Bispecific antibodies against claudin 18.2 and cd3 and uses thereof

Abstract

Novel bispecific antibodies and antibody fragments that specifically bind to Claudin 18.2 and CD3 and compositions containing the bispecific antibodies or antibody fragments are described. In addition, nucleic acids encoding the antibodies or antibody fragments thereof and host cells comprising the same, and related uses are described. Furthermore, therapeutic and diagnostic uses of these bispecific antibodies and antibody fragments are described.

Description

[0001] This invention relates to bispecific antibodies that specifically bind to Claudin18.2 and CD3, and compositions containing said antibodies. Furthermore, this invention relates to the nucleic acids encoding said antibodies and the host cells containing them, as well as related uses. This invention also relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments. Background of the Invention

[0003] Claudins are a family of proteins that are essential components of tight junctions in cells. They establish intercellular barriers that control the flow of molecules between cells. Claudins have four transmembrane domains, with their N-terminus and C-terminus both located in the cytoplasm. Different claudins are expressed in different tissues, and alterations in their function are associated with cancer development in various tissues. For example, claudin-1 is expressed in colon cancer and has prognostic value, claudin-18 is highly expressed in gastric and pancreatic cancers, and claudin-10 is highly expressed in hepatocellular carcinoma. As cell membrane surface proteins, claudins are useful targets for various therapeutic strategies.

[0004] Claudin-18 isotype 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker. Its expression in normal tissues is strictly limited to epithelial cells differentiated from the gastric mucosa, but not to gastric stem cell regions. CLDN18.2 is expressed in a significant proportion of primary gastric cancers and retains its expression level in metastatic gastric cancer tissues. In addition to gastric cancer, CLDN18.2 expression has also been found in pancreatic cancer, making it an ideal target molecule for the treatment of these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as new targeted therapy for advanced gastric cancer. J Hematol Oncol 10, 105 (2017). https: / / doi.org / 10.1186 / s13045-017-0473-4 ).

[0005] Regarding gastric cancer, in 2014, there were approximately 410,000 new cases and 290,000 deaths from gastric cancer in China, accounting for almost half of the global incidence and mortality, and this number continues to rise. However, there is a significant unmet clinical need for oncology treatment, making the development of drugs targeting Claudin18.2 essential.

[0006] CD3 is a homodimeric or heterodimeric antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed by the dimerization of two of four different chains: ε, ζ, δ, and γ. The CD3 dimer arrangement includes γ / ε, δ / ε, and ζ / ζ. Antibodies against CD3 have been shown to aggregate CD3 on T cells, thereby inducing T cell activation in a manner similar to that of peptide-loaded MHC molecules participating in the TCR. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. Furthermore, bispecific antibodies capable of binding CD3 and targeting tumor surface antigens have been proposed to connect tumor cells and T cells, thereby directly activating T cells and releasing granzymes, perforin, and cytokines to kill tumor cells, thus achieving the therapeutic goal of tumor suppression. CLDN18.2 is highly expressed in gastric cancer, pancreatic cancer, and gastroesophageal junction cancer, therefore, therapeutic goals can be achieved by developing bispecific antibodies that simultaneously bind CD3 and Claudin18.2. Invention Overview

[0008] In some aspects, the present invention provides a bispecific antibody comprising two binding domains, wherein the first binding domain specifically binds to CLDN18.2 and the second binding domain specifically binds to CD3.

[0009] In a preferred embodiment, the first binding domain of the present invention that specifically binds to CLDN18.2 is fully human, and / or the second binding domain is humanized.

[0010] In some respects, the bispecific antibody of the present invention is an IgG-like bispecific antibody.

[0011] In one aspect, the present invention relates to the following embodiments:

[0012] 1. A bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2 and the second antigen-binding domain specifically binds to CD3, wherein the first antigen-binding domain comprises the three complementary determinant regions A1-HCDR1, A1-HCDR2 and A1-HCDR3 contained in A1-VH as shown in SEQ ID NO:4, and the three complementary determinant regions A1-LCDR1, A1-LCDR2 and A1-LCDR3 contained in VL as shown in SEQ ID NO:9; the second antigen-binding domain comprises the three complementary determinant regions A2-HCDR1, A2-HCDR2 and A2-HCDR3 contained in A2-VH as shown in SEQ ID NO:30, 22 or 32, and the three complementary determinant regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL as shown in SEQ ID NO:27.

[0013] 2. The bispecific antibody of implementation scheme 1, wherein...

[0014] The first antigen-binding domain comprises A1-HCDR1, A1-HCDR2, and A1-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2, and 3, respectively, and A1-LCDR1, A1-LCDR2, and A1-LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7, and 8, respectively; and

[0015] The second antigen-binding domain contains

[0016] (i) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 20 and 29, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25 and 26, respectively; or

[0017] (ii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 20, and 21, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25, and 26, respectively; or

[0018] (iii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 31 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25 and 26, respectively.

[0019] 3. The antibody according to implementation scheme 1 or 2, wherein the first antigen-binding domain comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region

[0020] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:4; or

[0021] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:4; or

[0022] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:4, preferably, the amino acid alterations do not occur in the CDR region; and / or

[0023] Light chain variable region

[0024] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:9; or

[0025] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:9; or

[0026] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:9, preferably, the amino acid changes do not occur in the CDR region.

[0027] 4. An antibody according to any one of embodiments 1-3, wherein the second antigen-binding domain comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region

[0028] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 30, 22, or 32; or

[0029] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; or

[0030] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 30, 22, or 32, preferably, the amino acid changes do not occur in the CDR region.

[0031] and / or

[0032] Light chain variable region

[0033] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:27; or

[0034] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:27; or

[0035] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:27, preferably, the amino acid alterations do not occur in the CDR region.

[0036] 5. The antibody of any one of the implementation schemes 1-4, further comprising a heavy chain constant region and / or a light chain constant region.

[0037] 6. The antibody of any one of the implementation schemes 1-5 is an IgG-like bispecific antibody.

[0038] 7. An antibody according to any one of embodiments 1-6, wherein the heavy chain constant region of the antibody is derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0039] 8. An antibody according to any one of embodiments 1-6, comprising two heavy chain constant regions, wherein one heavy chain constant region A1-HC is connected to the heavy chain variable region A1-VH of the first antigen domain to form a heavy chain binding to CLDN18.2, and the other heavy chain constant region A2-HC is connected to the heavy chain variable region A2-VH of the second antigen binding domain to form a heavy chain binding to CD3, and comprising two light chain constant regions, wherein one light chain constant region A1-LC is connected to the light chain variable region A1-VL of the first antigen domain to form a light chain binding to CLDN18.8, and the other light chain constant region A2-LC is connected to the light chain variable region A2-VL of the second antigen binding domain to form a light chain binding to CD3.

[0040] 9. The antibody of implementation scheme 8, wherein A1-HC may be the same as or different from A2-HC, and / or A1-LC may be the same as or different from A2-LC.

[0041] 10. The antibody of implementation scheme 8, wherein A1-VH and A1-VL of the CLDN18.2 binding portion are fully human, and A2-VH and A2-VL of the CD3 binding portion are humanized.

[0042] 11. The antibody of implementation scheme 8, wherein the heavy chain of the CLDN18.2 binding portion

[0043] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:37;

[0044] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:37; or

[0045] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:37; and / or

[0046] Light chain in conjunction with CLDN18.2

[0047] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:38;

[0048] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:38; or

[0049] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:38.

[0050] 12. Antibodies according to implementation plan 8 or 11, wherein

[0051] Heavy chain in the CD3 junction

[0052] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 41, 39, or 42;

[0053] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:41, 39 or 42; or

[0054] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 41, 39, or 42; and / or

[0055] Light chain in the part that connects with CD3

[0056] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:40;

[0057] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:40; or

[0058] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:40.

[0059] 13. An isolated nucleic acid that encodes the light chain variable region or heavy chain variable region of an antibody of any one of embodiments 1 to 12, or the light chain or the heavy chain.

[0060] 14. A vector containing the nucleic acid of implementation scheme 13, preferably an expression vector.

[0061] 15. A host cell comprising the nucleic acid of embodiment 13 or the vector of embodiment 14, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, such as CHO-S cells or HEK293 cells) or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0062] 16. A method for preparing an antibody or antigen-binding fragment thereof that binds to CLDN18.2, the method comprising culturing a host cell of embodiment 15 under conditions suitable for expressing a nucleic acid encoding an antibody of any one of embodiments 1 to 12, optionally isolating the antibody or antigen-binding fragment thereof, and optionally further comprising recovering the antibody from the host cell.

[0063] 17. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof binding to CLDN18.2 according to any one of embodiments 1 to 12, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists), and optionally pharmaceutical excipients.

[0064] 18. A combination of drugs comprising an antibody or an antigen-binding fragment thereof of any one of embodiments 1 to 12, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0065] 19. A method for preventing or treating tumors in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof of any one of embodiments 1 to 12, or a pharmaceutical composition of embodiment 17, or a pharmaceutical combination of embodiment 18.

[0066] 20. The method of embodiment 19, wherein the tumor is cancer, preferably, the cancer has an elevated level (e.g., nucleic acid or protein level) of CLDN18.2, such as pancreatic cancer, gastric cancer, or gastroesophageal junction cancer.

[0067] 21. The method of any one of embodiments 19-20, wherein the method further comprises administering one or more therapies to the patient, such as treatment modalities and / or other therapeutic agents, preferably, the treatment modalities include radiotherapy or surgery, or the therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). Attached image description:

[0068] Figure 1 The HB37A6 antibody was shown to specifically bind to CLDN18.2 on the cell surface.

[0069] Figure 2 The results showed that the HB37A6 antibody did not bind to CLDN18.1 on the cell surface.

[0070] Figure 3 The binding of HB37A6 antibody to gastric cancer cell lines NUGC-4, KATO III-hCLDN18.2, and DAN-G-hCLDN18.2 was demonstrated.

[0071] Figure 4 The study demonstrated the antitumor effect of HB37A6 antibody in a mouse model of pancreatic cancer.

[0072] Figure 5 The antitumor effect of HB37A6 antibody in a mouse model of gastric cancer was demonstrated.

[0073] Figure 6 The binding affinity of CD3 monoclonal antibodies Hzsp34.24, Hzsp34.87 and Hzsp34.97 at the cellular level was demonstrated.

[0074] Figure 7 The T-cell activation capacity of CD3 antibodies Hzsp34.24, Hzsp34.87, and Hzsp34.97 was demonstrated.

[0075] Figure 8 A schematic diagram of the bispecific antibody structure used in the examples is shown.

[0076] Figure 9 The bispecific antibody of the present invention was shown to specifically kill CLDN18.2-positive gastric cancer cells NUGC-4.

[0077] Figure 10 The study demonstrated that the bispecific antibody specifically killed CLDN18.2-positive pancreatic cancer cells, DAN-GCLDN18.2.

[0078] Figure 11The results showed that the bispecific antibody did not cause nonspecific killing of CLDN18.2-negative cells.

[0079] Figure 12 This study demonstrates bispecific antibody-dependent T cell-mediated cytokine release in NUGC-4.

[0080] Figure 13 This study demonstrates bispecific antibody-dependent T cell-mediated cytokine release in DAN-G-CLDN18.2.

[0081] Figure 14 This study demonstrated CLDN18.2 expression-dependent bispecific antibody-mediated T cell activation.

[0082] Figure 15 The in vivo efficacy results of the bispecific antibody in the NUGC-4 humanized gastric cancer model are shown.

[0083] Figure 16 The in vivo efficacy results of the bispecific antibody in the DANS-G-CLDN18.2 pancreatic cancer humanized model are shown.

