Antibody-drug conjugates, methods for preparing same, and uses thereof

Abstract

To provide an anti-CLDN 18.2 antibody-drug conjugate, a preparation method and use thereof.SOLUTION: The present invention provides an antibody-drug conjugate (ADC), the structure of which is shown in the formula (D-L)γ-A, wherein, D is a fragment of a bioactive molecule; L is a linker; γ is an integer from 1 to 10; preferably, γ is an integer from 1 to 8; A is an antibody or antigen-binding fragment thereof that specifically binds to human CLDN 18.2.SELECTED DRAWING: Figure 12A

Description

[Technical Field]

[0001] This application claims priority based on Chinese Patent Application No. 202010410633.5, filed on May 15, 2020, the disclosure of which is hereby incorporated in its entirety into this application.

[0002] The present invention belongs to the field of biomedical science, and specifically relates to an anti-claudin 18.2 antibody-drug conjugate (ADC), a composition comprising the ADC, and uses thereof. [Background technology]

[0003] Surgical resection is the mainstream treatment for gastric cancer. For unresectable or recurrent gastric cancer, chemotherapy is the primary treatment, but with current technology, chemotherapy can only alleviate symptoms and extend survival. Currently, there is no widely recognized standard chemotherapy regimen for advanced gastric cancer. Regarding biomolecular drugs, with the exception of approved drugs such as trastuzumab, ramucirumab, and pembrolizumab, most other gastric cancer-targeted drugs have insufficient efficacy or are still in the early stages of clinical research. Major weaknesses of existing drugs include unmet clinical needs for non-surgical treatments, very limited treatment options for advanced or recurrent gastric cancer, very poor prognosis, and high mortality rates. Due to the high incidence of gastric cancer, there is currently a great demand for gastric cancer drugs.

[0004] Claudin 18.2 (CLDN18.2) is a member of the claudin protein family. Claudin family proteins are a type of protein that mediates tight junctions between cells. Different claudin subtypes are expressed in different tissues and associated with different types of cancer. In gastric mucosal cells, claudin 18.2 expression in normal tissues is limited. Claudin 18.2 is highly expressed in 70% of primary gastric adenocarcinomas and their metastases, and is also expressed in other cancers, such as pancreatic cancer, esophageal cancer, and non-small cell lung cancer (NSCLC). The highly specific expression of claudin 18.2 in tumor tissues makes it an excellent target for tumor immunotherapy. Summary of the Invention

[0005] The inventors of the present application have described an antibody with high specificity and high affinity for claudin 18.2 in PCT / CN2019 / 126495, the entire contents of which are incorporated herein by reference and made a part of this application. Through extensive creative research, the inventors of the present application have further invented a claudin 18.2 antibody-drug conjugate (ADC), which provides a safe and effective drug option for the treatment of tumors.

[0006] Specifically, the present inventors conjugated a high-affinity antibody that specifically binds to claudin 18.2 to a biologically active molecule via a linker to obtain a series of ADCs targeting claudin 18.2 and compositions containing the ADCs. The ADCs or compositions exhibit high killing activity against several tumor cells expressed by claudin 18.2. In vivo animal experiments showed that the ADCs effectively inhibited the growth of claudin 18.2-positive tumors, particularly gastric cancer and gastric adenocarcinoma, and were very safe to use.

[0007] Antibody-Drug Conjugates (ADCs) In one aspect, the present invention provides an ADC that specifically binds to human CLDN18.2 and has the structure of Formula (I): (DL) γ-A antibody-drug conjugate (ADC) represented by formula (I) is provided, wherein D is a fragment of a biologically active molecule; L is a linker; γ is an integer from 1 to 10; preferably, γ is an integer from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); and A is an antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2.

[0008] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2 comprises: (1) the following three heavy chain CDRs: CDR-H1, CDR-H2, and CDR-H3 contained in a VH (heavy chain variable region) set forth in SEQ ID NO: 13, 14, or 23; and / or the following three light chain CDRs: CDR-L1, CDR-L2, and CDR-L3 contained in a VL (light chain variable region) set forth in SEQ ID NO: 15 or 24; or (2) the following three heavy chain CDRs: CDR-H1 set forth in (1) or a variant thereof comprising an amino acid mutation, CDR-H2 set forth in (1) or a variant thereof comprising an amino acid mutation, and CDR-H3 set forth in (1) or a variant thereof comprising an amino acid mutation; and / or the following three light chain CDRs: CDR-L1 set forth in (1) or a variant thereof comprising an amino acid mutation, CDR-L2 set forth in (1) or a variant thereof comprising an amino acid mutation, and CDR-L3 set forth in (1) or a variant thereof comprising an amino acid mutation, wherein at least one of the three heavy chain CDRs and / or three light chain CDRs described in (2) contains an amino acid mutation compared to the corresponding CDR in (1), the amino acid mutation being a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); an antibody or antigen-binding fragment thereof containing the above mutation can still specifically bind to CLDN18.2, preferably human CLDN18.2, and preferably the substitution is a conservative substitution.

[0009] In some embodiments, the CDRs are defined according to the IMGT or AbM numbering system.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a framework region (FR) derived from a human immunoglobulin.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1-1) the following three heavy chain CDRs as defined by the IMGT numbering system: CDR-H1 having the sequence of SEQ ID NO: 1, CDR-H2 having the sequence of SEQ ID NO: 2 or 21, and CDR-H3 having the sequence of SEQ ID NO: 3; and / or the following three light chain CDRs as defined by the IMGT numbering system: CDR-L1 having the sequence of SEQ ID NO: 4, CDR-L2 having the sequence of SEQ ID NO: 5, and CDR-L3 having the sequence of SEQ ID NO: 6; or (1-2) the following three heavy chain CDRs: CDR-H1 as set forth in (1-1) or a variant thereof comprising an amino acid mutation, and CDR-H2 as set forth in (1-1) or a variant thereof comprising an amino acid mutation, and CDR-H3 as set forth in (1-1) or a variant thereof comprising an amino acid mutation; and / or The following three light chain CDRs: CDR-L1 or a variant thereof comprising an amino acid mutation as set forth in (1-1), CDR-L2 or a variant thereof comprising an amino acid mutation as set forth in (1-1), and CDR-L3 or a variant thereof comprising an amino acid mutation as set forth in (1-1), wherein at least one of the three heavy chain CDRs and / or the three light chain CDRs comprises an amino acid mutation compared to the corresponding CDR in (1-1), and the amino acid mutation is a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids); an antibody or antigen-binding fragment thereof comprising the above mutations still binds to human CLDN18.2, preferably with conservative substitutions; or (2-1) the following three heavy chain CDRs as defined by the AbM numbering system: CDR-H1 having the sequence of SEQ ID NO: 7, CDR-H2 having the sequence of SEQ ID NO: 8 or 22, and CDR-H3 having the sequence of SEQ ID NO: 9; and / or the following three light chain CDRs as defined by the AbM numbering system: CDR-L1 having the sequence of SEQ ID NO: 10, CDR-L2 having the sequence of SEQ ID NO: 11, and CDR-L3 having the sequence of SEQ ID NO: 12; or (2-2) the following three heavy chain CDRs: CDR-H1 as defined in (2-1) or a variant thereof comprising an amino acid mutation, CDR-H2 as defined in (2-1) or a variant thereof comprising an amino acid mutation, and CDR-H3 as defined in (2-1) or a variant thereof comprising an amino acid mutation; and / or The following three light chain CDRs: CDR-L1 or a variant thereof comprising an amino acid mutation as set forth in (2-1), CDR-L2 or a variant thereof comprising an amino acid mutation as set forth in (2-1), and CDR-L3 or a variant thereof comprising an amino acid mutation as set forth in (2-1); wherein at least one of the three heavy chain CDRs and / or the three light chain CDRs comprises an amino acid mutation compared to the corresponding CDR in (2-1), and the amino acid mutation is a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids); an antibody or antigen-binding fragment thereof comprising the mutations can still specifically bind to human CLDN18.2, and preferably, the substitutions are conservative substitutions.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a framework region (FR) derived from a human immunoglobulin.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a VH and / or VL, the CDRs of which are defined according to the IMGT numbering system, comprising: (1-1) a VH comprising the following three CDRs: CDR-H1 having the sequence of SEQ ID NO: 1, CDR-H2 having the sequence of SEQ ID NO: 2 or 21, and CDR-H3 having the sequence of SEQ ID NO: 3; and / or a VL comprising the following three CDRs: CDR-L1 having the sequence of SEQ ID NO: 4, CDR-L2 having the sequence of SEQ ID NO: 5, and CDR-L3 having the sequence of SEQ ID NO: 6; or (1-2): compared to the VH or VL described in (1-1), at least one CDR comprises a mutation that is one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, or any combination thereof), preferably the substitutions are conservative substitutions, and the antibody or antigen-binding fragment thereof comprising the mutations can still specifically bind to CLDN18.2, preferably human CLDN18.2; or (2) The following VH and / or VL, whose CDRs are defined according to the AbM numbering system: (2-1): VH comprising the following three CDRs: CDR-H1 having the sequence of SEQ ID NO: 7, CDR-H2 having the sequence of SEQ ID NO: 8 or 22, and CDR-H3 having the sequence of SEQ ID NO: 9; and / or VL comprising the following three CDRs: CDR-L1 having the sequence of SEQ ID NO: 10, CDR-L2 having the sequence of SEQ ID NO: 11, and CDR-L3 having the sequence of SEQ ID NO: 12. 3; or (2-2): at least one CDR comprises a mutation which is one or more amino acid substitutions, deletions or additions, or any combination thereof (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids, or any combination thereof), compared to the VH or VL described in (2-1), preferably the substitutions are conservative substitutions, and the antibody or antigen-binding fragment thereof comprising the mutations can still specifically bind to CLDN18.2, preferably human CLDN18.2.

[0014] In some embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof comprises a framework region (FR) derived from a human or mouse immunoglobulin.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a VH set forth in SEQ ID NO: 13 or 14, and / or a VL set forth in SEQ ID NO: 15; or (2) The VH contained in the antibody or antigen-binding fragment thereof has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH described in (1), and / or the VL contained in the antibody or antigen-binding fragment thereof has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VL described in (1), and the antibody or antigen-binding fragment thereof having identity to the VL or VH described in (1) can still specifically bind to CLDN18.2, preferably human CLDN18.2; or (3) Compared with the VH described in (1), the VH contained in the antibody or antigen-binding fragment thereof has one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids, or any combination thereof), and / or compared with the VL described in (1), the VL contained in the antibody or antigen-binding fragment thereof has one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids, or any combination thereof), preferably, the substitutions are conservative substitutions.An antibody or antigen-binding fragment thereof containing the mutations can still specifically bind to CLDN18.2, preferably human CLDN18.2.

