width (mm) of the tumor were observed and recorded at regular intervals, and the tumor growth volume (V) was calculated, using the formula V = (length x width). 2 ) / 2. Candidate antibody NA3-S was tested in a similar manner, using equimolar doses of IMAB362 antibody based on molecular weight, which are 5 mg / kg and 10 mg / kg, respectively, in terms of mass concentration.

[0236] The results of tumor inhibition by the antibodies are shown in Figures 9a-9b. As can be seen from the results, compared to the control antibody IMAB362, the candidate antibody NA1-S has a tumor growth inhibition effect at low concentration similar to that of the control antibody at high concentration at this mass dose, with a tumor growth inhibition rate of about 55%. The candidate antibody NA3-S has a tumor growth inhibition effect at low mass dose similar to that of the control antibody at high concentration, with almost all of them being able to inhibit tumor growth.

[0237] Example 10 Determination of the binding epitope of candidate antibodies on human CLDN18.2 Both the candidate antibody and the control antibody IMAB362 specifically bind to CLDN18.2 but do not bind to CLDN18.1. The extramembrane domains of CLDN18.2 and CLDN18.1 have eight amino acid differences only in the ECD1 domain. Therefore, it is speculated that the antigen-binding epitopes of the candidate antibody and the control antibody IMAB362 are located in the ECD1 domain. In order to confirm whether the antigen-binding sites of the candidate antibody and IMAB362 are identical, this embodiment employs a competitive binding method, the specific method of which is as follows:

[0238] CLDN18.2-HEK293T cells that had been passaged 2-4 times and were in good growth condition were used in the experiment. They were centrifuged at 300 g at 4 °C to remove the supernatant, and then the cells were resuspended in FACS buffer and counted to determine the cell density of 2 × 10 6 Adjust to cells / mL and add to a new 96-well round bottom plate at 100 μL per well, centrifuge at 300 g at 4° C. and remove the supernatant.

[0239] Take the candidate antibody NA1-S as an example, mix FACS buffer (1X PBS + 2% FBS), gradient dilute the competing antibody NA1-S (or IMAB362) with FACS buffer, and similarly dilute the biotin-labeled IMAB362 (or NA1-S) protein to 13.4 nM with FACS buffer, add 100 μL of gradient dilution solution and 100 μL of biotin-labeled protein dilution solution to a 96-well plate, gently pipette with a multichannel pipette to mix evenly, and incubate the 96-well plate at 4 ° C for 1 h. After incubation, the antibody and cell mixture is centrifuged at 4 ° C and 300 g to remove the supernatant, and then add 200 μL of FACS buffer to the corresponding wells to resuspend the cells, and then remove the supernatant by centrifuging at 4 ° C and 300 g. This step is repeated twice. Dilute PE-labeled streptavidin (eBioscience, 12-4317-87) 1:200 in FACS buffer, add 200 μL / well to the corresponding cells with a multichannel pipette, gently pipette to resuspend the cells, then incubate the cells at 4 °C in the dark for 30 min, and after incubation, centrifuge the cells at 4 °C and 300 g to remove the supernatant, add FACS buffer to resuspend the cells, and repeat this step twice. Finally, detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0240] As shown in Fig. 10a, the flow cytometry results show that the binding of IMAB362-biotin to CLDN18.2-HEK293T cells decreases with increasing NA1-S concentration, and similarly, as shown in Fig. 10c, the binding of NA1-S-biotin to CLDN18.2-HEK293T cells decreases with increasing IMAB362 concentration, all of which indicate that the antigen epitopes of NA1-S and IMAB362 on CLDN18.2 are similar. The results of NA3-S are similar to those of NA1-S, as shown in Figs. 10b and 10d.

[0241] In order to further understand the key amino acid sites in CLDN18.2 of NA1-S or NA3-S and IMAB362, based on the fact that CLDN18.2 and CLDN18.1 have 8 amino acid differences in ECD1, this embodiment constructs 8 mutant HEK293T cell lines, mutates the 8 amino acids with differences on CLDN18.2 to the corresponding amino acids in CLDN18.1, and then measures the difference in the binding ability of the antibody to be measured between the wild type and the mutant type to determine whether these amino acids are key amino acids for antibody binding. The specific method is as follows:

[0242] First, the expression of CLDN18.2 wild-type and mutant HEK293T cell lines was measured. The method was the same as that in 1.3.2. The cells were fixed and lysed according to the method in the specifications of the cell lysis kit (eBioscience, 88-8824-00). Then, the expression was determined based on the binding ability of the anti-CLDN18 antibody [34H14L15] to the C-terminal CLDN18 intracellular segment. The results are shown in Figure 10e. CLDN18.2 in each mutant cell line and the wild-type line both has high expression.