[0084] Figure 17 The pharmacokinetic (PK) of the bispecific antibody in mice was demonstrated. Invention Details

[0086] I. Definition

[0087] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0088] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0089] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0090] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0091] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0092] The term "CLAUDIN" or "CLDN" used in this article refers to the most important skeletal protein determining the structure of tight junctions between cells. Claudin proteins participate in adhesion and play a crucial role in tumor cell metastasis and invasion. Claudin proteins are widely distributed in mammalian epithelial and endothelial cells, primarily on the lateral surfaces of epithelial cells and the plasma membrane of basal cells. Different Claudin proteins exhibit specific expression in different tissues. The Claudin18 (CLDN18) gene, located at 3q22.3, has a molecular weight of 24 kDa, contains 261 amino acid residues, and belongs to the Claudins superfamily. Its protein structure includes two extracellular loops and four transmembrane regions. The two isoforms of human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1) and Claudin18.2 or CLDN18.2 (UniProt ID: P56856-2). In their primary structural sequences, they differ only in certain amino acid residues at positions from the N-terminal signal peptide to the extracellular loop 1 structure, particularly at the extracellular loop 1, where CLDN18.1 and CLDN18.2 differ by only 8 amino acids. The two isoforms of CLDN18 also exhibit very high interspecies sequence homology. The extracellular loop 1 of CLDN18.2 is completely sequence-identical in different species, including humans, mice, and macaques, with 84% homology between human and mouse CLDN18.2 proteins, indicating extreme conservation of the CLDN18.2 protein sequence (O. Tureci. et al., Gene 481:83-92, 2011). CLDN18.2 or any variants and isotypes thereof can be isolated from cells or tissues that naturally express them, or recombinantly generated using techniques well known in the art and / or those described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0093] As used herein, the terms "anti-CLDN18.2 antibody," "anti-CLDN18.2," "CLDN18.2 antibody," or "CLDN18.2-binding antibody" refer to antibodies that bind to (human) CLDN18.2 with sufficient affinity such that the antibody can be used as a therapeutic agent targeting (human) CLDN18.2. In one embodiment, the (human) CLDN18.2 antibody binds to (human) CLDN18.2 with high affinity in vitro or in vivo. In one embodiment, the (human) CLDN18.2 antibody does not bind to CLDN18.1. In one embodiment, the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), thin-layer biofilm interferometry (BLI), MSD assay, surface plasmon resonance (SPR), or flow cytometry.

[0094] As used herein, the term "CD3" refers to an antigen expressed on T cells as a portion of the multimolecular T cell receptor (TCR), which is composed of a homodimer or heterodimer formed from two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-εn (hCD3ε) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P07766.2. Human CD3-δ (hCD3δ) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P04234.1. In some embodiments, the CD3 referred to in this invention refers to CD3 derived from humans or cynomolgus monkeys.

[0095] As used herein, the terms "CD3-binding antibody" or "anti-CD3 antibody" encompass an antibody and its antigen-binding fragment that specifically recognize or bind to a single CD3 subunit (e.g., ε, δ, γ, or ζ), and a dimer complex (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers) that specifically recognizes two CD3 subunits, and an antibody and its antigen-binding fragment linked thereto. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3, bound CD3, and / or CD3 expressed on the cell surface. Soluble CD3 comprises native CD3 protein and recombinant CD3 protein variants, such as monomeric and dimer CD3 structures lacking a transmembrane region or otherwise not binding to the cell membrane. The present invention provides antibodies that bind to human and cynomolgus monkey CD3 with low or undetectable binding affinity, thereby enabling the activation of human and cynomolgus monkey T cells. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), biofilm thin-layer interferometry (BLI), MSD assay, or surface plasmon resonance (SPR) or flow cytometry.

[0096] The term "cell-surface expressed CD3" refers to one or more CD3 proteins that are expressed on the cell surface, either in vivo or in vitro, such that at least a portion of the CD3 protein is exposed to the extracellular space of the cell membrane and is readily accessible to the antigen-binding portion of an antibody. "Cell-surface expressed CD3" includes CD3 proteins contained within the functional T-cell receptor environment of the cell membrane. The term "cell-surface expressed CD3" also includes CD3 proteins expressed as homodimers or heterodimers (e.g., δ / ε, γ / ε, and ζ / ζ CD3 dimers) on the cell surface.

[0097] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2, and regulatory T cells (Tregs). Effector cells may also include natural killer cells, macrophages, granulocytes, plasma cells, or B cells (lymphocytes).

[0098] The term "multispecific antibody" refers to an antibody that is at least bispecific, meaning that the antibody contains at least a first binding domain and a second binding domain, wherein the first binding domain binds to one target or antigen and the second binding domain binds to another antigen or target. Therefore, antibodies according to the invention possess specificity for at least two different antigens or targets. Antibodies according to the invention also encompass multispecific antibodies containing multiple binding domains / binding sites, such as trispecific antibodies, wherein the antibody contains three binding domains.

[0099] Bispecific antibody forms include IgG-like and non-IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8:130). The most common IgG-like antibody type contains two Fab regions and one Fc region, with the heavy and light chains of each Fab region potentially derived from individual monoclonal antibodies. Non-IgG-like bispecific antibodies lack the Fc region; each of their antigen- or target-binding domains can be a Fab, a single-chain variable fragment (scFv), or a fusion protein mimicking the variable domains of two antibodies. The different binding domains are linked together via peptide linkers, chemical coupling, non-covalent linkages, or other means. These forms include bispecific T-cell adaptors (BiTEs).

[0100] The bispecific antibodies of the present invention can be prepared using any form of bispecific antibody or technique. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antibody. Specific exemplary bispecific forms that can be used in the context of the present invention include, but are not limited to, the following: scFv-based or bispecific antibody forms, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knocks-in-holes, ordinary light chains (e.g., ordinary light chains with knob-in-holes, etc.), CrossMab, CrossFab, (SEED)body, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab. 2 Bispecific form.

[0101] As used herein, the term "connector" refers to any molecule that enables the direct linking of different parts of a bispecific antibody. Examples of connectors that establish covalent links between different antibody parts include peptide connectors and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of polyethylene glycol and polypropylene glycol.

[0102] The term "peptide linker" according to the present invention refers to a sequence of amino acids that links an amino acid sequence of a first portion of an antibody to a second portion of the antibody. For example, a peptide linker can link a first (variable and / or binding) domain of an antibody to a second (variable and / or binding) domain. For example, a peptide linker can also link one portion of an antibody to another portion of the antibody, such as linking an antigen-binding domain to an Fc domain or a fragment thereof. Preferably, the peptide linker has a length sufficient to connect two entities in such a way that they maintain their conformation relative to each other, so as not to impede the desired activity.

[0103] Peptide linkers may or may not primarily include the following amino acid residues: Gly, Ser, Ala, or Thr. Useful linkers include glycine-serine polymers, including, for example, (GS). n (GSGGS) n (GGGGS) n (GGGS) n and (GGGGS) n G, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful connectors also include glycine-alanine polymers, alanine-serine polymers, and other flexible connectors.

[0104] According to the invention, the term "valence" indicates the presence of a specified number of binding sites in the antibody molecule. Therefore, the terms bivalent, trivalent, and tetravalent respectively indicate the presence of two, three, or four binding sites in the antibody construct. The bispecific antibody according to the invention is at least bivalent and can be multivalent, such as bivalent, trivalent, tetravalent, or hexavalent.

[0105] As used herein, the term "binding domain" refers to any portion of a bispecific antibody that binds to a specific target or antigen. A binding domain is an antigen-binding site. A binding domain can be, for example, the antibody or immunoglobulin itself or an antibody fragment. Such a binding domain may or may not have a tertiary structure independent of the remaining portion of BsAB and may bind to its target as a standalone entity.

[0106] In certain exemplary embodiments of the present invention, each antigen-binding domain of the bispecific antibody includes a heavy chain variable region (VH) and a light chain variable region (VL). In a bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, the VH, VL, or CDR of the first antigen-binding domain may be designated by the prefix "A1", and the VH, VL, or CDR of the second antigen-binding domain may be designated by the prefix "A2". For example, the heavy chain CDR (HCDR) of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the heavy chain CDR of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3. Similarly, the heavy chain variable region VH of the first antigen-binding domain is referred to herein as A1-VH, and the heavy chain variable region VH of the second antigen-binding domain is referred to herein as A2-VH. The light chain CDR (HCDR) of the first antigen-binding domain is referred to as A1-LCDR1, A1-LCDR2, and A1-LCDR3 in this paper; and the light chain CDR of the second antigen-binding domain is referred to as A2-LCDR1, A2-LCDR2, and A2-LCDR3 in this paper. Similarly, the light chain variable region (VL) of the first antigen-binding domain is referred to as A1-VL in this paper, and the light chain variable region (VL) of the second antigen-binding domain is referred to as A2-VL in this paper.

[0107] The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0108] "Antigen-binding fragment" refers to a molecule that is distinct from the intact antibody, contains a portion of the intact antibody, and binds the antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; bivalent antibodies or fragments thereof; or camelid antibodies.

[0109] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0110] "Antibody that binds to the same or overlapping epitopes as the reference antibody" means an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay; conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competitive assay.

[0111] An antibody that competes with a reference antibody for binding to its antigen is one that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competitive assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another; these assays include, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), and sandwich competitive assays.

[0112] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0113] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90%, or 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for measuring binding affinity and / or specificity.

[0114] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web at imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0115] For example, depending on the different CDR determination schemes, the residues of each CDR are as follows.

[0116]

[0117] CDRs can also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0118] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0119] Unless otherwise stated, in this invention, when referring to the position of a residue in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0120] In one embodiment, the heavy chain variable region (CDR) of the antibody of the present invention is determined according to the following rules:

[0121] VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are both determined according to the Kabat rule.

[0122] In one embodiment, the light chain variable region (CDR) of the antibody of the present invention is determined according to the Kabat rule.

[0123] In one embodiment, the heavy chain variable region CDR of the antibody of the present invention is determined according to the following rules: VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are both determined according to the Kabat rule; and the light chain variable region CDR is determined according to the Kabat rule.

[0124] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined by the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0125] Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs (within the same assignment system). However, although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining portion of the CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0126] The term "Fc region" is used herein to define the constant regions of CH2 and CH3 of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Native Fc regions can bind to different Fc receptors on the surface of immune cells, thereby eliciting CDC / ADCC / ADCP effector functions. Such effector functions generally require the Fc region to be coupled with a binding domain (e.g., an antibody variable domain). In some embodiments, the Fc region is mutated to enhance its CDC / ADCC / ADCP effector function. In some embodiments, the Fc region is mutated to weaken or delete its CDC / ADCC / ADCP effector function.

[0127] "IgG form antibody" refers to the IgG form to which the antibody's heavy chain constant region belongs. All antibodies of the same type have the same heavy chain constant region, while antibodies of different types have different heavy chain constant regions. For example, an IgG4 form antibody means its heavy chain constant region comes from IgG4, or an IgG1 form antibody means its heavy chain constant region comes from IgG1.

[0128] A "humanized" antibody is an antibody comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of nonhuman antibodies, and all or substantially all of the FRs correspond to those of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized.

[0129] The terms "human antibody," "fully human antibody," or "fully human-derived antibody" are used interchangeably to refer to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody generated by a human or human cell or derived from a non-human source, utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0130] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0131] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressants).

[0132] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0133] "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0134] The term "small molecule drug" refers to low-molecular-weight organic compounds capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0135] As used herein, the term "immunomodulator" refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. An immune response can be a humoral or cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0136] The term "effective amount" refers to such an amount or dose of the antibody, fragment, composition, or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention.

[0137] "Therapeutic effective amount" refers to the amount that, at the required dose and sustained for the required period of time, effectively achieves the desired therapeutic outcome. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody, antibody fragment, composition, or combination are less than the beneficial therapeutic effect. Relative to an untreated subject, "therapeutic effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 40%, and more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0138] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0139] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in their nucleic acid content and may contain mutations. This document includes mutant progeny with the same function or biological activity screened or selected from the initially transformed cells.

[0140] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent.

[0141] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0142] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some embodiments, the antibody is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0143] "Isolated nucleic acid encoding anti-CLDN18.2xCD3 bispecific antibody or a fragment thereof" refers to one or more nucleic acid molecules that encode the antibody heavy or light chain (or a fragment thereof, such as a variable region of the heavy chain or a variable region of the light chain), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0144] The following is a calculation of sequence identity between sequences.

[0145] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0146] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. A particularly preferred set of parameters (and, unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix employing a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. Alternatively, the percentage of identity between two amino acid or nucleotide sequences can be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). Additionally or alternatively, the nucleic acid and protein sequences described herein can be further used as “query sequences” to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0147] As used herein, the term "hybridization under stringent conditions (e.g., low-stringency, medium-stringency, high-stringency, or very high-stringency conditions)" describes the hybridization and washing conditions. Instructions for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which are incorporated herein by reference. Aqueous and non-aqueous methods are described in the references, and either method may be used. The preferred hybridization conditions mentioned herein are as follows: 1) Low-toughness hybridization conditions are performed at approximately 45°C in 6X sodium chloride / sodium citrate (SSC), followed by two washes at at least 50°C (for low-toughness conditions, the washing temperature can be increased to 55°C) in 0.2X SSC, 0.1% SDS; 2) Medium-toughness hybridization conditions are performed at approximately 45°C in 6X SSC, followed by one or more washes at 60°C in 0.2X SSC, 0.1% SDS; 3) High-toughness hybridization conditions are performed at approximately 45°C in 6X SSC, followed by one or more washes at 65°C in 0.2X SSC, 0.1% SDS; and preferably 4) Very high-toughness hybridization conditions are performed at 65°C in 0.5M sodium phosphate, 7% SDS, followed by one or more washes at 65°C in 0.2X SSC, 0.1% SDS. Very high-toughness condition (4) is the preferred condition and, unless otherwise stated, should be used.