[0016] In any of the above aspects, the antibody or antigen-binding fragment thereof can further comprise a constant region derived from a mammalian (e.g., murine or human) immunoglobulin or a variant thereof.

[0017] In some embodiments, the antibody may comprise: (1) a human immunoglobulin CH (heavy chain constant region) or variant thereof, wherein the variant comprises one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived; and / or (2) a human immunoglobulin CL (light chain constant region) or variant thereof, wherein the variant comprises one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived.

[0018] In some embodiments, the constant region is altered, e.g., mutated, to modify the characteristics of the anti-CLDN18.2 antibody (e.g., to alter one or more of the following characteristics: Fc receptor binding, antibody glycosylation, amount of cysteine ​​residues, function on effector cells, or complement). At least one amino acid residue in the constant region of the antibody can be substituted with another amino acid residue to alter function. For example, effector function can be altered (e.g., enhanced) by changing the antibody affinity for an effector ligand (e.g., FcR or complement C1q). In some embodiments, the constant region is altered (e.g., enhanced) to modify antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular phagocytosis (ADCP).

[0019] In some embodiments, the CH is an IgG heavy chain constant region, eg, an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain constant region of human IgG1.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CH set forth in SEQ ID NO: 16, or a variant thereof having up to 20 conservative amino acid substitutions (e.g., up to 15, 10, or 5 conservative amino acid substitutions, e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO: 16. A CH containing the mutations retains substantially the same function as SEQ ID NO: 16.

[0022] In some embodiments, CL is selected from a kappa or lambda light chain constant region.

[0023] In some embodiments, CL is a kappa light chain constant region (eg, a human kappa light chain).

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CL set forth in SEQ ID NO: 17, or a variant thereof having up to 20 conservative amino acid substitutions (e.g., up to 15, 10, or 5 conservative amino acid substitutions, e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO: 17. A CL containing the mutations retains substantially the same function as SEQ ID NO: 17.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CH set forth in SEQ ID NO:16 and / or a CL set forth in SEQ ID NO:17.

[0026] In some embodiments, the antibody comprises a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 13 and a CH set forth in SEQ ID NO: 16, and a light chain comprising a VL of the sequence set forth in SEQ ID NO: 15 and a CL set forth in SEQ ID NO: 17.

[0027] In some embodiments, the antibody comprises a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 14 and a CH set forth in SEQ ID NO: 16, and a light chain comprising a VL of the sequence set forth in SEQ ID NO: 15 and a CL set forth in SEQ ID NO: 17.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising an amino acid sequence selected from the group consisting of: (1-1) the sequence set forth in SEQ ID NO: 18; (1-2) a sequence having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 18; or (1-3) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 18; and (2) a light chain comprising an amino acid sequence selected from the group consisting of: (2-1) the sequence set forth in SEQ ID NO: 20; (2-2) A sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 20; or (2-3) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 20.

[0029] In some embodiments, the substitutions described in (1-2) and (2-2) above are conservative substitutions. CH containing the mutations retains substantially the same function as SEQ ID NO:16.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising an amino acid sequence selected from the group consisting of: (1-1) the sequence set forth in SEQ ID NO: 19; (1-2) a sequence having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 19; or (1-3) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 19; and (2) a light chain comprising an amino acid sequence selected from the group consisting of: (2-1) the sequence set forth in SEQ ID NO: 20; (2-2) A sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in SEQ ID NO: 20; or (2-3) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 20.

[0031] In some embodiments, the substitutions described in (1-2) and (2-2) are conservative substitutions. Preferably, an antibody or antigen-binding fragment thereof comprising a mutated or identical sequence can still specifically bind to human CLDN18.2.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof is selected from an scFv, Fab, Fab', (Fab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody, and multispecific antibody.

[0033] In some embodiments, (DL) γThe structure of -A is represented by the formula (II): {D-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A is shown in formula (II), wherein L1 is

[0034] [ka] wherein R1 and R2 are each independently hydrogen (e.g., protium or deuterium), a halogen, a carboxylic acid group, a sulfonic acid group, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl, cyano-substituted C 1-6 Alkyl (e.g., -CHCN), C 1-6 Alkoxy, C 2-10 Alkenyl, or C 2-10 alkynyl; Z1 is an amino acid or a peptide consisting of 2 to 10 amino acids; x1 and x2 are each independently 0, 1, 2, 3, 4, 5, or 6; L1 is connected to D at position 1 and to is connected to L2, and L2 is [ka] wherein y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; L2 is connected to L1 at the 1-position and to L3 at the 2-position; L3 is selected from 5- to 12-membered aromatic heterocycles; L4 is

[0035] [ka] where Z2 is C 1-6 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylene, and C 3-8 cycloalkylene; R is selected from hydrogen (e.g., protium or deuterium) and C 1-6 alkyl; Z3 is absent or C 1-6alkylene; or R3 and Z3 together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclyl; α is 0, 1, 2, 3, 4, 5, or 6, and L4 is connected to E at the 2-position and to L3 at the 1-position; E is [ka] wherein each R4 is independently hydrogen (e.g., protium or deuterium), β is 0, 1, 2, 3, 4, 5, or 6, and E is connected to A at the 2-position and to L4 at the 1-position; m1, m2, and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A, D, and γ are as defined above.

[0036] In some embodiments, (DL) γ The structure of -A is represented by the formula (III): {D-[(L1') m4 -L1-(L5) m5 -(L3) m2 -(L4) m3 -E]} γ -A is as shown in formula (III), wherein L1' is [ka] wherein R5 and R6 are each independently hydrogen (e.g., protium or deuterium) or C 1-6 x3 is 1, 2, 3, 4, 5 or 6; L1', if present, is connected to D at the 1-position and to L1 at the 2-position; L1 is

[0037] [ka] wherein R1 and R2 are each independently hydrogen (e.g., protium or deuterium), a halogen, a carboxylic acid group, a sulfonic acid group, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl, cyano-substituted C 1-6 Alkyl (e.g., -CHCN), C1-6 Alkoxy, C 2-10 Alkenyl, or C 2-10 Z1 is an amino acid or a peptide consisting of 2 to 10 amino acids; x1 and x2 are each independently 0, 1, 2, 3, 4, 5, or 6; L1 is connected to L1' at position 1 (if L1' is present) or to D at position 1 (if L1' is absent); L1 is connected to L5 at position 2; L5 is [ka] where R7 is hydrogen or C 1-6 is alkyl, or R7 is connected to the atom N on its γ-C to form a 5-6 membered heterocyclyl; x4 is 1, 2, 3, 4, 5 or 6; y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and L5 is connected to L1 at the 1-position and to L3 at the 2-position; L3 is selected from 5- to 12-membered aromatic heterocycles; L4 is [ka] where Z2 is C 1-6 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylene, and C 3-8 cycloalkylene; R3 is selected from hydrogen (e.g., protium and deuterium) and C 1-6 alkyl; Z3 is absent or C 1-6 alkylene; or R3 and Z3 together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclic radical; α is 0, 1, 2, 3, 4, 5, or 6; L4 is connected to E at the 2-position and to L3 at the 1-position; E is [ka] wherein each R4 is independently hydrogen (e.g., protium or deuterium); β is 0, 1, 2, 3, 4, 5, or 6; E is connected to A at position 2 and to L4 at position 1; m1, m2, m3, and m4 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A, D, and γ are as defined above.

[0038] In some embodiments, L1 in the ADC represented by Formula (II) or Formula (III) is [ka] wherein Z1 is an amino acid or a peptide consisting of 2 to 5 amino acids, wherein the amino acids are selected from Lys, Cit, Val, D-Val, Phe, Leu, Gly, Ala, and Asn; preferably, Z1 is selected from Cit, Lys, Cit-Val, and Ala-Val.

[0039] In some embodiments, L1 is [ka] is.

[0040] In some embodiments, L1 is [ka] is.

[0041] In some embodiments, L2 in the ADC represented by formula (II) is [ka] and m1 is 1.

[0042] In some embodiments, in the ADC represented by formula (II) or formula (III), L3 is a 5- to 6-membered aromatic heterocycle and m2 is 1.

[0043] In some embodiments, L3 is triazole and m2 is 1.

[0044] In some embodiments, L4 in the ADC represented by Formula (II) or Formula (III) is [ka] where Z2 is C 1-6 alkylene, Z3 is C 1-6 alkylene, and m3 is 1.

[0045] In some embodiments, L4 is [ka] and m3 is 1.

[0046] In some embodiments, L1' in the ADC represented by formula (III) is [ka] is.

[0047] In some embodiments, L5 in the ADC represented by formula (III) is [ka] where x4 is 1, 2, 3, 4, 5, or 6; and y1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0048] In some embodiments, L5 in the ADC represented by formula (III) is [ka] is.

[0049] In some embodiments, D in the antibody-drug conjugate represented by Formula (I) is [ka] [ka] or [ka] is.

[0050] In some embodiments, D in the antibody-drug conjugate represented by formula (II) is [ka] is.

[0051] In some embodiments, D in the ADC represented by formula (III) is [ka] or [ka] is.

[0052] In some embodiments, the D-[L-(L) m1 -(L3) m2 -(L4) m3 -E]- is [ka] is.

[0053] In some embodiments, D-[(L1') m4 -L1-(L5) m5 -(L3) m2 -(L4) m3 -E]- is [ka] or [ka] is.

[0054] In some embodiments, the structure of the ADC represented by formula (II) is: [ka] wherein γ is selected from an integer of 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and A is the above-mentioned antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2.

[0055] In some embodiments, the ADC represented by formula (II) has the following formula: [ka] where γ is an integer from 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and A is 2C6.9-hz21.

[0056] In some embodiments, the structure of the ADC represented by formula (III) is: [ka] wherein γ is selected from an integer of 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and A is the above-mentioned antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2.

[0057] In some embodiments, the ADC represented by formula (III) has the following formula: [ka] where γ is an integer from 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and A is 2C6.9-hz21.

[0058] In some embodiments, the structure of the ADC represented by formula (III) is: [ka] wherein γ is an integer of 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and A is the above-mentioned antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2.

[0059] In some embodiments, the ADC represented by formula (III) has the following formula: [ka] where γ is an integer from 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and A is 2C6.9-hz21.

[0060] The present invention also provides compositions comprising one or more of the above-described antibody-drug conjugates (ADCs), wherein the molar ratio (DAR value) of the fragment of the biologically active molecule (i.e., D in Formula (I)) to the antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 (i.e., A in Formula (I)) is a decimal or integer from 1 to 10 (e.g., a decimal or integer from 1 to 8, e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 3.20, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29 ... 0.4, 3.5, 3.6, 3.7, 3.79, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.95, 7.0, 7.03, 7.1, 7.12, 7.2, 7.3, 7.40, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0).