[0243] Based on this, referring to the method in 1.3.1, the binding intensity of the candidate antibodies NA1-S or NA3-S and IMAB362 in the CLDN18.2 wild-type and mutant HEK293T cell lines was measured, and the binding intensity of the candidate antibodies was divided by the expression level of each cell line, with the binding of the candidate antibodies in the wild-type line being set as 100%, and normalized to obtain the final relative binding intensity. As shown in Figures 10f to 10i, the three sites in CLDN18.2 ECD1 are Q29, Q47 and E56, which are very important for the binding of NA1-S and NA3-S to human CLDN18.2-HEK293T, respectively, and A42, E56 and G65 are the three important amino acids for IMAB362 to bind to CLDN18.2.

[0244] Example 11 Fab-ZAP cell killing effect mediated by candidate antibodies CLDN18.2 is highly expressed in many tumors and specifically expressed in the tight junction structures of gastric epithelial cells in normal tissues, making it a promising ADC drug target. In this experiment, the endocytosis activity of an antibody is detected by its cytotoxicity when Fab-ZAP endocytosis is mediated by an antibody. Fab-ZAP (Atsbio, IT-51-100) is a Fab fragment linked to the anti-Fc region of saporin, and saporin is a ribosome inhibitor that can cause cell death by inhibiting protein synthesis. After incubating Fab-ZAP and the CLDN18.2 antibody, the CLDN18.2 antibody is attached with Fab-ZAP. When the CLDN18.2 antibody is endocytosed, the Fab-ZAP enters the cell together with the antibody and kills the cell. Then, the endocytosis activity of the candidate antibody and the control antibody is detected and compared by detecting the activity of the cells with MTS (Promega, G3580). The specific method is as follows.

[0245] Logarithmically growing CLDN18.2-HEK293T cells are digested with trypsin (0.25% (w / v) Trypsin 0.53 mM EDTA) until no cell adhesion occurs and caps form, and digestion is terminated with complete medium. After the cells are thoroughly mixed evenly, the cells are counted to measure their viability. The cell density is adjusted to 4x10 4The cells / mL are adjusted, and 50 μL per well is added to the cell culture plate and placed in a cell incubator at 37 ° C. for 16 hours. At the same time, Fab-ZAP is diluted to 2 μg / mL with DMEM complete medium, and the candidate antibody NA1-S or NA3-S and the control antibody are further gradient-diluted using this as a diluent, and 50 μL is taken with a multichannel pipette and added to the cell culture plate, and mixed evenly with CLDN18.2-HEK293T by gently pipetting, and the cell culture plate is placed in a cell incubator at 37 ° C. and continues to incubate for 72 hours. Then, 20 μL of MTS is added to each well with a multichannel pipette, and mixed evenly by gently pipetting, and incubated at 37 ° C. for 2 to 4 hours. Finally, the cell plate is centrifuged at 1000 rpm for 5 hours in a tabletop centrifuge, and then the data is read from a microplate reader (Molecular Devices, SpectraMax190), and the detection wavelength is 492 nm.

[0246] The results are shown in Figure 11a-11b, and compared with the control antibody IMAB362, the candidate antibodies NA1-S and NA3-S can more effectively kill the target cell CLDN18.2-HEK293T by Fab-ZAP, with the IC50 of NA1-S and IMAB362 being 0.1nM and 0.24nM, respectively, and the IC50 of NA3-S and IMAB362 being 0.07nM and 0.32nM, respectively. This not only fully demonstrates that the candidate antibodies can enter cells via CLDN18.2, but also outperforms the control antibody IMAB362, and is likely to be used in the development of ADC antibody-drug conjugates.

[0247] Example 12 Humanization of the candidate antibody NA3-S Compared with mouse antibodies, alpaca-derived nano antibodies have high homology with human antibodies, but due to their special structure, in the humanization design process of NA3-S, the germline gene closest to humans was selected, and the maintenance of the antibody structure was also taken into consideration when performing reverse mutation, and a series of humanized antibodies were finally designed, among which NA3S-H1 is the most preferred molecule, and its antibody sequence is shown in the table below.