[0148] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0149] The terms “tumor” and “cancer” are used interchangeably in this article to cover both solid tumors and hematologic malignancies.

[0150] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. In some embodiments, cancers suitable for treatment with the antibodies of the present invention include gastric cancer, pancreatic cancer, or gastroesophageal junction cancer, including metastatic forms of those cancers.

[0151] The term "tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when used in this article.

[0152] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0153] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0154] The term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the anti-CLDN18.2xCD3 bispecific antibody of the present invention or a fragment thereof, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of two or more active agents. In some embodiments, the anti-CLDN18.2xCD3 bispecific antibody of the present invention or a fragment thereof and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in separate formulations, and their formulations may be the same or different.

[0155] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0156] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0157] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0158] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0159] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0160] II. Antibodies

[0161] In some embodiments, the anti-CLDN18.2xCD3 bispecific antibody of the present invention binds to CLDN18.2 (e.g., human CLDN18.2) with high affinity. In some embodiments, the antibody of the present invention specifically binds to CLDN18.2 (e.g., human CLDN18.2) but not to CLDN18.1 (e.g., human CLDN18.1). In some embodiments, the antibody of the present invention or its antigen-binding fragment has a higher binding affinity to human CLDN18.2 than known CLDN18.2 antibodies, such as Zolbetuximab (Zmab) antibody. In some embodiments, the anti-CLDN18.2 xCD3 bispecific antibody of the present invention binds to CD3 (e.g., human CD3 or cynomolgus monkey CD3) with a desired affinity (e.g., lower). In some embodiments, the antibody of the present invention is capable of binding to both human CD3 and cynomolgus monkey CD3. In some embodiments, the affinity of the antibody is determined by thin-layer interferometry or surface plasmon resonance assay.

[0162] In some embodiments, the anti-CLDN18.2 x CD3 bispecific antibody of the present invention is capable of binding to CLDN18.2 on the surface of tumor cells and CD3 on the surface of effector cells. In some embodiments, the binding is detected using flow cytometry.

[0163] In some embodiments, the antibodies of the present invention are capable of specifically killing tumor cells, such as tumor cells expressing CLDN18.2. In some embodiments, the antibodies of the present invention are capable of mediating the release of cytokines, such as IL-2, TNFα, and / or IFNgamma. In some embodiments, the antibodies of the present invention are capable of activating effector cells, such as T cells.

[0164] In some implementations, the effector cells are T cells, such as T lymphocytes, CD4+ T cells, or CD8+ T cells.

[0165] In some embodiments, the antibodies or antigen-binding fragments of the present invention can be used to treat cancer. In some embodiments, the antibodies or antigen-binding fragments of the present invention can effectively inhibit tumor growth, with tumor inhibition rates greater than or equal to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%.

[0166] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2 and the second antigen-binding domain specifically binds to CD3.

[0167] In a preferred embodiment of the present invention, the first antigen-binding domain includes three complementary determinant regions (A1-HCDRs) from the heavy chain variable region: A1-HCDR1, A1-HCDR2, and A1-HCDR3.

[0168] In a preferred embodiment of the present invention, the first antigen-binding domain includes three complementary determinant regions (A1-LCDRs) from the light chain variable region, namely A1-LCDR1, A1-LCDR2 and A1-LCDR3.

[0169] In some implementations, the first antigen-binding domain includes three complementarity-determining regions (A1-HCDR) from the heavy chain variable region and three complementarity-determining regions (A1-LCDR) from the light chain variable region.

[0170] In some aspects, the first antigen-binding domain includes a heavy chain variable region (A1-VH). In some aspects, the first antigen-binding domain includes a light chain variable region (A1-VL). In some aspects, the first antigen-binding domain includes both a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region includes three complementarity-determining regions (A1-HCDRs), HCDR1, HCDR2, and HCDR3, derived from the heavy chain variable region. In some embodiments, the light chain variable region includes three complementarity-determining regions (A1-LCDRs), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region.

[0171] In some implementations, A1-VH

[0172] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:4; or

[0173] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:4; or

[0174] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:4, preferably, the amino acid alterations do not occur in the CDR region.

[0175] In some implementations, A1-VL

[0176] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:9; or

[0177] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:9; or

[0178] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:9, preferably, the amino acid changes do not occur in the CDR region.

[0179] In some embodiments, the three complementary determination regions (A1-HCDRs) from A1-VH, A1-HCDR1, A1-HCDR2, and A1-HCDR3 of the present invention are

[0180] (i) the three complementary determining regions A1-HCDR1, A1-HCDR2 and A1-HCDR3 contained in VH as shown in SEQ ID NO:4, or

[0181] (ii) A sequence that, relative to (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three A1-HCDR regions.

[0182] In some embodiments, the three complementary determination regions (A1-LCDRs) from A1-VL, A1-LCDR1, A1-LCDR2, and A1-LCDR3 of the present invention are

[0183] (i) The three complementary determining regions A1-LCDR1, A1-LCDR2, and A1-LCDR3 contained in the VL shown in SEQ ID NO:9,

[0184] (ii) A sequence that, relative to (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three A1-LCDR regions.

[0185] In some embodiments, A1-HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:1, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:1.

[0186] In some embodiments, A1-HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:2, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:2.

[0187] In some embodiments, A1-HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:3, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:3.

[0188] In some embodiments, A1-LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:6, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6.

[0189] In some embodiments, A1-LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:7, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7.

[0190] In some embodiments, A1-LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:8, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8.

[0191] In a preferred embodiment of the present invention, the second antigen-binding domain includes three complementary determinant regions (A2-HCDRs) from the heavy chain variable region: A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0192] In a preferred embodiment of the present invention, the second antigen-binding domain includes three complementary determinant regions (A2-LCDRs) from the light chain variable region: A2-LCDR1, A2-LCDR2, and A2-LCDR3.

[0193] In some implementations, the second antigen-binding domain includes three complementarity-determining regions (A2-HCDR) from the heavy chain variable region and three complementarity-determining regions (A2-LCDR) from the light chain variable region.

[0194] In some aspects, the second antigen-binding domain includes a heavy chain variable region (A2-VH). In some aspects, the second antigen-binding domain includes a light chain variable region (A2-VL). In some aspects, the second antigen-binding domain includes both a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region includes three complementarity-determining regions (A2-HCDR), HCDR1, HCDR2, and HCDR3, derived from the heavy chain variable region. In some embodiments, the light chain variable region includes three complementarity-determining regions (A2-LCDR), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region.

[0195] In some implementations, A2-VH

[0196] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 22, 30, or 32; or

[0197] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:22, 30 or 32; or

[0198] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 22, 30, or 32, wherein the amino acid alterations do not occur in the CDR region.

[0199] In some implementations, A2-VL

[0200] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:27; or

[0201] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:27; or

[0202] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:27, preferably, the amino acid alterations do not occur in the CDR region.

[0203] In some embodiments, the three complementary determination regions (A2-HCDRs) from A2-VH, A2-HCDR1, A2-HCDR2, and A2-HCDR3 of the present invention are

[0204] (i) the three complementary determining regions A2-HCDR1, A2-HCDR2 and A2-HCDR3 contained in VH as shown in SEQ ID NO:22, 30 or 32, or

[0205] (ii) A sequence that, relative to the sequence in (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three A2-HCDR regions.

[0206] In some embodiments, the three complementary determination regions (A2-LCDRs) from A2-VL, A2-LCDR1, A2-LCDR2, and A2-LCDR3 of the present invention are

[0207] (i) The three complementary determining regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL as shown in SEQ ID NO:27,

[0208] (ii) A sequence that, relative to the sequence in (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three A2-LCDR regions.

[0209] In some embodiments, A2-HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:19, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:19.

[0210] In some embodiments, A2-HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:20 or 31, or comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:20 or 31.

[0211] In some embodiments, A2-HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:21 or 29, or comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:21 or 29.

[0212] In some embodiments, A2-LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:24, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:24.

[0213] In some embodiments, A2-LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:25, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:25.

[0214] In some embodiments, A2-LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:26, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:26.

[0215] In some embodiments, the bispecific antibody of the present invention further comprises a heavy chain constant region. In some embodiments, the bispecific antibody of the present invention further comprises a light chain constant region. In some embodiments, the bispecific antibody of the present invention further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the bispecific antibody of the present invention comprises two heavy chain constant regions, wherein one heavy chain constant region A1-HC is linked to the heavy chain variable region A1-VH of the first antigen domain to form a heavy chain binding to CLDN18.2, and the other heavy chain constant region A2-HC is linked to the heavy chain variable region A2-VH of the second antigen binding domain to form a heavy chain binding to CD3. In some embodiments, the bispecific antibody of the present invention comprises two light chain constant regions, wherein one light chain constant region A1-LC is linked to the light chain variable region A1-VL of the first antigen domain to form a light chain binding to CLDN18.2, and the other light chain constant region A2-LC is linked to the light chain variable region A2-VL of the second antigen binding domain to form a light chain binding to CD3. In one embodiment, the bispecific antibody of the present invention comprises A1-HC, A1-LC, A2-HC, and A2-LC. In some embodiments, A1-HC may be the same as or different from A2-HC. In some embodiments, A1-LC may be the same as or different from A2-LC. In some embodiments, A1-HC is different from A2-HC, and A1-LC is different from A2-LC.

[0216] In some embodiments, the heavy chain constant region HC of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1, such as the wild-type IgG1 heavy chain constant region or the mutant IgG1 heavy chain constant region (e.g. IgG1 LALA). In some embodiments, the antibody light chain constant region LC of the present invention is the lambda or kappa light chain constant region.

[0217] In some preferred embodiments, the heavy chain constant region HC of the present invention

[0218] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 5 or 23;

[0219] (ii) Contains or is composed of an amino acid sequence selected from or consisting of said amino acid sequence; or

[0220] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 5 or 23.

[0221] In some preferred embodiments, the heavy chain constant region HC of the present invention

[0222] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 33 or 34;

[0223] (ii) Contains or is composed of an amino acid sequence selected from or consisting of said amino acid sequence; or

[0224] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 33 or 34.

[0225] In some embodiments, the antibody light chain constant region LC of the present invention

[0226] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 10 or 28;

[0227] (ii) Contains or is composed of an amino acid sequence selected from or consisting of said amino acid sequence; or

[0228] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 10 or 28.

[0229] In some specific embodiments of the present invention, the bispecific antibody of the present invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein...

[0230] The first antigen-binding domain specifically binds to CLDN18.2, and the second antigen-binding domain specifically binds to CD3. The first antigen-binding domain includes the three complementary determinant regions A1-HCDR1, A1-HCDR2 and A1-HCDR3 contained in A1-VH as shown in SEQ ID NO:4, and the three complementary determinant regions A1-LCDR1, A1-LCDR2 and A1-LCDR3 contained in VL as shown in SEQ ID NO:9.

[0231] The second antigen-binding domain includes the three complementary determinant regions A2-HCDR1, A2-HCDR2 and A2-HCDR3 contained in A2-VH as shown in SEQ ID NO:22, 30 or 32, and the three complementary determinant regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL as shown in SEQ ID NO:27.

[0232] In some specific embodiments of the present invention, the bispecific antibody of the present invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2, and the second antigen-binding domain specifically binds to CD3.

[0233] The first antigen-binding domain comprises A1-HCDR1, A1-HCDR2, and A1-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2, and 3, respectively, and A1-LCDR1, A1-LCDR2, and A1-LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7, and 8, respectively; and

[0234] The second antigen-binding domain contains

[0235] (i) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 20 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25 and 26, respectively; or

[0236] (ii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 20, and 29, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25, and 26, respectively; or

[0237] (iii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 31 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25 and 26, respectively.

[0238] In some specific embodiments of the present invention, the bispecific antibody of the present invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2, and the second antigen-binding domain specifically binds to CD3.