[0061] The DAR value, also known as the drug-antibody ratio (referring to the average number of (DL) conjugated to each antibody), can be measured and calculated by methods known in the art. For example, the molecular weights of the conjugated antibody and (DL) measured by LC-MS, HIC, etc. are used to calculate the proportion of light and heavy chains conjugated with different numbers of (DL), and the DAR is calculated using the formula: DAR (light chain DAR1*1+...+light chain DAR n1 * n1 )*2+(heavy chain DAR1*1+...+DAR n2 * n2 The DAR value was calculated from the formula: n1 DARn2 means the proportion of light chains conjugated to n1 (DL)s among light chains, and DARn2 means the proportion of heavy chains conjugated to n2 (DL)s among heavy chains.

[0062] In some embodiments, the composition further comprises an antibody or antigen-binding fragment thereof described above.

[0063] In some embodiments, the composition comprises 2C6.9-TL001. In some embodiments, the composition is 2C6.9-TL001.

[0064] In some embodiments, the composition comprises 2C6.9-TL002. In some embodiments, the composition is 2C6.9-TL002.

[0065] In some embodiments, the composition comprises 2C6.9-TL003. In some embodiments, the composition is 2C6.9-TL003.

[0066] The antibodies or antigen-binding fragments thereof of the present invention can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibodies or antigen-binding fragments thereof does not adversely affect their binding to CLDN18.2 (particularly human CLDN18.2). Thus, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms.

[0067] Certain derivatized antibodies (e.g., bispecific antibodies) are produced by crosslinking two or more antibodies (of the same or different types). Methods for obtaining bispecific antibodies are well known in the art and include, but are not limited to, chemical crosslinking, cell engineering (hybrid hybridomas), or genetic engineering.

[0068] Another type of derivatized antibody is a labeled antibody. For example, the antibody or antigen-binding fragment thereof of the present invention is linked to a detectable label. The detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art, and examples include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa750), acridinium esters, magnetic beads (e.g., Dynabeads®), calorimetric labels, such as gold colloids or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and beads for binding to the label-modified avidin (e.g., streptavidin) described above. Examples of suitable markers include, but are not limited to, biotin. Patents that teach the use of such markers include, but are not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241 (each of which is incorporated herein by reference). Detectable labels such as those described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and thermal labels are detected by simply visualizing the dyed label.In certain embodiments, such markers may be adapted for use in immunological assays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.) In some embodiments, the above-described detectable labels may be linked to the antibodies or antigen-binding fragments thereof of the present invention by linkers of various lengths to reduce potential steric hindrance.

[0069] In certain embodiments, the antibody or antigen-binding fragment thereof included in the antibody-drug conjugates (ADCs) of the invention is one or more of the derivatized antibodies or antigen-binding fragments described above.

[0070] Therapeutic Methods and Pharmaceutical Compositions In another aspect, the present invention also provides pharmaceutical compositions.

[0071] In some embodiments, the pharmaceutical composition comprises one or more of the antibody-drug conjugates (ADCs) or compositions described above.

[0072] In some preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0073] In some preferred embodiments, the pharmaceutical composition further comprises another component having anti-tumor activity. In some embodiments, the ADC or composition and the other component having anti-tumor activity are contained in the same formulation or different formulations. Thus, the ADC or composition of the present invention and the other component having anti-tumor activity may be administered simultaneously, separately, or sequentially.

[0074] In some embodiments, the other component having anti-tumor activity is a biologically active polypeptide or an active fragment thereof, or a chemotherapeutic agent. In some embodiments, the biologically active polypeptide is selected from an immune checkpoint inhibitor (such as a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, or a LAG-3 antibody) or a cytokine (such as interferon, IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, or IL-21). In some embodiments, the chemotherapeutic agent is one or more selected from epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, and albumin-bound paclitaxel. In some embodiments, the other component having anti-tumor activity is a combination of epirubicin, oxaliplatin, and 5-fluorouracil, or a combination of oxaliplatin, leucovorin, and 5-fluorouracil.

[0075] In another embodiment, the ADC or composition in the pharmaceutical composition of the invention, when administered to a subject, is sufficient to: (a) induce apoptosis of tumor cells (particularly gastric cancer cells, e.g., gastric adenocarcinoma cells); (b) inhibit the proliferation of tumor cells (particularly gastric cancer cells, e.g., gastric adenocarcinoma cells); (c) induce and / or increase complement-dependent cytotoxicity (CDC); (d) induce and / or increase antibody-dependent cell-mediated cytotoxicity (ADCC); (e) inhibit the expression and activation of CLDN18.2; (f) inhibit CLDN18.2-mediated cell signaling pathways; or (g) any combination of (a)-(f).

[0076] In another aspect, the present invention provides the use of an antibody-drug conjugate, composition, or pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment and / or adjuvant treatment of a tumor.

[0077] In some embodiments, the tumor is selected from a solid tumor, a hematological malignancy, and a metastatic, refractory, or recurrent lesion of cancer.

[0078] In some embodiments, the tumor or cancer is selected from the group consisting of esophageal cancer, gastrointestinal cancer, gastric adenocarcinoma, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer, or NSCLC), liver cancer, stomach cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell tumor, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, and leukemia.

[0079] In some embodiments, the tumor is selected from gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, and lung cancer (e.g., NSCLC).

[0080] In some embodiments, the tumor is gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma, e.g., a locally advanced unresectable or metastatic tumor of gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma.

[0081] In some embodiments, the tumor is CLDN18.2 positive and further, the tumor is HER2 negative.

[0082] In some embodiments, the tumor is HER2-negative.

[0083] In another aspect, the present invention provides a method for preventing and / or treating a tumor in a subject. In another aspect, the present invention provides a method for delaying tumor progression in a subject. In another aspect, the present invention provides a method for reducing or inhibiting tumor recurrence in a subject. The method comprises administering an effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition of the present invention to a subject in need thereof.

[0084] In some embodiments, the methods also include administering a second therapy to the subject, wherein the second therapy is selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjunctive therapy, and any combination thereof. The compound is selected from the group consisting of:

[0085] In some embodiments, the second therapy can be applied separately or in combination with the above method; alternatively, the second therapy can be applied separately, in combination, simultaneously, or sequentially with the above method.

[0086] In some embodiments, the second therapy is chemotherapy. In some embodiments, the chemotherapy agent is selected from one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, and albumin-bound paclitaxel. In some embodiments, the chemotherapy agent is a combination of epirubicin, oxaliplatin, and 5-fluorouracil, or a combination of oxaliplatin, leucovorin, and 5-fluorouracil. In some embodiments, the combined administration regimen of oxaliplatin, leucovorin, and 5-fluorouracil is selected from FOLFOX4, FOLFOX6, or mFOLFOX6.

[0087] In some embodiments, the second therapy is an immunotherapy. In some embodiments, the drug for the immunotherapy is selected from an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, and an LAG-3 antibody) or a cytokine (e.g., an interference vegetarian, IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, and IL-21).

[0088] In some embodiments, the tumor is selected from a solid tumor, a hematological malignancy, and a metastatic, refractory, or recurrent lesion of cancer.

[0089] In some embodiments, the tumor or cancer is selected from the group consisting of esophageal cancer, gastrointestinal cancer, gastric adenocarcinoma, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., NSCLC), liver cancer, stomach cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell tumor, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, and leukemia.

[0090] In some embodiments, the tumor is selected from gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, and lung cancer (e.g., NSCLC).

[0091] In some embodiments, the tumor is gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma, e.g., a locally advanced unresectable or metastatic tumor of gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma.

[0092] In some embodiments, the tumor is CLDN18.2 positive and further, the tumor is HER2 negative.

[0093] In some embodiments, the tumor is HER2-negative.

[0094] The ADC, composition, or pharmaceutical composition of the present invention can be formulated into any pharmaceutical preparation known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention must be sterile and stable under the conditions of manufacture and storage. A preferred pharmaceutical preparation is an injection. The injection may be a sterile injectable solution. For example, a sterile injectable solution can be prepared using the following method: the required dose of the ADC or composition is dispersed in a suitable carrier, optionally with other desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. Furthermore, for ease of storage and use, a sterile lyophilized powder can be prepared using the sterile injectable solution (e.g., by vacuum drying or freeze-drying). The sterile, lyophilized powder can be dispersed in a suitable carrier, such as sterile, pyrogen-free water, before use.

[0095] Furthermore, the ADCs of the invention may be presented in pharmaceutical compositions in unit dosage form for ease of administration.

[0096] The ADCs, compositions, or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, intraocular, topical, parenteral, rectal, intrathecal, intracisternal, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic applications, the preferred route / mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the antibodies or antigen-binding fragments thereof, pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0097] Pharmaceutical compositions of the invention may contain a "therapeutically effective dose" or a "prophylactically effective dose" of an ADC or composition of the invention. A "prophylactically effective dose" refers to an amount sufficient to prevent, inhibit, or delay the onset of disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient already suffering from the disease. The therapeutically effective dose of an ADC or composition of the invention may vary depending on factors such as the severity of the disease being treated, the overall state of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, and the administration of drugs and other concurrently administered treatments.

[0098] In the present invention, the dosage regimen can be adjusted to provide the optimum intended response (e.g., a therapeutic or prophylactic response), e.g., it can be administered in a single dose, multiple doses over a period of time, or in proportionally reduced or increased doses depending on the exigencies of treatment.

[0099] A typical, non-limiting therapeutically or prophylactically effective dose range for an ADC or composition of the present invention is 0.02 to 100 mg / kg, e.g., 0.1 to 100 mg / kg, 0.1 to 50 mg / kg, or 1 to 50 mg / kg. It should be noted that dosages may vary depending on the type and severity of the condition being treated. Furthermore, one of ordinary skill in the art will understand that for any particular patient, a specific dosing regimen should be adjusted over time according to the patient's needs and a physician's professional assessment. Dosage ranges provided herein are for illustrative purposes only and are not intended to limit the use or scope of the pharmaceutical compositions of the present invention.

[0100] In the present invention, the subject may be a mammal, such as a human.

[0101] Abbreviation [Table 1]

[0102] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the experimental procedures used herein for cell culture, biochemistry, nucleic acid chemistry, and immunology are all common procedures widely used in the corresponding fields. At the same time, for a better understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0103] As used herein, the terms "ADC (antibody-drug conjugate)" or "conjugate" are interchangeable and refer to a substance obtained by conjugating a biologically active molecule to an antibody via a linker.