[0248] [Table 6]

[0249] Variable region sequence (SEQ ID NO:63): [ka]

[0250] where the italicized bold V (at position 5) and S (at position 6) are humanization sites. The nucleotide sequence encoding the variable region sequence is shown in SEQ ID NO:64.

[0251] To examine whether the affinity of NA3-S decreases after humanization, the binding ability of NA3-S and NA3S-H1 in CLDN18.2-HEK293T cells was compared using the method described in 1.3.1, and the results are shown in Figure 12. The humanized molecule NA3S-H1 and the parent antibody NA3-S have similar affinity and are superior to the control antibody IMAB362.

[0252] Example 13 Binding specificity of candidate antibody NA3S-H1 at high concentrations There are only eight amino acid differences in the candidate antibody binding region ECD1 of CLDN18.2 and CLDN18.1, and the latter is expressed in lung epithelial cells. Therefore, when antibody drugs bind non-specifically to CLDN18.1, they may cause severe lung damage or toxicity, limiting their clinical application. In this embodiment, antibodies at different concentrations (high concentration of 100 μg / mL) and CLDN18.1-HEK293 were incubated in vitro, and the method was as described in 1.3.1. The antibodies at different concentrations and the target cells CLDN18.1-HEK293 were incubated at 4°C for 1 h, followed by rinsing three times with the above FACS buffer, and 0.5 μg (0.5 mg / ml) of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) was added and incubated at 4°C for 1 h. After that, after rinsing three times with FACS buffer, 200 μL of FACS buffer is added to the cells to resuspend the cells, and finally, the cells are detected by a flow cytometer (Beckman, CytoFLEX AOO-1-1102) to record the binding intensity and the cell binding positive rate.

[0253] The results are shown in Figure 13a. In terms of binding strength, the binding of NA3S-H1 and the control antibody IMAB362 to CLDN18.1-HEK293 did not increase with increasing concentration, and was almost consistent with the binding strength of the isotype control. In terms of cell binding positivity in Figure 13b, even at a high concentration of 100 μg / mL, the positivity of NA3S-H1 and IMAB362 was low, ranging from 0.04 to 0.18%. When repeated three times, the positivity of NA3S-H1 was 0.145%, 0.17% and 0.173%, the positivity of IMAB362 was 0.042%, 0.128% and 0.041%, and the positivity of the isotype control hIgG1 was 0.063%, 0.233% and 0.115%. Summarizing the results in Figures 13a and 13b, neither NA3S-H1 nor the control antibody IMAB362 binds to CLDN18.1-HEK293 cells, and both can specifically bind to CLDN18.2, but not to CLDN18.1.

[0254] Example 14 Complement-dependent cytotoxicity (CDC) of candidate antibody NA3S-H1 This example compares the complement-dependent cytotoxicity of the humanized molecule NA3S-H1 and the control antibody IMAB362, which was measured with reference to the method of Example 7. Briefly, 5×10 4 CLDN18.2-KATOIII cells are mixed with 5-fold diluted rabbit serum, and 50 μL of gradient diluted candidate antibody NA3S-H1 or control antibody IMAB362 are added, and incubated at 37 ° C for 3 h. Then, 30 μL of MTS reagent (Promega, G3580) is added, thoroughly mixed, and incubated in a constant temperature incubator at 37 ° C and 5% CO2 for 4 h. During this time, the color change of the medium is observed, and finally, the OD value at a wavelength of 492 nm is measured by a microplate reader. 10% Triton X-100 and target cells are used as a complete lysis control, only target cells are used as a blank negative control, and rabbit complement and target cells are used as a background negative control, and are used to estimate the cell killing effect of the antibody.

[0255] The results are shown in FIG. 14 and show that NA3S-H1 has a higher CDC cell killing effect than the control antibody IMAB362, with an EC50 of 10.41 nM for NA3S-H1 and an EC50 of 149.4 nM for IMAB362.