[0239] The first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or A1-VH composed of said amino acid sequence, and comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 90% identity with it, or A1-VL composed of said amino acid sequence;

[0240] The second antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:22, 30 or 32, or an amino acid sequence having at least 90% identity with it, or an A2-VH composed of said amino acid sequence, and comprises the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or an A2-VL composed of said amino acid sequence.

[0241] In some specific embodiments of the present invention, the bispecific antibody of the present invention is an IgG-like bispecific antibody, that is, it comprises a heavy chain and a light chain that binds to the CLDN18.2 binding site, and a heavy chain and a light chain that binds to the CD3 binding site. In some embodiments, a knock-in-hol sequence is present in the CH3 region of the two heavy chains (Shane Atwell et al., Journal of Molecular Biology, 1997; A. Margaret Merchant et al., Nature Biotechnology, 1998), thus forming a pestle-and-mortar structure. In some embodiments, the structure of the bispecific antibody of the present invention is described below. Figure 8 .

[0242] In some implementations, the heavy chain in conjunction with CLDN18.2

[0243] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:37;

[0244] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:37; or

[0245] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:37.

[0246] In some implementations, the light chain in conjunction with the CLDN18.2 portion

[0247] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:38;

[0248] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:38; or

[0249] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:38.

[0250] In some implementations, the heavy chain associated with the CD3 binding portion

[0251] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 39, 41, or 42;

[0252] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:39, 41 or 42; or

[0253] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 39, 41, or 42.

[0254] In some implementations, the light chain that binds to the CD3 portion

[0255] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:40;

[0256] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO:40; or

[0257] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:40.

[0258] In one embodiment of the invention, the amino acid alteration described herein includes amino acid substitution, insertion, or deletion. Preferably, the amino acid alteration described herein is an amino acid substitution, and more preferably a conservative substitution.

[0259] In a preferred embodiment, the amino acid alteration described in this invention occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid alteration described in this invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid alteration described in this invention occurs in the Fc region of the antibody heavy chain constant region, and in a preferred embodiment, the amino acid alteration in the Fc region weakens or deletes the antibody's ADCC and / or CDC activity.

[0260] In some implementations, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the table below:

[0261]

[0262]

[0263] In some embodiments, the substitution occurs in the CDR region of the antibody. Typically, the resulting variant has modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity) and / or will have some biological properties that are substantially retained by the parent antibody. An exemplary substitution variant is an affinity-matured antibody.

[0264] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to create Fc region variants that alter one or more functional properties of the antibody, such as serum half-life, complement binding, complement-dependent cytotoxicity, Fc receptor binding, and / or antibody-dependent cytotoxicity. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) containing amino acid alterations (e.g., substitutions) at one or more amino acid positions.

[0265] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0266] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0267] In some embodiments, in the bispecific antibody of the present invention, the first antigen-binding domain, or heavy chain and light chain combination, that specifically binds to CLDN18.2 is fully human, and the second antigen-binding domain, or heavy chain and light chain combination, that binds to CD3 is humanized.

[0268] III. The nucleic acid of the present invention and the host cell containing it.

[0269] In one aspect, the present invention provides a nucleic acid encoding a variable region or heavy or light chain of any of the above-described bispecific antibodies. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector, such as pcDNA3.1. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In another embodiment, the host cell is prokaryotic.

[0270] In one aspect, the nucleic acid of the present invention may be a nucleic acid comprising an amino acid sequence encoding an amino acid sequence encoding an antibody light chain variable region and / or a heavy chain variable region, or a nucleic acid comprising an amino acid sequence encoding an antibody light chain and / or a heavy chain.

[0271] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:4, 9, 22, 27, 30, 32, 37-42, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:4, 9, 22, 27, 30, 32, 37-42.

[0272] The present invention also covers nucleic acids that hybridize under stringent conditions or that have one or more substitutions (e.g., conservative substitutions), deletions, or insertions with the following nucleic acids: nucleic acids comprising nucleic acid sequences encoding amino acid sequences selected from any one of SEQ ID NO:4, 9, 22, 27, 30, 32, 37-42; or nucleic acids comprising nucleic acid sequences encoding amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acid sequences selected from any one of SEQ ID NO:4, 9, 22, 27, 30, 32, 37-42.

[0273] In one embodiment, one or more vectors containing the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, kinases, λ phages, or yeast artificial chromosomes (YAC). In one embodiment, the vector is pcDNA3.1.

[0274] In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing the vector encoding the antibody include prokaryotic or eukaryotic cells as described herein. For example, the antibody may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

[0275] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains whose glycosylation pathways have been “humanized” result in the production of antibodies with partial or complete human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F, or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. Mammalian host cell lines suitable for antibody production are known in the art.

[0276] IV. Production and purification of the antibody molecules of the present invention

[0277] In one embodiment, a method for preparing the antibody molecule of the present invention is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0278] To recombinantly generate the antibody molecules of the present invention, nucleic acids encoding antibodies (such as those described above, such as any one or more polypeptide chains) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0279] The antibody molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibody molecules of this invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0280] V. Determination Method

[0281] The antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. On one hand, the antigen-binding activity of the antibodies of the present invention can be tested, for example by known methods such as ELISA, Western blotting, etc. Binding to CLDN18.2 and / or CD3 can be determined using methods known in the art, exemplary methods of which are disclosed herein. In some embodiments, radioimmunoassay (RIA), thin-layer interferometry, MSD assay, surface plasmon resonance (SPR), or flow cytometry are used for measurement.

[0282] On the other hand, competitive assays can be used to identify antibodies that compete with any bispecific antibodies disclosed herein for binding to CLDN18.2 and / or CD3. In some embodiments, such competitive antibodies bind to epitopes that are the same as or overlap with the epitopes bound by the bispecific antibodies disclosed herein (e.g., linear or conformational epitopes).

[0283] This invention also provides an assay for identifying antibodies with biological activity. Biological activity may include, for example, binding to CLDN18.2 and / or CD3 (e.g., binding to human CLDN18.2 and / or CD3), binding to cells expressing CLDN18.2 and / or CD3, activation of T cells, stimulation of cytokine secretion, inhibition and killing of tumor cells, etc. Antibodies possessing such biological activity in vivo and / or in vitro are also provided.

[0284] In some embodiments, the antibodies of the present invention are tested for such biological activity.

[0285] Cells used for any of the above-described in vitro assays include cell lines that naturally express CLDN18.2 and / or CD3, or that are engineered to express or overexpress CLDN18.2 and / or CD3. Such cell lines also include cell lines transfected with encoding DNA that expresses CLDN18.2 and / or CD3 and is not normally expressed in CLDN18.2 and / or CD3. In some embodiments, these cells are gastric cancer cells or pancreatic cancer cells. In some embodiments, these cells are CHO cells expressing CLDN18.2. In some embodiments, these cells are cell lines NUGC-4, KATO III, and DAN-G, such as KATO III and DAN-G cell lines overexpressing CLDN18.2. In some embodiments, these cells are T cells, such as human T lymphocytes, such as Jurkat cells.

[0286] It is understood that any of the above-described assays can be performed using the combination of the antibodies of the present invention and other active agents.

[0287] VI. Pharmaceutical Composition

[0288] In some embodiments, the present invention provides an antibody comprising any antibody or composition thereof described herein, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example a pharmaceutical composition, comprises an antibody or fragment thereof of the present invention, and a combination of one or more other therapeutic agents.

[0289] The present invention also includes bispecific antibodies or compositions thereof comprising the present invention (including pharmaceutical compositions). These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0290] As used in this article, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0291] For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Swen, Pharmaceutical Press, London, Chicago.

[0292] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0293] A drug comprising the antibody described herein can be prepared by mixing the antibody of the present invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0294] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). The active ingredients are suitably combined in amounts effective for the intended use.

[0295] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0296] VII. Drug combinations and medicine boxes

[0297] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the antibody of the present invention, and one or more other therapeutic agents (e.g., therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists)).

[0298] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0299] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0300] - A first container containing a pharmaceutical composition comprising the bispecific antibody of the present invention;

[0301] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0302] VIII. Applications and Methods

[0303] In one aspect, the present invention provides a method for preventing or treating tumors (e.g., cancer) in a subject, comprising administering to the subject an effective amount of the present invention’s bispecific antibody against CLDN18.2 x CD3 or a fragment thereof (preferably an antigen-binding fragment), a pharmaceutical composition, a pharmaceutical combination, or a kit.

[0304] In some implementations, the tumor (e.g., cancer) patient has (e.g., elevated levels, such as nucleic acid or protein levels) CLDN18.2.

[0305] In some embodiments, the tumor, such as cancer, includes solid tumors, hematologic malignancies, and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. Cancer can be in the early, intermediate, or late stage, or metastatic stage.

[0306] In some implementations, the tumor treatment will benefit from inhibiting nucleic acid or protein levels of CLDN18.2.

[0307] In one specific embodiment, the antibody of the present invention can kill tumor cells and / or inhibit tumor cell proliferation, such as tumor cells expressing CLDN18.2, such as gastric cancer cells or pancreatic cancer cells.

[0308] In another specific embodiment, the anti-CLDN18.2 x CD3 bispecific antibody of the present invention can activate T cells.

[0309] Therefore, the antibodies of the present invention are suitable for the prevention or treatment of any tumor or cancer in which an effector mechanism requiring cytotoxic T cells is needed, or any tumor or cancer requiring T cell recruitment.

[0310] In some implementations, the tumor is a tumor immune escape.

[0311] In some embodiments, the tumor is cancer, such as stomach cancer or pancreatic cancer, and the subject can be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual with or at risk of having the disease described herein). In one embodiment, the subject has or is at risk of having the disease described herein. In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0312] In other respects, the present invention provides the use of antibody molecules or pharmaceutical compositions or drug combinations or kits in the manufacture or preparation of medicaments for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0313] In some embodiments, the antibody molecules or pharmaceutical compositions or kits of the present invention may delay the onset of symptoms and / or symptoms associated with the disease.

[0314] In some embodiments, the antibody molecules or pharmaceutical compositions of the present invention may also be administered in combination with one or more other therapies, such as treatments and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0315] In some implementation schemes, treatment methods include surgery; radiotherapy; local irradiation; or focused irradiation.

[0316] In some implementations, the therapeutic agent is selected from chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0317] Exemplary immunomodulators include immunosuppressants or anti-inflammatory agents. In some embodiments, immunomodulators also include immune checkpoint inhibitors or agonists. Other exemplary antibodies include antibodies that specifically bind to immune checkpoints.

[0318] In some implementations, the antibody combinations described herein can be administered separately, for example, as individual antibodies.

[0319] Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which case the antibody of the present invention may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceuticals.

[0320] The drug composition can be administered via known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (internal parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; via a continuous release system or via an implantable device. In some embodiments, the composition can be administered by bolus injection, continuous infusion, or via an implantable device.

[0321] The composition can also be applied topically via an implantable membrane, a sponge, or another suitable material on which the desired molecules are absorbed or encapsulated. In some embodiments, when an implantable device is used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecules via diffusion, timed release of a large pellet, or continuous administration.

[0322] IX. Methods and compositions for diagnosis and detection

[0323] In some embodiments, any antibody provided herein can be used to detect the presence of CLDN18.2 in a biological sample. The term "detection," as used herein, includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is derived from lesions associated with hyperplastic or cancerous lesions.

[0324] In one embodiment, a bispecific antibody is provided for use in a diagnostic or detection method. In another aspect, a method is provided for detecting the presence of CLDN18.2 in a biological sample. In some embodiments, the method comprises detecting the presence of the CLDN18.2 protein in a biological sample. In some embodiments, CLDN18.2 is human CLDN18.2. In some embodiments, CD3 is human CD3. In some embodiments, the method comprises contacting a biological sample with an antibody as described herein under conditions that allow it to bind to CLDN18.2, and detecting whether a complex is formed between the antibody and CLDN18.2. The formation of the complex indicates the presence of CLDN18.2. This method may be an in vitro or in vivo method. In one embodiment, the antibody of the present invention is used to select subjects suitable for treatment with the bispecific antibody of the present invention, for example, where CLDN18.2 is a biomarker for selecting said subject.

[0325] In one embodiment, the antibodies of the present invention can be used to diagnose tumors, such as cancer, for example, to evaluate (e.g., monitor) the treatment or progression of the disease described herein in an individual, its diagnosis, and / or staging. In some embodiments, labeled bispecific antibodies are provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0326] In some embodiments provided herein, the sample is obtained prior to treatment with the antibody of the present invention. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0327] In some embodiments, the sample is formalin-fixed and paraffin-coated (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical excision), or a fine-needle aspirate.