[0104] The linker can be connected to the antibody via various chemical bonds. For example, in some embodiments, the linker is connected by forming a thioether bond with the sulfhydryl group of the antibody. In the structural formula of some specific ADC molecules (e.g., ADC molecules 2C6.9-TL001, 2C6.9-TL002, or 2C6.9-TL003), -S- merely represents the thioether bond formed between the linker and the sulfhydryl group of the antibody, and does not mean that -S- is part of the linker.

[0105] The structural formula of ADC in this application is (DL) γ -A, where D is a biologically active molecule fragment; L is a linker; A is an antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2; and γ, which refers to the number of (DL)s attached to each antibody molecule, is selected from an integer of 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. However, during the process of ADC preparation, each antibody molecule may be attached with a different number of (DL). Therefore, the ADC product is generally a composition of antibodies attached with different numbers of (DL). In practice, DAR is often used to represent the average number of (DL)s attached to an antibody.

[0106] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε heavy chains, thus defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in binding between antibodies and antigens but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the respective antigen-binding sites.The assignment of amino acids in each region or domain may follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)) or Chothia and Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883, or the related work of AbM, Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272).

[0107] In this context, unless the context clearly indicates otherwise, reference to the term "antibody" includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0108] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are involved in antigen binding. The exact boundaries of these amino acid residues can be determined according to various numbering systems known in the art, e.g., the Kabat numbering system (Kab The definitions can be based on the definitions in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991), the Chothia numbering system (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or Martin's related work (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). Martin's definition method integrates parts of the definitions of Kabat and Chothia and is based on the Oxford Molecular It was first applied in antibody modeling software (Martin A. C. R. Protein sequence and structure analysis of antibody variable domains [M] / / Antibody engineering. Springer, Berlin, Heidelberg, 2010:33-51). For a given antibody, a person skilled in the art would easily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0109] The CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined according to the Kabat, Chothia, IMGT, or AbM numbering system.

[0110] As used herein, the term "framework region" or "FR" residues refers to amino acid residues other than the above-defined CDR residues in antibody variable regions.

[0111] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide that is an antibody fragment, e.g., a polypeptide that is a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen that the full-length antibody binds and / or that retains the ability to compete with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA techniques or enzymatic or chemical cleavage of intact antibodies can be used to produce antigen-binding fragments of antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0112] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains," where a "full-length heavy chain" or "heavy chain" refers to a polypeptide chain consisting, from N- to C-terminus, of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and if the full-length antibody is of the IgE isotype, it may also contain a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting, from N- to C-terminus, of VH, CH1, HR, CH2, and CH3. A "full-length light chain" or "light chain" is a polypeptide chain consisting, from N- to C-terminus, of a light chain variable region (VL) and a light chain constant region (CL). The two pairs of full-length antibody chains are connected by a disulfide bond between the CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention can be derived from a single species, such as human; it can also be a chimeric or humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites, each formed by a pair of VH and VL, which specifically recognize and bind to the same antigen.

[0113] As used herein, the term "Fd fragment" refers to an antibody fragment consisting of the VH domain and the CH1 domain; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; and the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in an F(ab')2 fragment consisting of an intact light chain and a heavy chain Fd fragment (consisting of the VH domain and the CH1 domain).

[0114] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single antibody arm. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. Six CDRs are generally considered to be capable of conferring antigen-binding specificity to an antibody. However, even a variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to an antigen, although the affinity may be lower than that of the complete binding site.

[0115] As used herein, the term "Fc fragment" refers to an antibody fragment formed by the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain of an antibody bound via disulfide bonds. The Fc fragment of an antibody has many different functions, but is not involved in antigen binding.

[0116] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL domain and a VH domain, wherein the VL and VH domains are connected via a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56; and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0117] As used herein, the term "diabody" refers to a diabody whose VH and VL domains are expressed on a single polypeptide chain, but which uses a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0118] Each of the above-described antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen.

[0119] As used herein, a "bispecific antibody" refers to a complex formed by a first antibody (fragment) and a second antibody (fragment) or antibody mimetic via coupling arms. Coupling modes include, but are not limited to, chemical reaction, gene fusion, protein fusion, polypeptide fusion, and enzymatic reaction. "Multispecific antibodies" include, for example, triabodies and tetrabodies, where the former are antibodies with three different antigen-binding specificities and the latter are antibodies with four different antigen-binding specificities.

[0120] As used herein, "antibody mimics" refer to substances that specifically bind to antigens like antibodies but do not have the antibody structure. They are typically artificial peptides or proteins with a molar mass of approximately 3 to 20 kDa, such as designed ankyrin repeat proteins (DARPins) and phenomers. Designed ankyrin repeat proteins (DARPins) are linked to IgG antibodies, scFv-Fc antibody fragments, or combinations thereof, as in Chinese Patent Application Publication No. 104341529. Anti-IL-17a phenomers bind to anti-IL-6R antibodies, as in International Publication No. 2015141862.

[0121] In this regard, antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods) and screened for specificity in the same manner as intact antibodies are screened.

[0122] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably to refer to a group of highly homologous antibody molecules, i.e., naturally occurring antibodies. "Monoclonal" refers to an antibody or antibody fragment derived from a population of antibody molecules that are identical except for natural mutations that may occur. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, in comparison to monoclonal antibodies, usually contain at least two or more different antibodies, which usually recognize different epitopes on the antigen. In addition, the modifier "monoclonal" merely indicates the character of the antibody as being obtained from a highly homogeneous antibody population and should not be construed as requiring that the antibody be produced by any particular method.

[0123] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, e.g., U.S. Patent Application No. 4,816,567), or phage antibody library technology (see, e.g., Clackson et al., Nature, 352:624-628, 1991, or Marks et al., J. Mol. Biol., 222:581-597, 1991).

[0124] For example, monoclonal antibodies can be prepared as follows: A mouse or other suitable host animal is first immunized with an immunogen (with an adjuvant, if necessary). The immunogen or adjuvant is usually injected subcutaneously or intraperitoneally. To enhance the immunogenicity of the antigen in the host, the immunogen can be pre-bound to a specific known protein, such as serum albumin or soybean trypsin inhibitor. The adjuvant can be Freund's adjuvant or MPL-TDM. After the animal is immunized, its body produces lymphocytes that secrete antibodies that specifically bind to the immunogen. Lymphocytes can also be obtained by in vitro immunization. The lymphocytes of interest are collected and fused with myeloma cells using an appropriate fusing agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The hybridoma cells prepared as described above can be seeded in an appropriate culture medium, preferably containing one or more substances capable of inhibiting the growth of unfused parent myeloma cells. For example, in the case of parent myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), adding hypoxanthine, aminopterin, and thymine (HAT medium) to the culture medium inhibits the growth of HGPRT-deficient cells. Preferred myeloma cells should have a high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium. Among these, mouse myeloma cells, such as MOP-21 or MC-11 mouse tumor-derived lines (The Salk Institute Cell Distribution Center, San Diego, CA, USA) and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, MD, USA), are preferred.Furthermore, studies have reported the use of human myeloma and human-mouse heteromyeloma cell lines to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). Culture medium for growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for measuring the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). For example, the affinity of a monoclonal antibody can be measured using the Scatchard assay described in Munson et al., Anal. Biochem., 107:220 (1980). After determining the specificity, affinity, and reactivity of the antibodies produced by the hybridoma, the desired cell line can be subcloned by the standard limiting dilution method described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. Suitable culture media can be DMEM or RPMI-1640, etc. Furthermore, hybridoma cells can be grown in animals in the form of ascites tumors. Monoclonal antibodies secreted by the subcloned cells can be isolated from cell culture medium, ascites, or serum using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0125] Monoclonal antibodies can also be obtained by recombinant genetic engineering techniques. DNA molecules encoding the heavy and light chain genes of a monoclonal antibody can be isolated from hybridoma cells by PCR amplification using nucleic acid primers that specifically bind to the heavy and light chain genes of the monoclonal antibody. The resulting DNA molecules are inserted into an expression vector, which is then transfected into host cells (e.g., E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins) and cultured under appropriate conditions to obtain the desired recombinant antibody.

[0126] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, the specific antigen (the target molecule recognized by the antibody) or its antigenic epitope can be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography. Purification of immunoglobulins can be found, for example, in D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0127] As used herein, the term "mouse antibody" refers to an antibody prepared by fusing B cells of an immunized mouse with myeloma cells, selecting mouse hybrid fusion cells capable of indefinite proliferation and antibody secretion, followed by screening, antibody preparation, and antibody purification, or an antibody secreted by plasma cells formed by B cell differentiation and proliferation after an antigen enters the mouse body. In the case of an antibody produced under the stimulation of a specific antigen, the production of the antibody is due to the interaction of various immune cells triggered by the antigen entering the human body, which leads to the differentiation and proliferation of B cells into plasma cells, which can produce and secrete the antibody.

[0128] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass) and another portion of its light and / or heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different antibody class or subclass), yet still retains binding activity for an antigen of interest (Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). For example, the term "chimeric antibody" can include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a mouse antibody), while the heavy and light chain variable regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0129] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology with that of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity, and the ability to enhance the immune response. The donor antibody can be an antibody derived from a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) that has the desired properties (e.g., antigen specificity, affinity, reactivity, the ability to increase immune cell activity and / or the ability to enhance the immune response).

[0130] Humanized antibodies are particularly advantageous because they can not only retain the expected properties of non-human donor antibodies (e.g., mouse antibodies), but also effectively reduce the immunogenicity of non-human donor antibodies (e.g., mouse antibodies) in human subjects. However, due to compatibility issues between the CDRs of the donor antibody and the FRs of the recipient antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to improve immune cell activity, and / or ability to enhance immune responses) are generally inferior to those of non-human donor antibodies (e.g., mouse antibodies).

[0131] Thus, although researchers in the field have conducted extensive research and made some progress with respect to antibody humanization (see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21:397-402 (2000)), the prior art does not provide detailed guidance on how to fully humanize a donor antibody so that the produced humanized antibody not only has the highest degree of humanization but also retains as much of the expected properties of the donor antibody as possible. Engineers must conduct much creative research to search, investigate, and modify a particular donor antibody to obtain a humanized antibody that not only has a high degree of humanization (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% humanization), but also retains the expected properties of the particular donor antibody.

[0132] In the present invention, the framework region (FR) of the humanized antibody of the present invention may contain both amino acid residues of a human acceptor antibody and amino acid residues of a corresponding non-human donor antibody so that the humanized antibody can retain the properties of the donor antibody as much as possible (including, for example, antigen specificity, affinity, reactivity, the ability to improve immune cell activity and / or the ability to enhance the immune response).