[0256] Example 15 NK cell cytotoxicity (ADCC) mediated by humanized antibody NA3S-H1 This example compares the cytotoxicity mediated by humanized antibody NA3S-H1 and a control antibody, and employs the method of Example 8, in which gradient-diluted candidate antibodies are incubated with a certain ratio of target cells and human PBMC cells for 4 hours, and then the release of lactate dehydrogenase in the cell supernatant is detected at a wavelength of 492 nm using the LDH lactate dehydrogenase kit (Takara, MK401) method, thereby measuring the killing effect of NK cells mediated by the antibodies on target cells.

[0257] Figures 15a and 15b show the antibody-mediated NK cell killing effect against CLDN18.2-KATOIII or CLDN18.2-HEK293T, and the results show that the ADCC effects mediated by the candidate antibody NA3S-H1 and the control antibody IMAB362 are similar.

[0258] Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from its spirit or central characteristics. It is to be understood that the above description of the present invention discloses only exemplary embodiments thereof, and that other variations are contemplated to fall within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments detailed herein. Rather, the scope and content of the present invention should be indicated by reference to the appended claims.

Claims

1. 1. An antibody comprising at least one immunoglobulin single variable domain, the immunoglobulin single variable domain binds to CLDN18.2, the immunoglobulin single variable domain is a VHH, The immunoglobulin single variable domain comprises a CDR1, a CDR2 and a CDR3 of any of the following groups: (a) CDR1 has the amino acid sequence set forth in SEQ ID NO: 30, CDR2 has the amino acid sequence set forth in SEQ ID NO: 31, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 32; and (b) CDR1 has the amino acid sequence shown in SEQ ID NO:1, CDR2 has the amino acid sequence shown in SEQ ID NO:2, and CDR3 has the amino acid sequence shown in SEQ ID NO:

3.

2. The immunoglobulin single variable domain comprises: (a) an amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90% identical to SEQ ID NO:7; and (b) an amino acid sequence set forth in SEQ ID NO:47, or an amino acid sequence that is at least 90% identical to SEQ ID NO:47; The antibody of claim 1, comprising any one selected from the following:

3. 3. The antibody of claim 1 or 2, wherein the immunoglobulin single variable domain comprises or consists of the sequence of SEQ ID NO: 7, 47 or 63.

4. 4. The antibody of claim 1, wherein the immunoglobulin single variable domain is fused to another molecule, the other molecule being an immunoglobulin Fc domain or a fluorescent protein.

5. The antibody described in claim 4, wherein the antibody is a chimeric antibody comprising a VHH derived from a camelid and an Fc domain of human IgG1 or IgG4, or a humanized antibody comprising a VHH obtained after humanizing a VHH derived from a camelid and an Fc domain of human IgG1 or IgG4.

6. The antibody described in claim 5, which is a chimeric antibody comprising an alpaca-derived VHH and a human IgG1 Fc domain.

7. An antibody described in any one of claims 1 to 6, which binds to extracellular domain 1 (ECD1) of human CLDN18.

2.

8. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody of any one of claims 1 to 7.

9. 9. The isolated nucleic acid molecule of claim 8, comprising or consisting of any one of SEQ ID NOs: 22, 58 and 64.

10. 10. An expression vector comprising the isolated nucleic acid molecule of claim 8 or 9.

11. A host cell comprising the expression vector of claim 10.

12. A pharmaceutical composition comprising at least one antibody according to any one of claims 1 to 7 and a pharma- ceutically acceptable carrier, The drug composition, wherein the immunoglobulin single variable domain is fused to the Fc domain of IgG.

13. Culturing a host cell containing an expression vector encoding the antibody; isolating the antibody from the culture supernatant; A method for preparing the antibody according to any one of claims 1 to 7, comprising:

14. An antibody according to any one of claims 1 to 7 for treating a disease associated with CLDN18.2 in a subject, comprising: An antibody, wherein the immunoglobulin single variable domain is fused to an IgG Fc domain.

15. The antibody described in claim 14, wherein the CLDN18.2-associated disease includes a disease involving cells expressing CLDN18.2 or a disease associated with cells expressing CLDN18.

2.

16. The antibody according to claim 14 or 15, wherein the disease associated with CLDN18.2 is cancer.

17. The antibody of claim 16, wherein the cancer comprises bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, cancer of the genitals and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.

18. The antibody of claim 17, wherein the cancer is gastric cancer.

19. A kit for treating or diagnosing a disease associated with CLDN18.2, comprising a container containing an antibody according to any one of claims 1 to 7, The immunoglobulin single variable domain is fused to the Fc domain of an IgG.

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