[0328] In some implementations, CLDN18.2 is detected before treatment, for example, before the start of treatment or before a treatment after a treatment interval.

[0329] In some embodiments, a method for treating the disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) for the presence of CLDN18.2, thereby determining a CLDN18.2 value; comparing the CLDN18.2 value with a control value; and if the CLDN18.2 value is greater than the control value, administering to the subject a therapeutically effective amount of the bispecific antibody of the present invention, optionally in combination with one or more other therapies, thereby treating the disease.

[0330] These and other aspects and embodiments of the invention are described in the accompanying drawings (briefly described below) and the following detailed description of the invention, and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art. Example

[0331] Example 1: Construction of a stable expression cell line

[0332] Preparation of human CLDN18.2 overexpression cell line

[0333] Use according to the manufacturer's instructions. CHO- The kit (Invitrogen, A1369601) was used to construct a cell line stably expressing human Claudin18.2 (hereinafter referred to as CLDN18.2). First, the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pCHO1.0 to form a plasmid. The constructed plasmid was then transfected into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) using chemical transfection and electrotransfection methods, respectively. The transfected cells underwent two rounds of pressure selection to obtain cell pools expressing CLDN18.2. Then, the cells that highly expressed CLDN18.2 were sorted out using a flow cytometer (MoFlo XDP, Beckman Coulter), and the monoclonal cell lines CHO-hCLDN18.2 and HEK293-hCLDN18.2 that stably expressed CLDN18.2 were obtained by dilution.

[0334] Preparation of human CLDN18.1 overexpressing cell lines

[0335] Use according to the manufacturer's instructions. CHO- A cell line stably expressing human Claudin18.1 (hereinafter referred to as CLDN18.1) was constructed using a kit (Invitrogen, A1369601). First, the full-length human CLDN18.1 (UniProt ID: P56856-1) gene was constructed into the vector pCHO1.0 (Invitrogen, A1369601) to form a plasmid. The constructed plasmid was then chemically transfected into CHO-S cells (Invitrogen, A1369601). The transfected cells underwent two rounds of pressure selection to obtain cell pools expressing CLDN18.1. Then, cells highly expressing CLDN18.1 were sorted using a flow cytometry system (MoFlo XDP, Beckman Coulter), and a dilution method was used to obtain the stable CLDN18.1 monoclonal cell line CHO-hCLDN18.1.

[0336] Construction of tumor cell lines overexpressing CLDN18.2

[0337] The full-length human CLDN18.2 gene (UniProt ID: P56856-2) was constructed into the vector pWPT-GFP (Addgene, 12255), replacing the GFP sequence. This vector, along with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), was co-transfected into HEK293T (ATCC, CRL-3216) cells for viral packaging. Culture supernatants were collected after 48 and 72 hours of culture, and the lentivirus was concentrated using PEG8000. The concentrated virus was then used to transfect pancreatic cancer DAN-G cells and gastric cancer KATO III cells. Cells expressing CLDN18.2 were then sorted using a flow cytometry system (MoFlo XDP, Beckman Coulter) to obtain stably transfected CLDN18.2 tumor cell lines: DAN-G-hCLDN18.2 and KATO III-hCLDN18.2.

[0338] Example 2: Generation of CLDN18.2 monoclonal antibodies

[0339] This invention employs hybridoma technology, using cells (CHO-hCLDN18.2) obtained in Example 1 to immunize H2L2 fully human antibody-transgenic mice (purchased from Harbour BioMed). Then, spleen cells from these mice were electrofused with myeloma cells. The supernatant was then collected and screened using flow cytometry (FACS) to identify hybridoma cells specifically expressing anti-CLDN18.2 antibodies, whose secreted antibodies did not bind to CLDN18.1. The test cells (HEK293-hCLDN18.2) obtained in Example 1 were counted and diluted to 1×10⁻⁶. 6 Cells / ml, add 100 μl / well to a U-bottom 96-well plate. Centrifuge at 500g for 5 min, remove cell culture medium. Add the supernatant from the hybridoma 96-well plate to the U-bottom plate and resuspend the cells, 100 μl per well, incubate on ice for 30 min. Centrifuge at 500g for 5 min, remove supernatant, wash cells once with PBS. Add 100 μl of FITC-labeled anti-mouse Fab secondary antibody (1:500 diluted in PBS) to each well, and add 100 μl of FITC-labeled anti-human Fab secondary antibody to the positive control antibody. Incubate on ice in the dark for 30 min. Centrifuge at 500g for 5 min, remove supernatant, wash cells once with PBS. Resuspend cells in 50 μl of 1×PBS, and analyze by FACS. Positive clones are then screened again for CHO-hCLDN18.1 using the same method. After two rounds of screening, the fully human antibody clone HB37A6 was obtained.

[0340] Example 3: Preparation of recombinant CLDN18.2 monoclonal antibody

[0341] The anti-CLDN18.2 monoclonal antibody HB37A6 (see CN202010570517.X) and the control antibody zolbetuximab (abbreviated as Zmab, sequence derived from INN117) were expressed as full-length monoclonal antibodies in HEK293 cells (Invitrogen, A14527).

[0342] First, expression vectors were constructed by placing the heavy chain variable region and light chain variable region (see sequence information) of HB37A6 and the control antibody at the N-terminus of the heavy chain constant region (SEQ ID NO: 5) and light chain kappa constant region (SEQ ID NO: 10) of human IgG1, respectively. These were then incorporated into the pcDNA3.1 expression vector containing the N-terminal signal peptide to obtain the light and heavy chain expression vectors. The obtained light and heavy chain expression vectors were co-transfected into HEK293 cells using PEI (Polysciences Inc, 23966). After 7 days of culture, the supernatant was collected. The supernatant was purified using a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), then ultrafiltered and the medium was changed to PBS (Gibco, 70011-044). The concentration was detected using the A280 method, and the purity was determined by SEC-HPLC. An antibody solution with a purity greater than 95% was obtained, yielding the recombinant CLDN18.2 monoclonal antibody HB37A6.

[0343] The specific transfection and purification process is as follows:

[0344] Expi293 cells (Invitrogen, A14527) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 × 10⁻⁶ cells / day before transfection. 6 Cells / ml. The cell density on the day of transfection was approximately 3 × 10⁻⁶ cells / ml. 6 Cells / ml. Take 1 / 10 of the final volume of Opti-MEM medium (Gibco, 31985-070) as transfection buffer, add an appropriate amount of plasmid at 1.0 μg / ml for cell transfection, and mix well. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the plasmid to PEI ratio is 1:3 in 293F cells), mix well, and incubate at room temperature for 20 min to obtain a DNA / PEI mixture. Slowly add the DNA / PEI mixture to the cells while gently shaking the flask, then incubate at 36.5℃ in an 8% CO2 incubator. After seven days, obtain the cell culture medium, collect the cell supernatant, and purify it.

[0345] The Protein A column (Hitrap Mabselect Sure, GE, 11-0034-95) used for purification was treated with 0.1M NaOH for 2 hours. Glass bottles and other equipment were washed with distilled water and then dried at 180°C for 4 hours. Before purification, the collected cell material was centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered through a 0.22 μm filter. The Protein A column was equilibrated with 10 column volumes of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). The filtered supernatant was added to the purification column and equilibrated with 10 column volumes of binding buffer. 5 ml of elution buffer (0.1 M citric acid + sodium citrate, pH 3.5) was added, and the eluent was collected. 80 μl of 2M Tris-HCl was added to every 1 ml of eluent. The collected antibody was ultrafiltered and concentrated, then exchanged for PBS (Gibco, 70011-044), and the concentration was determined.

[0346] Example 4: Determination of CLDN18.2 antibody affinity by SPR method

[0347] The equilibrium dissociation constant (K0) of HB37A6 bound to human CLDN18.2 was determined using surface plasmon resonance (SPR). D Following the manufacturer's instructions, human Claudin 18.2 (GenScrip, P50251802) was conjugated onto the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino-conjugation kit (GE Healthcare, BR-1006-33). After conjugation, 1M ethanolamine was injected to block the remaining activation sites. Affinity and kinetic constants were obtained by detecting the binding and dissociation of the chip surface antigen with the antibody in the mobile phase using a Biacore (GE Healthcare, T200) analyzer. Serially diluted antibodies (0-100 nM) were flowed sequentially from low to high concentration onto the chip surface, with a binding time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). Data were analyzed using Biacore T200 software with a 1:1 binding model for kinetic analysis. As shown in Table 1, HB37A6 showed better affinity than the control antibody Zmab.

[0348] Table 1. Affinity constants (equilibrium dissociation constants) for SPR assay of CLDN18.2 antibody

[0349]

[0350] Example 5: Binding specificity of CLDN18.2 antibody to CLDN18 cells

[0351] The binding of the above-mentioned anti-CLDN18.2 monoclonal antibody HB37A6 and control antibody Zmab to the CHO-S cell lines (i.e., CHO-hCLDN18.2 and CHO-hCLDN18.1 prepared as described in Example 1) stably transfected with human CLDN18.2 and human CLDN18.1 obtained in Example 1 was determined by flow cytometry (FACS).

[0352] Specifically, the cells to be tested (CHO-hCLDN18.2 and CHO-hCLDN18.1) obtained in Example 1 were counted and diluted to 1×10⁻⁶. 6 Cells / ml, add 100 μl / well to a 96-well U-bottom plate. Centrifuge at 500g for 5 min, remove cell culture medium. Add anti-CLDN18.2 monoclonal antibody HB37A6 and control antibody Zmab to the U-bottom plate and resuspend cells, 100 μl per well. The initial antibody concentration is 900 nM, then 3-fold diluted sequentially for a total of 10 concentration points. Incubate on ice for 30 min. Remove supernatant at 500g for 5 min, wash cells once with PBS. Add 100 μl of PE-labeled goat anti-human Fc secondary antibody (SouthernBiotech, J2815-5H87B) to each well. Incubate on ice in the dark for 30 min. Remove supernatant at 500g for 5 min, wash cells once with PBS. Resuspend cells in 50 μl of 1×PBS, and analyze using FACS. Analyze experimental data using GraphPad Prism software. Figure 1 and Figure 2 .like Figure 1 and Figure 2 As shown, the antibodies specifically bind to human CLDN18.2 but not to human CLDN18.1.

[0353] Example 6: Binding of CLDN18.2 antibody to tumor cell lines

[0354] Referring to Example 5, the binding of HB37A6 to the gastric cancer cell line NUGC-4 (JCRB cell bank, JCRB0834), the gastric cancer cell line KATO III-hCLDN18.2, and the pancreatic cancer cell line DAN-G-hCLDN18.2 was determined by FACS. Figure 3 The results showed that the fully human antibody HB37A6 had good tumor cell-specific binding, which was superior to the control antibody Zmab.

[0355] Example 7: In vivo antitumor effect of CLDN18.2 antibody

[0356] 1. Antibody activity against DAN-G-CLDN18.2 tumor-bearing mouse model

[0357] The antitumor effect of HB37A6 antibody in NOD-SCID mice with human pancreatic cancer (60 female NOD-SCID mice (15-18g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was tested. The human pancreatic cancer cells DAN-G-hCLDN18.2 constructed in Example 1 were routinely passaged for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) to obtain a cell suspension with a cell density of 12×10^5 cells / ml. The cell suspension was mixed with Matrigel gel 1:1 to prepare a cell suspension with a cell concentration of 6×10^5 cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously injected into the right abdominal region of NOD-SCID mice to establish a DAN-G-CLDN18.2 tumor-bearing mouse model.

[0358] Five days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of 43.36 mm were selected. 3 ~89.47mm 3 Mice within the range were grouped in a serpentine pattern according to tumor size (8 mice per group).

[0359] Each mouse was administered hIgG (Equitech-Bio, batch number 160308-02), HB37A6, and the control antibody Zmab at a dose of 10 mg / kg per administration, on days 5, 9, 12, and 16 post-inoculation. Tumor volume was monitored 2-3 times per week. Tumor volume was measured using calipers to determine the maximum long axis (L) and maximum wide axis (W) of the tumor. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0360] 2. Antibody activity against NUCG-4 tumor-bearing mouse model

[0361] The HB37A6 antibody was selected to test its antitumor effect in NOG mice with human gastric cancer (100 female NOG mice (15-18g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). PBMC cells (Allcells) were resuscitated, centrifuged to collect the cells, and dispersed in PBS (1×) at a cell density of 2.5×10^6 cells / ml. On day 0, 0.2ml of the cell suspension was injected intravenously into the eye vein of NOG mice to establish a NOG humanized mouse model.