[0133] Humanized antibodies of the present invention can be prepared based on the sequences of the mouse monoclonal antibodies prepared above. DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0134] To prepare chimeric antibodies, the variable regions of murine immunoglobulins are synthesized using methods known in the art (e.g., U.S. Patent No. 4,629,493 to Cabilly et al.). ,816,567) to a human immunoglobulin constant region. For example, DNA encoding VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. For example, DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region, CL, to obtain a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0135] To prepare humanized antibodies, murine CDR regions can be grafted onto human framework sequences using any method known in the art (see U.S. Pat. No. 5,225,539 by Winter; U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 by Queen et al.; and Lo, Benny, KC, eds., Antibody Engineering: Methods and Protocols, Vol. 248, Humana Press, NJ, 2004). Alternatively, transgenic animals can be used that are capable of producing fully human antibody libraries after immunization without producing endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and that transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of such transgenic animals include the HuMAb mouse (Medarex, Inc.), which has a human immunoglobulin gene minilocus encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences and targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368(6474):856-859); or the "KM Mouse™," which carries a human heavy chain transgene and a human light chain transchromosome (see Patent Application WO 02 / 43478).Other methods for humanizing antibodies include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech, 14:309).

[0136] As used herein, the term "degree of humanization" refers to an index used to evaluate the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be evaluated by predicting the homology of the variable region sequence to a human V domain, for example, using DomainGapAlign on the IMGT website.

[0137] As used herein, the term "homologous antibody" refers to an antibody variant in which the amino acid sequences of the heavy and light chain variable regions contained therein are homologous to the amino acid sequences of an antibody or antigen-binding fragment thereof provided herein, and which variant retains the desired functional properties of the anti-CLDN18.2 antibody of the present invention.

[0138] Methods for aligning sequences for comparison are well known in the art. Various procedures and alignment algorithms are described in Smith TF and Waterman MS, Adv. Appl. Math., 2:482, 1981; Higgins DG and Sharp PM, CABIOS 5:151, 1989. Altschul SF et al., Nature Genet., 6:119, 1994 provides a detailed scheme for sequence alignment and homology calculation.

[0139] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which the antibody is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or the half-maximal effective concentration (EC50) of the interaction.

[0140] The specific binding properties between two molecules can be measured using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen-binding site / antigen complex. Both the "association rate constant" (ka or k) and the "dissociation rate constant" (k or k) can be calculated from the concentration and the actual association and dissociation rates (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k / k is the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990;59:439-473). D The k, k and k values ​​can be measured by any effective method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., ForteBio Octet method). The dissociation constant can also be measured by surface plasmon resonance (e.g., Biacore) or Kinexa.

[0141] As used herein, the term "identity" refers to the degree of correspondence between two polypeptides or two nucleic acids. If two sequences for comparison have the same base or amino acid monomer subunit at a particular site (e.g., two DNA molecules each have an adenine at a particular site, or two polypeptides each have a lysine at a particular site), the two molecules are identical at that site. The percent identity between two sequences is a function of the number of identical sites shared by the two sequences times 100 relative to the total number of sites for comparison. For example, if 6 out of 10 sites in two sequences are identical, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 sites are identical). Generally, the comparison of two sequences is performed to maximize identity. Such alignments can be performed using computer programs such as the Align program (DNAstar, Inc.), which is based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage identity between two amino acid sequences can be determined by the Needleman and Wunsch algorithm (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0142] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence, such that the resulting antibody variant retains the biological activity of the original sequence, such as specifically binding to CLDN18.2. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, valine, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Set USA 94:412-417 (1997) (incorporated herein by reference)).

[0143] The 20 conventional amino acids included herein are expressed in the usual manner. For example, see Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Sinauer Associates, Sunderland, Massachusetts (1991)), which is incorporated herein by reference. In this disclosure, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. In this disclosure, amino acids are also generally represented by one-letter and three-letter abbreviations as known in the art. For example, alanine can be represented by A or Ala.

[0144] As used herein, "pharmaceutically acceptable carriers and The term "and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerin). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or their degradation products (e.g., lactalbumin hydrolysate).

[0145] As used herein, the term "prophylaxis" refers to a method performed to prevent or delay the onset of a disease or disorder or condition (e.g., a tumor, an infectious disease, or an autoimmune disease) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., not further worsening), delay or prolongation of the onset of the disease, improvement or alleviation of the disease state, and alleviation of symptoms (partial or complete). Furthermore, the term "treatment" can also refer to prolonging survival compared to expected survival (if not receiving treatment).

[0146] As used herein, the term "subject" refers to a mammal, e.g., a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., a human) is suffering from or at risk of suffering from a tumor, an infectious disease, or an autoimmune disease.

[0147] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor, an infectious disease, or an autoimmune disease) refers to an amount sufficient to prevent, prevent, or delay the onset of the disease (e.g., a tumor, an infectious disease, or an autoimmune disease), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, the effective amount for therapeutic use depends on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition (e.g., age, weight, and sex), the mode of drug administration, and other therapies administered simultaneously.

[0148] As used herein, the term "immune cells" includes cells that have hematopoietic origin and play a role in the immune response, such as lymphocytes, e.g., B cells and T cells; natural killer cells; myeloid cells, e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0149] As used herein, the term "immune response" refers to the effects of immune cells (e.g., lymphocytes, antigen-presenting cells, phagocytes, or granulocytes) and soluble macromolecules (including antibodies, cytokines, and complement) produced by immune cells or the liver, which result in the selective damage or destruction of, or removal from the body of, invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation. In the present invention, an "antigen-specific T cell response" refers to an immune response generated by T cells when the T cells are stimulated with a T cell-specific antigen. Non-limiting examples of responses generated by T cells upon antigen-specific stimulation include T cell proliferation and the production of cytokines such as IL-2.

[0150] As used herein, the term "effector function" refers to a biological activity attributable to the Fc region of an antibody (native sequence or amino acid sequence variant Fc region), which varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance of antibodies and antigen-antibody complexes, etc. Methods for altering antibody effector functions are known in the art, for example, by introducing mutations into the Fc region.

[0151] The terms "cancer" and "tumor" are used interchangeably to refer to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes blood cancers, particularly hematological malignancies.

[0152] The term "hematological malignancy" or "hematological tumor" includes lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as splenic cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphoma and T-cell lymphoma. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Hematological malignancies also include leukemia, such as secondary leukemia or acute lymphocytic leukemia. Hematological malignancies also include myeloma (e.g., multiple myeloma), and other blood and / or B-cell or T-cell related cancers.

[0153] As used herein, the term "pharmaceutically acceptable" means that the molecule itself, molecular fragment, or composition does not cause adverse allergic or other untoward reactions when properly administered to an animal or human. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or components thereof include sugars such as lactose, starch, cellulose, and their derivatives, vegetable oils, gelatin, polyols such as propylene glycol, alginic acid, etc.

[0154] In this context, combination therapy includes the use of an ADC or composition or pharmaceutical composition encompassed by the invention in combination with one or more therapeutic agents of a second therapy (e.g., chemotherapeutic agents) or other prophylactic or therapeutic modalities (e.g., radiation therapy).

[0155] Exemplary therapeutic agents for second therapy include chemotherapeutic agents (e.g., antimitotic agents), alkylating agents (e.g., nitrogen mustards), antimetabolites (e.g., folic acid analogs), natural products (e.g., vinca alkaloids), miscellaneous drugs (e.g., platinum coordination complexes), hormones and antagonists (e.g., adrenal corticosteroids), immunomodulators (e.g., bropirimine, Upjohn), etc. Other anti-cancer treatments include other antibodies that specifically target cancer cells.

[0156] In this type of combination therapy, the various therapeutic agents often have different, complementary mechanisms of action, and the combination therapy may result in a synergistic effect. The combination therapy includes a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects tumor / cancer cells (e.g., inhibits or kills them). The combination therapy may reduce the likelihood of developing drug-resistant cancer cells. The combination therapy may allow for a reduced dosage of one or more of the drugs to reduce or eliminate adverse effects associated with one or more of the drugs. Such a combination therapy may have a synergistic therapeutic or preventative effect against the underlying disease, disorder, or symptom.

[0157] In this context, "combination" includes therapies that can be administered separately, e.g., formulated separately for separate administration (e.g., provided in a kit), and therapies that can be administered together in a single formulation (i.e., "co-formulation"). In some embodiments, the ADCs, compositions, or pharmaceutical compositions of the invention can be administered sequentially. In other embodiments, they can be administered simultaneously. The ADCs of the invention can be used in any combination with at least one other (active) agent.

[0158] HER2 negative refers to the absence of significant amounts of HER2 protein on the cell surface, including IHC1+ or IHC2+ / FISH negative, as well as a range of IHC0-1+ and IHC1+-2+.

[0159] As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0160] As used herein, "C 1-6 The term "alkyl" refers to a straight or branched chain alkyl (group) containing 1 to 6 carbon atoms, e.g., "C 1-4 Alkyl" and "C 1-3 Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, and 1,2-dimethylpropyl.

[0161] As used herein, "C 1-6 The term "alkylene" refers to a divalent group obtained by the loss of two hydrogen atoms from a straight- or branched-chain alkane containing from 1 to 6 carbon atoms, e.g., "C 1-4 alkylene" and "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, prop-1,3-ylene, but-1,4-ylene, pent-1,5-ylene, and hex-1,6-ylene.

[0162] As used herein, "C 2-10 The term "alkenyl" refers to a straight or branched chain alkenyl having at least one double bond and 2 to 10 carbon atoms, e.g., "C 2-6 alkenyl" and "C 2-4 Examples include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, Including, but not limited to, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, and 1,4-cyclohexadienyl.

[0163] As used herein, "C 2-10 The term "alkenylene" refers to a divalent group obtained by the loss of two hydrogen atoms from an olefin containing 2 to 10 carbon atoms, e.g., "C 2-8 alkenylene" and "C 4-6 Examples include, but are not limited to, pentene-1,5-ylene, 2-pentene-1,5-ylene, and hexene-1,6-ylene.

[0164] As used herein, "C 2-10 The term "alkynyl" refers to a straight or branched chain alkynyl having at least one triple bond and 2 to 10 carbon atoms, e.g., "C 2-6 alkynyl" and "C 2-4 Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, and 5-methyl-2-hexynyl.

[0165] As used herein, "C 2-10 The term "alkynylene" refers to a divalent group obtained by the loss of two hydrogen atoms from an alkyne containing 2 to 10 carbon atoms, e.g., "C 2-8 alkynylene" and "C 4-6 Examples include, but are not limited to, pentyn-1,5-ylene, 2-pentyn-1,5-ylene, and hexyn-1,6-ylene.

[0166] As used herein, "C 1-6 The term "alkoxy" refers to C1-6 It refers to a group having the structure alkyl-O-, where C 1-6 Alkyl is as defined above. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and hexoxy.

[0167] As used herein, the term "5-12 membered heteroaryl" refers to an aromatic cyclic group containing 5 to 12 ring members, wherein one of the ring members is at least a heteroatom selected from N, O, and S. Specific examples include, but are not limited to, 5- to 10-membered heteroaryls, 5- to 10-membered nitrogen-containing heteroaryls, and 5- to 6-membered oxygen-containing heteroaryls, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazole, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, and 1,2,4,5-tetrazinyl.

[0168] The present invention is not limited to the particular methodology, protocols, cell lines, vectors / carriers, or reagents described herein, which may vary. Furthermore, the terminology used herein is not intended to limit the scope of the invention, but is used only to describe particular embodiments.

[0169] In this specification (both in the specification and in the claims), the singular forms "a," "an," "the," and "said" include their plural forms unless the context clearly dictates otherwise. For example, "host cells" includes a plurality of host cells. In addition, Chinese does not have corresponding grammatical rules for singular and plural forms of English, and the singular and plural forms of nouns must be determined according to the context or actual situation. Therefore, in the English translation, the prefix "one or more" to the noun is considered correct. [Brief explanation of the drawings]

[0170] [Figure 1A] Figure 1 shows the detection of HEK293T-claudin18.2 monoclonal stable cell lines by flow cytometry. [Figure 1B] 1 shows the detection of L929-claudin18.2 monoclonal stable cell lines by flow cytometry. [Figure 1C] 1 shows the detection of KATOIII-claudin18.2 monoclonal stable cell lines by flow cytometry. [Figure 1D] 1 shows the detection of NCI-N87-claudin18.2 monoclonal stable cell lines by flow cytometry. [Figure 1E] Figure 1 shows the detection of HEK293T-claudin18.1 monoclonal stable cell lines by Western blot. [Figure 2] 1 shows the determination of affinity of 2C6.9-hz21 and IMAB362 by flow cytometry. [Figure 3] 1 shows the measurement of the affinity of binding of 2C6.9-hz21 and IMAB362 to L929-claudin18.2 cells. [Figure 4] 1 shows the measurement of the specificity of 2C6.9-hz21 by flow cytometry. [Figure 5] Figure 1 shows the measurement of the CDC killing activity of 2C6.9-hz21 and IMAB362 against HEK293T-claudin18.2 cells. [Figure 6]1 shows measurement of the ADCC activity of 2C6.9-hz21 and IMAB362 against HEK293T-claudin18.2 cells. [Figure 7] HPLC-SEC spectrum of 2C6.9-TL001 (DAR: 3.79) is shown. [Figure 8] HPLC-SEC spectrum of 2C6.9-TL001 (DAR: 7.12) is shown. [Figure 9] 1 shows the results of a test of the affinity of 2C6.9-TL001 (DAR: 7.12) for claudin 18.2 on the cell membrane surface. [Figure 10A] 1 shows the test results of the cytotoxic effect of 2C6.9-TL001 (DAR: 7.12) on HEK293T-claudin18.2. [Figure 10B] 1 shows the test results of the cytotoxic effect of 2C6.9-TL001 (DAR: 3.79) on HEK293T-claudin18.2. [Figure 10C] 1 shows the test results of the cytotoxic effect of 2C6.9-TL001 (DAR: 7.12) on HEK293T-claudin18.1. [Figure 10D] 1 shows the results of testing the cytotoxic effects of 2C6.9-TL002 and 2C6.9-TL003 on HEK293T-claudin18.2. [Figure 10E] 1 shows the results of testing the cytotoxic effects of 2C6.9-TL002 and 2C6.9-TL003 on HEK293T-claudin18.1. [Figure 11A] 1 shows the test results of the cytotoxic effect of 2C6.9-TL001 (DAR: 7.12) on NUGC-4 cells. [Figure 11B] 1 shows the test results of the cytotoxic effect of 2C6.9-TL001 (DAR: 3.79) on NUGC-4 cells. [Figure 11C] 1 shows the test results of the cytotoxic effects of 2C6.9-TL002 and 2C6.9-TL003 on NUGC-4 cells. [Figure 12A]The changes in tumor volume in each group of mice in a subcutaneously implanted NCI-N87-claudin18.2 cell tumor model in Balb / c nude mice are shown (*: P<0.05; ****: P<0.0001). [Figure 12B] 1 shows the changes in body weight of mice in each group in a subcutaneously implanted NCI-N87-claudin18.2 cell tumor model in Balb / c nude mice. [Figure 12C] Figure 1 shows a comparison of the in vivo efficacy of 2C6.9-TL001 and 2C6.9 mAb plus chemotherapy over 11 days in the CDX model (****: P<0.0001). [Figure 12D] Figure 1 shows a comparison of the in vivo efficacy of 2C6.9-TL001 and 2C6.9 monoclonal antibody plus chemotherapy over 21 days in the CDX model (****: P<0.0001). [Figure 12E] The changes in tumor volume within 21 days in mice treated with 2C6.9-TL001 with different DAR values ​​in the CDX (NUGC-4) model are shown (****: P<0.001). [Figure 12F] 1 shows the weight changes within 21 days in mice of each group treated with 2C6.9-TL001 with different DAR values ​​in the CDX (NUGC-4) model. [Figure 12G] Figure 1 shows the change in tumor volume within 17 days in each group of mice in the subcutaneous HuPrime® gastric cancer GA0006 PDX model in Balb / c nude mice (****: P<0.0001). [Figure 12H] Figure 1 shows the weight change within 17 days for mice in each group in the subcutaneous HuPrime® gastric cancer GA0006 PDX model in Balb / c nude mice. [Figure 12I] Figure 1 shows the change in tumor volume within 24 days in each group of mice in the subcutaneous HuPrime® gastric cancer GA0006 PDX model in Balb / c nude mice (****: P<0.0001). [Figure 12J] Figure 1 shows the weight change within 24 days for mice in each group in the subcutaneous HuPrime® gastric cancer GA0006 PDX model in Balb / c nude mice. [Figure 12K] The change in tumor volume in each group of mice in a Balb / c nude mouse subcutaneously implanted with NCI-N87-claudin18.2 cells is shown (***: P<0.001; ****: P<0.0001). [Figure 12L] This shows the changes in body weight of mice in each group in a Balb / c nude mouse subcutaneously transplanted NCI-N87-claudin18.2 cell tumor model. [Figure 12M] The figure shows the change in tumor volume in each group of mice in a Balb / c nude mouse subcutaneously implanted with HEK293T-claudin18.2 cells (**: P<0.01; ****: P<0.0001). [Figure 12N] This shows the changes in body weight of mice in each group in a Balb / c nude mouse subcutaneously transplanted HEK293T-claudin18.2 cell tumor model. [Figure 12O] The graph shows the change in tumor volume in each group of mice in a Balb / c nude mouse subcutaneously implanted with NUGC-4 cells (****: P<0.0001). [Figure 12P] This shows the changes in body weight of mice in each group in a Balb / c nude mouse subcutaneous NUGC-4 cell transplantation tumor model. [Figure 13] HPLC-SEC spectrum of 2C6.9-TL001 (DAR: 7.40) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0171] Hereinafter, the embodiments of the present invention will be described in detail with reference to examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Specific conditions not shown in the examples are conventional conditions or conditions suggested by manufacturers. Reagents or equipment used without indicating the manufacturer are all commercially available conventional products. [Example]

[0172] [Example 1] Synthesis of biologically active molecule (TOXIN-1) [ka] To a solution of belotecan hydrochloride (3 g, 6.38 mmol) and triethylamine (2.58 g, 25.54 mmol) in dichloromethane (40 mL), methanesulfonyl chloride (462 mg, 12.77 mmol, approximately 70% purity) was added dropwise, and the resulting mixture was allowed to react at room temperature for 2 hours. After suction filtration, the filter cake was washed three times with dichloromethane (3 mL) to obtain 2.2 g of (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (TOXIN-1).

[0173] The structural characterization data are as follows: 1 H NMR(400MHz,DMSO-d6)δ 8.32(d,J=8.4Hz,1H),8.20(dd,J=8.4,1.2Hz,1H),7.93-7.84(m,1H),7.79(t,J= 7.6Hz,1H),7.35(s,1H),6.56(s,1H),5.44(d,J=9.2Hz,4H),3.98(p,J=6.7Hz,1H) ,3.50(t,J=8.0Hz,2H),3.42-3.35(m,2H),3.00(s,3H),1.93-1.82(m,2H),1.15(d,J=6.7Hz,6H),0.88(t,J=7.3Hz,3H). ESI-MS(m / z):512.2[M+H] + . [α] D 20 is +28.19° (c=0.101g / 100mL, CH3CN).

[0174] [Example 2] Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-4) [ka]

[0175] Step 1: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid methyl ester (Compound 3-2) Methyl 5-hexynoate (500 mg, 3.97 mmol) and 5-bromo-2-methylthiopyrimidine were dissolved in N,N-dimethylformamide (3 mL) at room temperature, and triethylamine (3 mL), cuprous iodide (75 mg, 0.4 mmol), and palladium(II) bis(triphenylphosphine) dichloride (279 mg, 0.4 mmol) were added successively. The mixture was then heated to 95 °C under nitrogen protection and reacted with stirring for 6 hours. The reaction was quenched with water. The reaction mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered to remove the drying agent, concentrated in vacuo, and purified by preparative liquid chromatography to give 300 mg of the title compound. ESI-MS (m / z): 251.3 [M+H] + .

[0176] Step 2: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-3) Compound 3-2 (200 mg, 0.8 mmol) was dissolved in a mixture of tetrahydrofuran and water (4 mL / 4 mL) at room temperature, and lithium hydroxide monohydrate (235 mg, 5.6 mmol) was added. The resulting mixture was stirred at room temperature for 4 hours, then diluted with water and extracted with ethyl acetate (20 mL x 2). The aqueous phase was adjusted to pH 3 with 1N hydrochloric acid and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated in vacuo to give 120 mg of the title compound.

[0177] Step 3: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (compound 3-4) Compound 3-3 (20 mg, 0.085 mmol) was dissolved in dichloromethane (4 mL) at room temperature, and m-chloroperbenzoic acid (22 mg, 0.127 mmol) was added. The resulting mixture was reacted overnight with stirring at room temperature and purified by preparative liquid chromatography to give 20 mg of the title compound. ESI-MS (m / z): 269.1 [M+H] + .