[0362] NUGC-4 cells were routinely resuscitated and passaged for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) at a density of 12×10^6 cells / ml. The PBS was then mixed 1:1 with Matrigel gel to prepare a cell suspension with a concentration of 6×10^6 cells / ml. On day 5, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of NOG humanized mice to establish a NUGC-4 tumor-bearing mouse model.

[0363] On day 1 after tumor cell inoculation, mice were randomly divided into groups of 7 mice each. Each mouse was administered hIgG (Equitech-Bio, batch number 160308-02), HB37A6, and the control antibody Zmab at a dose of 10 mg / kg on days 1, 5, 8, and 12 post-inoculation. Tumor volume and body weight were monitored 2-3 times per week. Tumor volume was measured using calipers to determine the maximum long axis (L) and maximum wide axis (W) of the tumor. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance. The relative tumor inhibition rate (TGI%) was calculated on day 26 after vaccination using the following formula:

[0364] TGI% = 100% * (Tumor volume in control group – Tumor volume in treatment group) / (Tumor volume in control group – Tumor volume in control group before drug administration).

[0365] 3. Results

[0366] The results are as follows Figure 4 As shown, both HB37A6 and the control antibody Zmab inhibited tumor growth in a human pancreatic cancer DANG-CLDN18.2 mouse model, with a TGI of 28% for HB37A6 and 24% for Zmab. Figure 5 As shown, HB37A6 exhibited better anti-tumor effects than the control antibody Zmab in the NUGC-4 mouse model of human gastric cancer, with a TGI of 31% for HB37A6 and 0% for Zmab.

[0367] Example 8: Preparation of anti-CD3 monoclonal antibody

[0368] The murine CD3 antibody sp34 (US Pat. No. 8, 236, 308; J. Immunol. Methods., 1994, 178: 195) and the humanized and affinity-optimized anti-CD3 monoclonal antibodies HzSP34.24, HzSP34.87, and HzSP34.97, as well as the full-length monoclonal antibody, were expressed in HEK293 cells (Invitrogen, A14527).

[0369] The heavy chain variable regions and light chain variable regions (see sequence information) of HzSP34.24, HzSP34.87, HzSP34.97, and sp34 were placed at the N-terminus of the IgG1 heavy chain constant region (SEQ ID NO: 23) and the light chain lambda constant region (SEQ ID NO: 28). These were then constructed into the pcDNA3.1 expression vector containing an N-terminal signal peptide to obtain the light and heavy chain expression vectors. The obtained light and heavy chain expression vectors were co-transfected into HEK293 cells using PEI (Polysciences Inc, 23966), and the culture supernatant was collected after 7 days of culture. The supernatant was purified using a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), followed by ultrafiltration and buffer exchange into PBS (Gibco, 70011-044). The concentration was determined using the A280 method, and purity was measured using SEC-HPLC to obtain antibody solutions with a purity greater than 95%, yielding recombinant anti-CD3 monoclonal antibodies HzSP34.24, HzSP34.87, HzSP34.97, and murine CD3 antibody sp34. For detailed transfection and purification procedures, please refer to Example 3.

[0370] Example 9. Binding of anti-CD3 monoclonal antibody to human CD3

[0371] Jurkat cells are immortalized human T lymphocytes that express the human CD3 complex. This cell line was used to detect the binding of the antibody of the present invention to this cell.

[0372] The detailed procedure was as follows: Jurkat cells (Promega, J1621) were seeded into U-shaped 96-well plates, 2 × 10⁶ cells per well. 5 The antibodies to be tested (anti-CD3 monoclonal antibodies HzSP34.24, HzSP34.87, HzSP34.97 and mouse CD3 antibody sp34) were added to the corresponding cell wells according to a series of concentration gradients (the initial concentration of antibody molecules was 500 nM, and it was diluted 3-fold). The cells were incubated at 4°C for 30 minutes, and then the unbound portions were washed with PBS. Goat anti-human Fc PE fluorescent secondary antibody (SouthernBiotech, J2815-5H87B) was added, and the cells were incubated at 4°C for 15 minutes. The cells were then detected by flow cytometry (FACSCELESTA, BD).

[0373] result:

[0374] like Figure 6The results showed that the humanized CD3 antibody HzSP34.24 and the mouse antibody sp34 exhibited considerable affinity at the cellular level, while the affinity of Hzsp34.87 and Hzsp34.97 for CD3 at the cellular level was weakened to varying degrees.

[0375] Example 10. Detection of T cell activation function of CD3 antibody

[0376] This invention uses Jurkat NFAT (Nuclear Factor of Activated T) reporter cells (Promega, J1621) to detect the T cell activation function of anti-CD3 monoclonal antibodies HzSP34.24, HzSP34.87, HzSP34.97 and murine CD3 antibody sp34. These cells are engineered Jurkat T cells. When these cells are activated through the TCR-CD3 pathway, they release luciferase substrate into the experimental system via the downstream signal NFAT, thereby allowing the detection of the degree of T cell activation.

[0377] The detailed procedure in this embodiment is as follows: Using a white 96-well flat plate, 4 × 10⁶ Jurkat NFAT cells are placed in each well. 4 Each antibody molecule was mixed with its corresponding concentration (starting at 500 nM, diluted 3-fold serially) and incubated at 37°C for 6-8 hours. Then, 100 μL of Bio-Glo (Promega, G7940) was added to each well, and the samples were detected using a microplate reader (Spectra, Molecular Devices). Results are as follows: Figure 7 As shown, the murine CD3 antibody sp34, the humanized CD3 antibody HzSP34.24, Hzsp34.87 and Hzsp34.97 all have T cell activation capabilities.

[0378] Example 11. Construction and preparation of bispecific antibodies

[0379] Following the combinations in Table 2, the sequences of the antigen-binding regions of the anti-CLDN18.2 monoclonal antibody HB37A6 and three different anti-human CD3 monoclonal antibodies HzSP34.24, HzSP34.87, and HzSP34.97 were used to construct bispecific antibodies 030, 032, and 033 targeting CLDN18.2×CD3 in a "1+1" configuration, respectively. See the schematic diagram of their antibody structures below. Figure 8Specifically, the heavy chain variable region sequence specifically targeting the antigen-binding domain of CLDN18.2 is SEQ ID NO:4, and the light chain variable region sequence is SEQ ID NO:9; the heavy chain variable region sequences specifically targeting the antigen-binding domain of CD3 are SEQ ID NO:22, SEQ ID NO:30, and SEQ ID NO:32, and the light chain variable region sequence is SEQ ID NO:27. The Fc segment of the antibody is selected from the IgG1LALA sequence of the club-and-socket structure (A. Margaret Merchant et al., Nature Biotechnology, 1998). Therefore, the heavy chain of the CLDN18.2 terminal portion of the bispecific antibody is SEQ ID NO:37, and the light chain is SEQ ID NO:38. The heavy chains of the CD3 terminal portion of the bispecific antibody are SEQ ID NO:39, 41, and 42, and the light chain is SEQ ID NO:40.

[0380] The plasmid construction process for the bispecific antibody was as follows: the heavy chain sequence (SEQ ID NO:37) and light chain sequence (SEQ ID NO:38) of CLDN18.2, the heavy chain sequence (SEQ ID NO:39, 41, and 42) of CD3, and the light chain sequence (SEQ ID NO:40) of CD3 were inserted into the vector pcDNA3.1 (Invitrogen, V790-20) to obtain heavy chain and light chain plasmids at the CLDN18.2 end, and heavy chain and light chain plasmids at the CD3 end, respectively. Then, using PEI (Polysciences, 23966), the heavy chain and light chain plasmids at the CLDN18.2 end and the heavy chain and light chain plasmids at the CD3 end were transiently transfected into Expi293 cells (Invitrogen, A14527) to express the three antibody molecules at the CLDN18.2 end and the CD3 end. Seven days later, the cell fermentation broth was obtained, filtered and clarified, and captured separately using a Hitrap Mabselect Sure Protein A column (GE Healthcare, 11-0034-95) to obtain antibodies at the CLDN18.2 and CD3 ends. After concentration determination using the A280 method, the antibodies at both ends were mixed at a molar ratio of 1:1. An appropriate amount of reducing agent GSH was added, and the reaction was carried out overnight at room temperature. The reducing agent was removed by ultrafiltration, terminating the reaction. Fine purification was then performed using MonoS cation exchange chromatography (GE Healthcare, 17-5168-01). Solution A was 20 mM sodium phosphate buffer (pH 6.6), and solution B was 20 mM sodium phosphate buffer containing 1 M sodium chloride (pH 6.6), with an elution gradient of 0-50% (30 column volumes). The eluted protein solution was ultrafiltered and the buffer was replaced with PBS (Gibco, 70011-044). Molecular weight was determined by mass spectrometry, and purity was identified by SEC-HPLC. The obtained 030, 032 and 033 bispecific antibodies were used in the following examples.

[0381] Table 2. List of CD3 / CLDN18.2 bispecific antibodies

[0382] 030 HB37A6 HzSP34.24 032 HB37A6 HzSP34.87 033 HB37A6 HzSP34.97

[0383] Example 12. Bispecific antibody affinity assay

[0384] The equilibrium dissociation constant (KD) of the bispecific antibody of this invention binding to human CD3 protein was determined using biomembrane thin-layer interferometry (BLI). The affinity determination by BLI was performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitopebinning. MAbs, 2013.5(2): pp. 270-8).

[0385] Half an hour before the experiment, based on the sample quantity, an appropriate number of AHC (18-5060, Fortebio) sensors were immersed in SD buffer (1x PBS, BSA 0.1%, Tween-20 0.05%). 100 μl of SD buffer, each bispecific antibody, and human CD3 protein (CT026H0323H, Beijing Yiqiao Shenzhou) were added to 96-well black polystyrene semi-mass microplates (Greiner, 675076). Detection was performed using a Fortebio Octet Red96. The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: Run steps: Baseline, Loading ~1nm, Baseline, Association, and Dissociation; the run time for each step depended on the sample binding and dissociation rates, the rotation speed was 1000 rpm, and the temperature was 30℃. KD values ​​were analyzed using ForteBio Octet analysis software.

[0386] The affinities of the bispecific antibodies are shown in Table 3. Among them, molecule 030 has the highest affinity at the CD3 end, at 7.4 nM. The CD3-terminal affinities of molecules 032 and 033 decrease sequentially, to 89 nM and 440 nM, respectively.

[0387] Table 3. CD3-terminal affinity of bispecific antibodies

[0388] 030 6.927E+5 0.005164 7.455E-9 032 8.066E+5 0.07183 8.906E-8 033 3.788E+5 0.1689 4.459E-7

[0389] The equilibrium dissociation constant (KD) of human CLDN18.2 binding was determined using surface plasmon resonance (SPR). Following the manufacturer's instructions, the antigen human Claudin18.2 (GenScrip, P50251802) was conjugated onto the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino-conjugation kit (GE Healthcare, BR-1006-33). After conjugation, 1M ethanolamine was injected to block the remaining activation sites. Affinity and kinetic constants were obtained by detecting the binding and dissociation between the antigen on the chip surface and the various bispecific antibodies in the mobile phase using a Biacore (GE Healthcare, T200) device. Serially diluted antibodies (0-100 nM, 2-fold dilution) were flowed sequentially from low to high concentration onto the chip surface, with a binding time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). Data were analyzed using Biacore T200 software with a 1:1 binding model for kinetic analysis. The results are shown in Table 4. The bispecific antibodies 030, 032, and 033 used the same clone HB37A6 at the CLDN18.2 end, exhibiting consistent and very strong affinity at 0.57 nM.

[0390] Table 4. Affinity of the 2-end of CLDN18 for bispecific antibodies

[0391]

[0392] Example 13. In vitro T cell killing experiment

[0393] The bispecific antibodies 030, 032, and 033 obtained in Example 11 were used for in vitro T-cell killing experiments. Human peripheral blood mononuclear cells (PBMCs, Allcells, or Saily) were resuspended in complete culture medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03), and the PBMCs were adjusted to 2 × 10⁶ cells / mL. 6 per ml.

[0394] NuGC-4 or Claudin18.2-overexpressing DAN-G tumor target cells (DAN-G-hCLDN18.2) and non-target cells (L363 (DSMZ, ACC49)) were labeled with Far-Red (Invitrogen) for 10 min, washed twice, and resuspended in complete culture medium. The cell concentration was adjusted to 2 × 10⁻⁶ cells / year.5 per ml.