[0178] Example 3 (4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl)((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound TL001) [ka]

[0179] Step 1: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-propargyl-hex-5-ynamide (Compound 3-5) Propynylamine (189 mg, 3.4 mmol) and Compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL) at 25 °C, and then N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-aza-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.63 g, 4.25 mmol) were added successively. The resulting mixture was reacted with stirring for 2 hours. The reaction solution was concentrated in vacuo, and the residue was purified with a flash silica gel column (ethyl acetate / petroleum ether = 3 / 1) to give 700 mg of the title compound. ESI-MS (m / z): 306.1 [M+H] + .

[0180] Step 2: Synthesis of (4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl)((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound 33-1) TOXIN-1 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL) at 25 °C under nitrogen protection, cooled to 0 °C, and a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added, followed by the slow dropwise addition of a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL). After the addition, the resulting mixture was reacted with stirring at 0 °C for 20 min and purged with nitrogen for 20 min. Next, a solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added, and the mixture was stirred at 0° C. for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative liquid chromatography to obtain 500 mg of the title compound. ESI-MS (m / z): 1597.5 [M+H] + .

[0181] Step 3: Synthesis of ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontanamido)benzyl)carbonate (Compound 33-2) Compound 33-1 (80 mg, 0.05 mmol) and fragment 3-5 (23 mg, 0.075 mmol) were dissolved in a 2.0 mL:0.5 mL solution of dimethyl sulfoxide and water at room temperature, and cuprous bromide (11 mg, 0.08 mmol) was added. The resulting mixture was stirred for 1 hour and purified by preparative high-performance liquid chromatography to give 30 mg of the title compound. ESI-MS (m / z): 815.9 [(M-273) / 2+H]. + .

[0182] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl)((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound TL001) Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL), followed by the addition of trifluoroacetic acid (0.2 mL). The resulting mixture was reacted at room temperature for 30 minutes and purified by preparative HPLC to obtain 20.0 mg of the trifluoroacetic acid salt of the title compound. The structure is characterized as follows:

[0183] 1 H NMR(400MHz,DMSO-d6)δ 10.18(s,1H),9.10(s,2H),8.38(t,J=5.56Hz,1H),8.32(d,J=8.40Hz,1H),8.22-8.20(m,2H),8.09(t,J=5.68Hz,1H),7.91-7.87(m,2H),7.82-7.78(m,1H),7.69(brs,3H),7.61(d,J=8.56Hz,2H),7.32(d,J=8.56Hz,2H),7.06(s,1H),5.56(d,J=16.96Hz,1H),5.51(d,J=16.96Hz,1H),5.47(d,J=19.28Hz,1H),5.42(d,J=19.28Hz,1H),5.14(d,J=12.20Hz,1H),5.07(d,J=12.16Hz,1H),4.48(t,J=5.24Hz,2H),4.46-4.43(m,1H),4.29(d,J=5.60Hz,2H),4.08-3.95(m,5H),3.79(t,J=5.28Hz,2H),3.51-3.43(m,32H),3.40(s,3H),3.39-3.35(m,2H),3.30-3.26(m,2H),3.00(s,3H),2.82-2.74(m,2H),2.56(t,J=7.08Hz,2H),2.29(t,J=7.36Hz,2H),2.23-2.13(m,2H),1.82(p,J=7.24Hz,2H),1.78-1.63(m,2H),1.61-1.49(m,2H),1.42-1.27(m,2H),1.15(d,J=6.80Hz,3H),1.13(d,J=6.76Hz,3H),0.90(t,J=7.32Hz,3H). ESI-MS(m / z):816.0[M / 2+H] + . [α] D 20 は、-19.55°(c=1.000g / 100mL、CH3CN)である。

[0184] Example 4 Synthesis of ((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolino[1,2-b]quinolin-9-yl)(4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)butyramido)propionamido)benzyl)ethane-1,2-diylbis(methylcarbamate)trifluoroacetate (TL002) [ka]

[0185] Step 1: Synthesis of (S)-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indozino[1,2-b]quinolin-9-yl(4-nitrophenyl)carbonate) (S)-4,11-diethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indozino[1,2-b]quinoline-3,14(4H,12H)-dione (150 mg, 0.37 mmol) was dissolved in dichloromethane (15 mL) and diisopropylethylamine (96.81 mg, 0.74 mmol) was added, followed by bis(4-nitrophenyl)carbonate (1 A solution of 27.92 mg (0.41 mmol) of methylpropanol in dichloromethane (15 mL) was added, and the resulting mixture was reacted at 25° C. for 3 hours. The reaction solution was concentrated to give 207 mg of the title compound, which was used directly in the next reaction. ESI-MS (m / z): 558.1 [M+H] + .

[0186] Step 2: Synthesis of (S)-tert-butyl (4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolino[1,2-b]quinolin-9-yl)ethane-1,2-diylbis(methylcarbamate) (S)-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)(4-nitrophenyl)carbonate (207 mg, 0.33 mmol) was dissolved in dichloromethane (10 mL), diisopropylethylamine (130.87 mg, 1.00 mmol) and tert-butyl N-methyl-N-[2-(methylamino)ethyl]carbamate (71.34 mg, 0.37 mmol) were added, and the resulting mixture was stirred at 25 °C for 12 hours. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 9 / 1) to give 207 mg of the title compound. ESI-MS (m / z): 607.3 [M+H] + .

[0187] Step 3: Synthesis of (S)-N-methyl-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indozino[1,2-b]quinolin-9-yl)(2-(methylamino)ethyl)carbamate trifluoroacetate (S)-tert-butyl(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indozino[1,2-b]quinolin-9-yl)ethane-1,2-diylbis(methylcarbamate) (207 mg, 0.19 mmol) was dissolved in dichloromethane (8 mL), trifluoroacetic acid (2 mL) was added, and the resulting mixture was reacted at 25°C for 2 hours. The reaction solution was concentrated to give 200 mg of the title compound, which was used directly in the next reaction. ESI-MS (m / z): 507.2 [M+H] + .

[0188] Step 4: Synthesis of 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolino[1,2-b]quinolin-9-yl)ethane-1,2-diylbis(methylcarbamate) (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)(4-nitrophenyl)carbonate (150 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (3 mL) and added with 1-hydroxybenzotriazole (34.03 mg, 0.25 mmol) and diisopropyl ether. Isopropylethylamine (48.82 mg, 0.37 mmol) was added, followed by (S)-N-methyl-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)(2-(methylamino)ethyl)carbamate trifluoroacetate (102.11 mg, 0.12 mmol), and the resulting mixture was reacted at 25°C for 16 hours. The reaction solution was purified by preparative HPLC, and the collected fractions were lyophilized to give 44 mg of the title compound. ESI-MS (m / z): 1400.7 [M+H] + .

[0189] Chromatography column: Waters XBridge Prep C18 OBD 19 mm x 150 mm x 5.0 μm. Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid).

[0190] [Table 2]

[0191] Step 5: Synthesis of ((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolino[1,2-b]quinolin-9-yl)(4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)butyramido)propionamido)benzyl)ethane-1,2-diylbis(methylcarbamate) trifluoroacetate 4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]chino (Phosphorin-9-yl)ethane-1,2-diylbis(methylcarbamate) (44 mg, 0.30 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-propyl)hexamide (14.09 mg, 0.045 mmol) were dissolved in a solution of dimethyl sulfoxide (3 mL) and water (0.75 mL), and cuprous bromide (8.65 mg, 0.06 mmol) was added. The resulting mixture was reacted at 25°C for 1 hour. The reaction solution was purified by preparative HPLC, and the collected fractions were lyophilized. The resulting solution was then dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.2 mL) was added, stirred at 25°C for 0.5 hours, purified by preparative HPLC, and the collected fractions were lyophilized to give 16 mg of the title compound. ESI-MS (m / z): 853.6 [M / 2+H] + .

[0192] Chromatography column: Waters SunFire Prep C18 ODS 5 μm 19 × 50 mm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0193] [Table 3]

[0194] Example 5 Synthesis of (4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)butyramido)propionamido)benzyl)(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)N-ethyl-N-isopropylcarbamate trifluoroacetate (TL003) [ka]

[0195] Step 1: Synthesis of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)-3-methylbutyramide At room temperature, ((9H-fluoren-9-yl)methyl)(((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (2.00 g, 3.88 mmol) was dissolved in N,N-dimethylformamide (12 mL), piperidine (3 mL) was added, and the resulting mixture was reacted at 25 ° C. for 2 hours. Then, the reaction solution was poured into water to precipitate a solid, which was filtered and purified by preparative HPLC. The collected fractions were lyophilized to obtain 810 mg of the title compound. ESI-MS(m / z): 294.0 [M + H] + .

[0196] Chromatography column: Waters SunFire Prep C18 ODS 8 μm 45 × 450 mm Mobile phase A: acetonitrile; Mobile phase B: water

[0197] [Table 4]

[0198] Step 2: Synthesis of tert-butyl 4-(4-((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)amino-3-methyl-1-oxobut-2-yl)amino-4-oxobutyl)piperidine-1-carboxylate At room temperature, (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)-3-methylbutyramide (810 mg, 2.76 mmol), 4-(1-(tert-butoxycarbonyl)piperidin-4-yl)butyric acid, and 2-ethoxy-1-ethoxycarbo-1,2-dihydroquinoline were dissolved in dichloromethane (8 mL) and methanol (8 mL), and the mixture was reacted at 45° C. for 2 hours. The reaction solution was concentrated in vacuo, and the residue was purified by silica gel chromatography (eluent: dichloromethane / methanol=15 / 1) to give 1.31 g of the title compound. ESI-MS (m / z): 546.8 [M+H] + .

[0199] Step 3: Synthesis of (S)—N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)-3-methyl-2-(4-(piperidin-4-yl)butyrylamido)butyramide trifluoroacetate At room temperature, tert-butyl 4-(4-((S)-1-((S)-1-((4-(hydroxymethyl)phenylamino)-1-oxoprop-2-yl)amino-3-methyl-1-oxobut-2-yl)amino-4-oxobutyl)piperidine-1-formate (1.3 g, 2.14 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (5 mL) was added, and the mixture was reacted at 25° C. for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in acetonitrile (30 mL). Potassium carbonate (1.22 g, 8.85 mmol) was added, and the mixture was reacted at 25° C. for 2 hours. The reaction mixture was suction filtered, the filter cake was washed with acetonitrile, and the filtrate was collected and concentrated under reduced pressure to give 900 mg of the title compound. ESI-MS (m / z): 446.9 [M+H] + .