[0395] PBMCs were mixed with bispecific antibodies 030, 032, and 033 (030 and 032 were initially diluted to 1 nM, and 033 was initially diluted to 400 nM; all antibodies were diluted 5-fold, resulting in 10 concentration points). The mixtures were incubated at 37°C for 30 min, and then 50 μl of tumor target cells (1 × 10⁻⁶ cells) were added at an effector-to-target ratio of 10:1. 4 (Number of cells) was added to 50 μl of PBMC effector cells. After incubation at 37°C for 24 hours, the cells were centrifuged and resuspended in propidium iodide (PI, Invitrogen) at a final concentration of 10 μg / ml. Cells positive for both Far-Red and PI were detected using flow cytometry (BD, FACSCELESTA). The tumor target cell killing ratio was calculated using FACSDiva software (BD, Celestsa).

[0396] from Figure 9 The results showed that molecules 030 and 032 exhibited very strong killing activity in NUGC-4 gastric cancer cells, with EC50 values ​​both less than 1 pM. From... Figure 10 The results showed that on CLDN18.2-overexpressing pancreatic cancer cells DANN-G-hCLDN18.2, the EC50 values ​​of both were even less than 0.1 pM. The killing activity of 033 molecule was weaker on both tumor cell lines, weaker than 030 and 032, approximately 1000 times lower, but still achieved maximum killing (nearly 100% cell lysis). However, in cases of CLDN18.2 negative expression, 030, 032, and 033 molecules did not exhibit non-specific killing activity. Figure 11 This indicates that molecules 030, 032, and 033 all exhibit tumor cell-specific killing dependent on CLDN18.2 expression. Furthermore, the killing effect of the bispecific antibody of this invention is related to the abundance of CLDN18.2 on the cell surface; within a certain expression abundance range, the higher the expression level of CLDN18.2 on the cell surface, the better the killing effect.

[0397] Example 14. In vitro cytokine release experiment

[0398] Human peripheral blood mononuclear cells (PBMCs, Allcells, or Saily) were resuspended in complete culture medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03), and the PBMCs were adjusted to 2 × 10⁶ cells / mL. 6Cells / ml. The concentration of DAN-G-hCLDN18.2 in NUCGC-4 or DAN-G tumor target cells overexpressing Claudin18.2 was adjusted to 2 × 10⁻⁶ cells / ml. 5 per ml.

[0399] PBMCs were mixed with bispecific antibodies 030, 032, and 033 respectively and incubated at 37°C for 30 min. Then, at an effector-to-target ratio of 10:1, 50 μl of tumor target cells (1×10⁻⁶) were added. 4 (Number of cells) was added to 50 μl of PBMC effector cells. The cells were incubated at 37°C for 24 hours, centrifuged, and the cell supernatant was collected. Cytokines were detected using the Human Th1 / Th2 / Th17 Kit (BD, catalog number 560484). After incubation at room temperature for 3 hours, the cells were analyzed using flow cytometry (BD, FACSCELESTA), and the release of cytokines in the supernatant was analyzed using FCAP Array software (BD).

[0400] from Figure 12 and Figure 13 The results showed that molecules 030, 032, and 033 could mediate the massive release of IL-2, TNFα, and IFNgamma cytokines in gastric cancer cells NUGC-4 and pancreatic cancer cells DANG-hCLDN18.2, and were positively correlated with the affinity of the CD3 terminus.

[0401] Example 15. In vitro T cell activation experiment

[0402] Human peripheral blood mononuclear cells (PBMCs, Allcells, or Saily) were resuspended in complete culture medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03), and the PBMCs were adjusted to 2 × 10⁶ cells / mL. 6 per ml.

[0403] The concentration of DAN-G-CLDN18.2 in NUGC-4 or DAN-G tumor target cells overexpressing Claudin18.2 was adjusted to 2×10⁻⁶. 5 PBMCs were mixed with bispecific antibodies 030, 032, and 033 respectively and incubated at 37°C for 30 min. Then, at an effector-to-target ratio of 10:1, 50 μl of tumor target cells (1 × 10⁻⁶ cells / ml) were added. 4(Number of cells) were added to 50 μl of PBMC effector cells. After incubation at 37°C for 24 hours, the cells were centrifuged, the supernatant was removed, and the cells were incubated at 4°C for one hour with BV421 anti-human CD3 (Biolegend, 317344), PerCP / Cy5.5 mouse anti-human CD4 (BD, 552838), APC / Cy7 anti-human CD8a (Biolegend, 300926), PE anti-human CD25 (Biolegend, 302606), and FITC anti-human CD69 (Biolegend, 310904). The cells were then washed three times with 1×PBS. The proportion of CD25 and CD69 double-positive cells in CD4+ and CD8+ T cells was detected by flow cytometry (BD, FACSCELESTA). The proportion of CD25 and CD69 double-positive cells in CD4+ and CD8+ T cells was calculated using FACSDiva software (BD, Celestsa), which represents the activation rate of CD4+ and CD8+ T cells.

[0404] according to Figure 14 As shown, molecules 030, 032, and 033 can specifically activate T cells when co-cultured with gastric cancer cells NUGC-4, and the activation ability is positively correlated with the affinity of the CD3 terminus.

[0405] Example 16. In vivo pharmacodynamic experiment—gastric cancer model

[0406] This study investigated the antitumor effect of a bispecific antibody against NUGC-4 tumor-bearing mice in female NOG mice. Forty-nine female NOG mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected.

[0407] PBMC cells (Allcells) were resuscitated, centrifuged, and dispersed in PBS (1×) to obtain a cell density of 2×10⁶ cells / year. 7 / ml of cell suspension. 200μl of cell suspension was injected intravenously into mice via the orbital vein with PBMC cells, 4×10 6 / Only.

[0408] NUGC-4 cells were routinely resuscitated and passaged for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) at a density of 6×10⁶ cells / cells. 7 Cells / ml were mixed with Matrigel gel at a 1:1 ratio to prepare a cell concentration of 3×10⁶ cells / ml. 7 Cells / ml suspension. On day 3, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of NOG humanized mice to establish a NUCG-4 tumor-bearing mouse model.

[0409] On day 7 after cell inoculation, the maximum width and length axes of mouse tumors were measured using calipers, and the tumor volume was calculated. Tumors with a volume of 53.35 mm² were selected. 3 ~168.07mm 3 Mice were divided into serpentine groups (n=6 per group) based on tumor volume. Each mouse was intravenously injected with the bispecific antibodies 030, 032, and 033 of this invention, as well as the negative control h-IgG (Equitech-Bio, batch number 160308-02), at doses of 0.3 mg / kg and 1 mg / kg, respectively, once weekly for a total of four administrations. Tumor volume was measured twice weekly. Tumor inhibition rate (TGI%) was calculated using the following formula:

[0410] TGI% = 100% * (Tumor volume in control group – Tumor volume in treatment group) / (Tumor volume in control group – Tumor volume in control group before drug administration).

[0411] according to Figure 15 As shown, in a NUCG-4 human gastric cancer mouse model, 030 and 032 achieved a TGI of 100% at a low dose of 0.3 mg / kg, and even achieved a CR (complete remission) of 50% at a high dose of 1 mg / kg (3 out of 6 mice achieved complete tumor disappearance). 033, however, showed almost no efficacy at low doses, and at a dose of 1 mg / kg, the TGI reached 20%. Throughout the experiment, neither the experimental nor control groups experienced a decrease in body weight.

[0412] Example 17. In vivo efficacy experiment—pancreatic cancer model

[0413] This study investigated the antitumor effect of a bispecific antibody against DAN-G-Claudin18.2 tumor-bearing mice in female NOG mice. Forty-nine NOG mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were injected intravenously with PBMC cells at a dose of 4 × 10⁴ cells. 6 / animal, inoculation volume 200ul / animal (as shown in Example 18). This is recorded as day 0.

[0414] The human pancreatic cancer cells DAN-G-CLDN18.2 constructed in Example 1 were routinely passaged for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) to obtain a cell density of 10×10⁻⁶ cells / mL. 6 A cell suspension was prepared at a concentration of 5 × 10⁶ cells / ml. The cell suspension was mixed with Matrigel gel at a 1:1 ratio to prepare a cell concentration of 5 × 10⁶ cells / ml. 6Cells / ml of cell suspension. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of NOD-SCID mice to establish a humanized pancreatic cancer model with CLDN18.2 overexpression and DNA-G expression.

[0415] Seven days after tumor cell inoculation, the maximum width and length axes of mouse tumors were measured using calipers to calculate tumor volume. Mice were then grouped in a serpentine pattern based on tumor volume, with tumors exceeding 46.42 mm² grouped as the largest. 3 ~120.64mm 3 Mice within the range were grouped in a serpentine pattern according to tumor size (6 mice per group).

[0416] Each mouse was intravenously injected with the bispecific antibodies 030, 032, and 033 of this invention, as well as the negative control h-IgG (Equitech-Bio, batch number 160308-02), at intraperitoneal doses of 0.3 mg / kg and 1 mg / kg, respectively, once a week for a total of four administrations. Tumor volume was measured twice a week. The tumor inhibition rate (TGI%) was calculated using the following formula:

[0417] TGI% = 100% * (Tumor volume in control group – Tumor volume in treatment group) / (Tumor volume in control group – Tumor volume in control group before drug administration). Figure 16 As shown, in the CLDN18.2-overexpressing DNA-G pancreatic cancer humanized model, molecules 030 and 032 achieved a TGI of 100% at both doses. Molecule 033 also achieved TGIs of 42% (0.3 mg / kg) and 76% (1 mg / kg), respectively, which may be related to the high expression level of CLDN18.2 in DNA-G-CLDN18.2 pancreatic cancer cells. Throughout the experiment, the body weight of mice in both the experimental and control groups did not decrease.

[0418] Example 18. Mouse PK Experiment

[0419] This study investigated the pharmacokinetic properties of 030, 032, and 033 in female Balb / C mice via tail vein injection (Vitaliva). Blood samples were collected via ocular sampling at 0.086 hr, 0.5 hr, 2 hr, 6 hr, 24 hr, 48 hr, 4 days, 7 days, 14 days, and 21 days post-administration. The blood samples were centrifuged at 3000 rpm for 10 min at 4°C, and serum was collected. Antibody levels in the serum were measured using ELISA, and the half-lives of 030, 032, and 033 in mice were calculated.

[0420] Experimental results are as follows Figure 17As shown, the half-lives of 030, 032, and 033 in mice all reached p-values ​​similar to those of normal monoclonal antibodies. This further demonstrates that the bispecific antibodies constructed in this invention do not affect the antibody half-life. Sequence information:

[0421]

[0422]

[0423]

[0424]

[0425] sequence list <110> Innovent Biologics (Suzhou) Co., Ltd. <120> Bispecific antibodies against Claudin18.2 and CD3 and their applications <130> PF 210767PCT <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <400> 1 Gly Phe Thr Phe Ser Ser Tyr Val Met Ser 1 5 10 <210> 2 <211> 17 <212> PRT <213> artificial <220> <223> synthesis <400> 2 Thr Ile Ser His Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 13 <212> PRT <213> artificial <220> <223> synthesis <400> 3 Asp Ala Pro Tyr Tyr Asp Ile Leu Thr Gly Tyr Arg Tyr 1 5 10 <210> 4 <211> 122 <212> PRT <213> artificial <220> <223> synthesis <400> 4 Glu Val Gln Leu Leu Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asn Trp Val 35 40 45 Ser Thr Ile Ser His Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Asp Ala Pro Tyr Tyr Asp Ile Leu Thr Gly Tyr Arg Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 6 <211> 11 <212> PRT <213> artificial <220> <223> synthesis <400> 6 Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala 1 5 10 <210> 7 <211> 7 <212> PRT <213> artificial <220> <223> synthesis <400> 7 Lys Ala Ser Ser Leu Glu Ser 1 5 <210> 8 <211> 9 <212> PRT <213> artificial <220> <223> synthesis <400> 8 Gln Gln Tyr Donkey Ser Tyr Ser Tyr Thr 1 5 <210> 9 <211> 107 <212> PRT <213> artificial <220> <223> synthesis <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> artificial <220> <223> synthesis <400> 10 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 11 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <400> 11 Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn 1 5 10 <210> 12 <211> 16 <212> PRT <213> artificial <220> <223> synthesis <400> 12 Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 <210> 13 <211> 9 <212> PRT <213> artificial <220> <223> synthesis <400> 13 Ser Trp Arg Gly Asn Ser Phe Asp Tyr 1 5 <210> 14 <211> 118 <212> PRT <213> artificial <220> <223> synthesis <400> 14 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 15 <211> 17 <212> PRT <213> artificial <220> <223> synthesis <400> 15 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 16 <211> 7 <212> PRT <213> artificial <220> <223> synthesis <400> 16 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 17 <211> 9 <212> PRT <213> artificial <220> <223> synthesis <400> 17 Gln Asn Asp Tyr Ser Tyr Pro Phe Thr 1 5 <210> 18 <211> 113 <212> PRT <213> artificial <220> <223> synthesis <400> 18 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Light <210> 19 <211> 10 <212> PRT <213> artificial <220> <223> synthesis <400> 19 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn 1 5 10 <210> 20 <211> 19 <212> PRT <213> artificial <220> <223> synthesis <400> 20 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Light Asp <210> 21 <211> 14 <212> PRT <213> artificial <220> <223> synthesis <400> 21 His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 22 <211> 125 <212> PRT <213> artificial <220> <223> synthesis <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 23 <211> 330 <212> PRT <213> artificial <220> <223> synthesis <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 24 <211> 14 <212> PRT <213> artificial <220> <223> synthesis <400> 24 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 25 <211> 7 <212> PRT <213> artificial <220> <223> synthesis <400> 25 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic <400> 26 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 27 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 27 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 28 <211> 106 <212> PRT <213> Artificial <220> <223> Synthetic <400> 28 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 29 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 29 His Tyr Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 30 <211> 125 <212> PRT <213> artificial <220> <223> synthesis <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Tyr Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 31 <211> 19 <212> PRT <213> artificial <220> <223> synthesis <400> 31 Arg Ile Arg Ser Lys Ala Gly Gly Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Light Asp <210> 32 <211> 125 <212> PRT <213> artificial <220> <223> synthesis <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ala Gly Gly Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 330 <212> PRT <213> artificial <220> <223> synthesis <400> 33 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 34 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 34 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 35 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 36 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 36 Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 37 <211> 452 <212> PRT <213> Artificial <220> <223> Synthetic <400> 37 Glu Val Gln Leu Leu Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asn Trp Val 35 40 45 Ser Thr Ile Ser His Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Asp Ala Pro Tyr Tyr Asp Ile Leu Thr Gly Tyr Arg Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 38 <211> 214 <212> PRT <213> Artificial <220> <223> Synthetic <400> 38 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 39 <211> 455 <212> PRT <213> Artificial <220> <223> Synthetic <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 40 <211> 215 <212> PRT <213> Artificial <220> <223> Synthetic <400> 40 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 41 <211> 455 <212> PRT <213> Artificial <220> <223> Synthetic <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Tyr Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 42 <211> 455 <212> PRT <213> Artificial <220> <223> Synthetic <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ala Gly Gly Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455

Claims

1. A bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2, and the second antigen-binding domain specifically binds to CD3. The first antigen-binding domain comprises three heavy chain variable region complementarity-determining regions (CDRs): A1-HCDR1, A1-HCDR2, and A1-HCDR3, and three light chain variable region CDRs: A1-LCDR1, A1-LCDR2, and A1-LCDR3. A1-HCDR1, A1-HCDR2, and A1-HCDR3 are the three heavy chain CDRs contained in A1-VH as shown in SEQ ID NO:4, and A1-LCDR1, A1-LCDR2, and A1-LCDR3 are the three light chain CDRs contained in VL as shown in SEQ ID NO:

9. A1-HCDR1 is determined according to the AbM rule; and A1-HCDR2, A1-HCDR3, A1-LCDR1, A1-LCDR2, and A1-LCDR3 are determined according to the Kabat rule. The second antigen-binding domain comprises three heavy chain variable region complementarity-determining regions A2-HCDR1, A2-HCDR2, and A2-HCDR3, and three light chain complementarity-determining regions A2-LCDR1, A2-LCDR2, and A2-LCDR3, wherein A2-HCDR1, A2-HCDR2, and A2-HCDR3 are the three complementarity-determining regions contained in A2-VH as shown in SEQ ID NO: 30, 22, or 32, and A2-LCDR1, A2-LCDR2, and A2-LCDR3 are the three complementarity-determining regions contained in A2-VL as shown in SEQ ID NO: 27; wherein A2-HCDR1 is determined according to the AbM rule; and A2-HCDR2, A2-HCDR3, A2-LCDR1, A2-LCDR2, and A2-LCDR3 are determined according to the Kabat rule.

2. The bispecific antibody of claim 1, wherein The first antigen-binding domain comprises A1-HCDR1, A1-HCDR2, and A1-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2, and 3, respectively, and A1-LCDR1, A1-LCDR2, and A1-LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7, and 8, respectively; and The second antigen-binding domain contains (i) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 20, and 29, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25, and 26, respectively; or (ii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 20, and 21, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25, and 26, respectively; or (iii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 31 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25 and 26, respectively.

3. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (i) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 4; or (ii) Contains one or more, but no more than 10, amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 4; and Light chain variable region (i) Contains an amino acid sequence that has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 9; or (ii) An amino acid sequence comprising one or more, but not more than 10, amino acid substitutions compared to an amino acid sequence selected from SEQ ID NO:

9.

4. The bispecific antibody of claim 3, wherein the amino acid substitution is a conservative amino acid substitution.

5. The bispecific antibody of claim 3, wherein the first antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

9.

6. The bispecific antibody of claim 3, wherein the first antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:

9.

7. The bispecific antibody according to any one of claims 1, 2, and 4-6, wherein the second antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (i) Contains an amino acid sequence that has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; or (ii) An amino acid sequence comprising one or more, but not more than 10, amino acid substitutions compared to an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; and Light chain variable region (i) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 27; or (ii) Contains one or more, but no more than 10, amino acid substitutions compared to the amino acid sequence of SEQ ID NO:

27.

8. The bispecific antibody of claim 7, wherein the amino acid substitution is a conservative amino acid substitution.

9. The bispecific antibody of claim 7, wherein the second antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, 22 or 32, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

27.

10. The bispecific antibody of claim 7, wherein the second antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 30, 22 or 32, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

27.

11. The bispecific antibody according to any one of claims 1, 2, 4-6 and 8-10, further comprising a heavy chain constant region and / or a light chain constant region.

12. The bispecific antibody according to any one of claims 1, 2, 4-6 and 8-10, wherein it is an IgG-like bispecific antibody.

13. The bispecific antibody according to any one of claims 1, 2, 4-6 and 8-10, wherein the heavy chain constant region of the antibody is derived from IgG1 or IgG2 or IgG3 or IgG4.

14. The bispecific antibody of claim 13, wherein the heavy chain constant region of the antibody is derived from IgG1.

15. The bispecific antibody according to any one of claims 1, 2, 4-6, 8-10, and 14, comprising two heavy chain constant regions, wherein one heavy chain constant region A1-HC is linked to the heavy chain variable region A1-VH of the first antigen domain to form a heavy chain binding to CLDN18.2, and the other heavy chain constant region A2-HC is linked to the heavy chain variable region A2-VH of the second antigen binding domain to form a heavy chain binding to CD3, and comprising two light chain constant regions, wherein one light chain constant region A1-LC is linked to the light chain variable region A1-VL of the first antigen domain to form a light chain binding to CLDN18.2, and the other light chain constant region A2-LC is linked to the light chain variable region A2-VL of the second antigen binding domain to form a light chain binding to CD3.

16. The bispecific antibody of claim 15, wherein A1-HC may be the same as or different from A2-HC, and / or A1-LC may be the same as or different from A2-LC.

17. The bispecific antibody of claim 15, wherein A1-VH and A1-VL of the CLDN18.2 binding portion are fully human, and A2-VH and A2-VL of the CD3 binding portion are humanized.

18. The bispecific antibody of claim 15, wherein the heavy chain of the CLDN18.2 binding moiety... (i) Contains an amino acid sequence that has at least 85% identity with the amino acid sequence of SEQ ID NO:37; or (ii) An amino acid sequence comprising one or more, but not exceeding 20, amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 37; and Light chain in conjunction with CLDN18.2 (i) Contains an amino acid sequence that has at least 85% identity with the amino acid sequence of SEQ ID NO:38; or (ii) An amino acid sequence having one or more, but not more than 20, amino acid substitutions compared to the amino acid sequence of SEQ ID NO:

38.

19. The bispecific antibody of claim 18, wherein the amino acid substitution is a conservative amino acid substitution.

20. The bispecific antibody of claim 18, wherein the heavy chain of the CLDN18.2 binding portion comprises the amino acid sequence of SEQ ID NO:37, and the light chain of the CLDN18.2 binding portion comprises the amino acid sequence of SEQ ID NO:

38.

21. The bispecific antibody of claim 18, wherein the heavy chain of the CLDN18.2 binding portion consists of the amino acid sequence of SEQ ID NO:37, and the light chain of the CLDN18.2 binding portion consists of the amino acid sequence of SEQ ID NO:

38.

22. The bispecific antibody of claim 15, wherein... Heavy chain in the CD3 junction (i) Contains an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 41, 39, or 42; or (ii) Contains an amino acid sequence having one or more, but not more than 20, amino acid substitutions compared to an amino acid sequence selected from SEQ ID NO: 41, 39, or 42; and Light chain in the part that connects with CD3 (i) Contains an amino acid sequence that has at least 85% identity with the amino acid sequence of SEQ ID NO:40; or (ii) Contains one or more, but no more than 20, amino acid substitutions compared to the amino acid sequence of SEQ ID NO:

40.

23. The bispecific antibody of claim 22, wherein the amino acid substitution is a conservative amino acid substitution.

24. The bispecific antibody according to any one of claims 16-23, wherein the heavy chain of the CD3-binding portion comprises the amino acid sequence of SEQ ID NO: 41, 39 or 42, and the light chain of the CD3-binding portion comprises the amino acid sequence of SEQ ID NO:

40.

25. The bispecific antibody according to any one of claims 16-23, wherein the heavy chain of the CD3-binding portion consists of the amino acid sequence of SEQ ID NO: 41, 39 or 42, and the light chain of the CD3-binding portion consists of the amino acid sequence of SEQ ID NO:

40.

26. An isolated nucleic acid encoding the light chain variable region and the heavy chain variable region of the bispecific antibody of any one of claims 1 to 25.

27. An isolated nucleic acid encoding the light and heavy chains of the bispecific antibody of any one of claims 1 to 25.

28. A vector comprising the nucleic acid of claim 26 or 27.

29. The carrier of claim 28, wherein the carrier is an expression carrier.

30. A host cell comprising the nucleic acid of claim 26 or 27 or the vector of claim 28 or 29.

31. The host cell of claim 30, wherein the host cell is prokaryotic or eukaryotic.

32. The host cell of claim 30, wherein the host cell is selected from yeast cells or mammalian cells.

33. The host cell of claim 30, wherein the host cell is selected from 293 cells or CHO cells.

34. The host cell of claim 30, wherein the host cell is selected from CHO-S cells or HEK293 cells.

35. A method for preparing a bispecific antibody that specifically binds to CLDN18.2 and CD3, the method comprising culturing a host cell according to any one of claims 30-34 under conditions suitable for expressing a nucleic acid encoding a bispecific antibody of any one of claims 1 to 25.

36. The method of claim 35, wherein the method further comprises separating the antibody or its antigen-binding fragment.

37. The method of claim 35 or 36, wherein the method further comprises recovering the antibody from the host cell.

38. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 25.

39. The pharmaceutical composition of claim 38, further comprising pharmaceutical excipients.

40. The pharmaceutical composition of claim 38 or 39, wherein the pharmaceutical composition further comprises one or more therapeutic agents other than the bispecific antibody of any one of claims 1 to 25.

41. A pharmaceutical combination product comprising a bispecific antibody of any one of claims 1 to 25, and one or more therapeutic agents other than the bispecific antibody of any one of claims 1 to 25.

42. Use of the bispecific antibody of any one of claims 1 to 25, or the pharmaceutical composition of any one of claims 38 to 40, or the pharmaceutical combination product of claim 41 in the preparation of a medicament for the treatment of pancreatic cancer, gastric cancer, or gastroesophageal junction cancer.

43. The use of claim 42, wherein the drug is further administered to a patient in combination with one or more therapies.

44. The use as described in claim 43, wherein the therapy is a treatment method.

45. The use as described in claim 44, wherein the treatment method includes radiotherapy or surgery.

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