[0200] Step 4: Synthesis of (S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramide)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)-3-methylbutyramide (S)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)-3-methyl-2-(4-(piperidin-4-yl)butyrylamido)butyramide trifluoroacetate (487 mg, 0.78 mmol) and 26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl p-toluenesulfonate (619 mg, 0.94 mmol) were dissolved in acetonitrile (20 mL) at room temperature, and potassium carbonate (655 mg, 4.69 mmol) was added. The reaction mixture was reacted at 16°C for 6 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 8 / 1) to obtain the title compound. 586 mg of the product was obtained. ESI-MS (m / z): 868.5 [M+H] + .

[0201] Step 5: Synthesis of (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)(4-nitrophenyl)carbonate At room temperature, (S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramide)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)-3-methylbutyramide (585 mg, 0.64 mmol) was dissolved in dichloromethane (30 mL), and N,N-diisopropylethylamine (334.31 mg, 2.56 mmol) was added, followed by dropwise addition of a solution of di(p-nitrobenzene)carbonate (602.35 mg, 1.92 mmol) in dichloromethane (30 mL). The resulting mixture was reacted at 25 ° C. for 6 hours. The reaction solution was concentrated under reduced pressure, methyl tert-butyl ether was added to the residue, and the mixture was filtered to obtain 760 mg of the title compound. ESI-MS(m / z): 1033.4 [M + H] + .

[0202] Step 6: Synthesis of (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)(N-(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolino[1,2-b]quinolin-11-yl)ethyl)-N-isopropyl)carbamate (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)(4-nitrophenyl)carbonate (150 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (3 mL) at room temperature, and 1-hydroxybenzotriazole (34.03 mg, 0.25 mmol) and N,N- Diisopropylethylamine (48.82 mg, 0.37 mmol) was added, followed by (S)-4-ethyl-4-hydroxy-11-(2-(isopropylamino)ethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione hydrochloride (59.79 mg, 0.12 mmol). The reaction solution was stirred overnight at room temperature and then purified by preparative HPLC. The collected fractions were lyophilized to give 64 mg of the title compound. ESI-MS (m / z): 1327.6 [M+H] + .

[0203] Step 7: Synthesis of (4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)butyramido)propionamido)benzyl)(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolino[1,2-b]quinolin-11-yl)N-ethyl-N-isopropylcarbamate trifluoroacetate At room temperature, (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)(N-(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropyl)carbamate 64 mg, 0.046 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-ynyl)hexamide (21.63 mg, 0.069 mmol) were dissolved in dimethyl sulfoxide (4 mL) and water (1 mL), and cuprous bromide (13.27 mg, 0.092 mmol) was added. The resulting mixture was reacted at 25°C for 1 hour. The reaction solution was purified by preparative HPLC, and the collected fractions were lyophilized to give 35 mg of the title compound. ESI-MS (m / z): 1632.8 [M+H] + .

[0204] Chromatography column: Waters SunFire Prep C18 OBD 5 μm 19 × 150 mm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0205] [Table 5]

[0206] [Example 6] Preparation of monoclonal antibodies targeting human claudin 18.2 The monoclonal antibodies targeting human claudin 18.2 in the present invention are humanized monoclonal antibodies, including 2C6.9-hz11 and 2C6.9-hz21, whose CDRs, variable region sequences, and constant region sequences are shown in Table 1. [Table 6]

[0207] 6.1 Construction and Identification of Human Claudin 18.2 and Human Claudin 18.1 Overexpressing Cell Lines 6.1.1 Construction of Human Claudin 18.2 and Human Claudin 18.1 Overexpressing Cell Lines To measure the specificity and function of the anti-human claudin 18.2 antibody, the complete coding sequences of human claudin 18.2 (gene accession number: NM_001002026.2, synthesized by Nanjing Genscript Biotech Corporation) and human claudin 18.1 (gene accession number: NM_016369.3, synthesized by Nanjing Genscript Biotech Corporation) were cloned into the lentiviral vector pLVX-IRES-puro, and viruses were prepared using a lentiviral packaging system according to published methods (Mohammadi Z et al., Mol Biotechnol. 2015 Sep;57(9):793-800). The resulting viruses were used to infect HEK293T, L929, KATOIII, and NCI-N87 cells. Monoclonal stable cell lines of HEK293T-claudin18.1, HEK293T-claudin18.2, L929-claudin18.2, KATOIII-claudin18.2, and NCI-N87-claudin18.2 were obtained by puromycin screening and single clone selection. BaF / 3 cells (DSMZ, catalog no. ACC300) were transfected with plasmids encoding human claudin18.2 or human claudin18.1 using the 4D-Nucleofector X transfection kit (Lonza, catalog no. V4XC-3012). 48 hours after transfection, cells were screened by the addition of 1.25 mg / mL hygromycin (Thermo Fisher Scientific catalog no. 10687010). After 12 days of screening, a single clone was selected, thereby obtaining the monoclonal cell lines BaF / 3-Claud18.1 and BaF / 3-Claud18.2.

[0208] 6.1.2 Detection of Human Claudin 18.2 and Human Claudin 18.1 Overexpressing Cell Lines. Western blot was used to detect HEK293T-claudin 18.1 (detection antibody: Proteintech, 66167-1-Ig), and FACS was used to detect other cell lines (flow cytometer: Beckman, CytoFlex; detection antibody: IMAB362, the sequence of which is taken from China Patent Application Publication No. 101312989). As shown in Figure 1A-1D, the FACS results demonstrated that HEK293T-claudin 18.2, L929-claudin 18.2, KATOIII-claudin 18.2, and NCI-N87-claudin 18.2 monoclonal cell lines were obtained with high positive rates (close to 100%) and good homogeneity, and were used in the following experiments. Western blot results (Figure 1E) demonstrated that all three HEK293T-claudin18.1 stable cell lines overexpressed human claudin18.1, where a single clone, HEK293T-claudin18.1-1C2, showed a higher expression level than the others and was used in the following experiments.

[0209] 6.2 Preparation of Mouse Anti-Human Claudin 18.2 Monoclonal Antibody. DNA / cell immunization was performed in wild-type mice to generate mouse anti-human claudin 18.2 monoclonal antibodies. Each Balb / c mouse was injected via the tail vein with 100 μg of a plasmid containing the complete coding sequence of human claudin 18.2. After the fourth and sixth immunizations, serum titers were measured by FACS. Mice with high serum titers were boosted with the BaF / 3-claudin 18.2-overexpressing cell line 3–5 days before fusion. PEG-mediated fusion of mouse splenocytes and the mouse myeloma cell line Sp2 / 0 (ATCC, catalog no. CRL-1581) was performed using a standard fusion protocol, followed by HAT pressure selection. FACS screening was performed 10–14 days after fusion.

[0210] Approximately 6,000 hybridoma supernatants were screened using flow cytometry (available from Sartorius as the Model iQue Screener Plus). 43 positive hybridomas that bound to the HEK293T-claudin18.2 cell line were obtained and subcloned. Fourteen hybridomas that specifically bound to human claudin18.2 but not to human claudin18.1 were selected by FACS using the HEK293T-claudin18.2 and HEK293T-claudin18.1 cell lines. Single clones were obtained by limiting dilution and subclone selection.

[0211] The human gastric cancer cell line NUGC4 (purchased from the JCRB Cell Bank, Japan, catalog number: JCRB0834) endogenously expresses claudin 18.2 and is widely used to evaluate binding between antibodies and endogenous claudin 18.2 and to develop functional assays. We evaluated candidate clones using NUGC4 cells and ultimately selected seven subclones. After further affinity detection, we selected 2C6.9M for variable region amplification and humanization.

[0212] To detect the antibody subtype of the candidate hybridoma clone, 2C6.9M was identified using the Pierce Rapid Isotyping Kit (Thermo Fisher SCI, Catalog No. 26179). The identification results indicated that the heavy chain was an IgG1 subtype and the light chain was a kappa subtype.

[0213] Hybridoma cells were grown, and approximately 8,000 cells were harvested and lysed. First-strand cDNA was synthesized using a cDNA reverse transcription kit (Thermo Fisher Sci. Cat. No. 18080-200). VH and VK (VL kappa) genes were amplified from cDNA by PCR using primers. The PCR products were purified with a DNA purification kit (Qiagen, Cat. No. 28104) and ligated into the TOPO vector (Thermo Fisher Sci. Cat. No. K457540). Approximately 12 clones were harvested from each ligation reaction and sequenced. The sequences were then analyzed using the Vector The sequences were analyzed using Sequencer NTI11.5 (Thermo Fisher Sci.) and Sequencer 5.4.6 (Genecodes). The variable region sequences and CDR sequences of the resulting mouse antibody 2C6.9M are shown in Table 2.

[0214] [Table 7]

[0215] 6.3 Humanization of 2C6.9M Mouse Antibody The mouse antibody 2C6.9M was humanized using a CDR-grafting method. Briefly, the humanization process included the following steps: the amino acid sequence of the mouse monoclonal antibody was aligned with that of a human germline antibody to identify a human germline framework with high homology and good physicochemical properties; the affinity to HLA-DR was measured, and then a human germline framework with low affinity to HLA-DR was selected; the six CDR regions of the mouse antibody were then grafted onto the selected heavy and light chain frameworks.

[0216] Specifically, the heavy and light chain CDRs of the murine antibody 2C6.9M were grafted onto the frameworks (FRs) of the corresponding humanization templates, which are the human germline sequences IGHV4-59*01 (IMGT Reference No. AB019438) and IGKV4-1*01 (IMGT Reference No. Z00023), respectively.

[0217] Furthermore, computer simulations were performed using molecular docking to analyze the amino acid sequences of the variable region and the surrounding framework to determine the spatial and steric binding configuration of the antibody. By calculating electrostatic forces, van der Waals forces, hydrophobic interactions, and entropy, we identified key amino acid residues in the mouse antibody sequence that could interact with claudin-18.2 or maintain the spatial structure. These mouse amino acids were then maintained after transplantation. In other words, a series of back mutations were incorporated into the FR region residues of the humanization template to maximize the affinity of the mouse antibody in the humanized antibody.

[0218] The variable region sequences of mouse antibody 2C6.9M are shown in SEQ ID NOs: 23 and 24, and its CDR sequences are shown in SEQ ID NOs: 1 to 12. To avoid isomerization without affecting affinity, the amino acid sequence of 2C6.9 CDR-H2 was modified in the present invention. The modified sequences are shown in SEQ ID NOs: 21 and 22. Finally, two humanized antibodies were constructed and designated 2C6.9-hz11 and 2C6.9-hz21, respectively. The heavy chain constant region of each antibody is the human wild-type IgG1 heavy chain constant region (SEQ ID NO: 16), and the light chain constant region of each antibody is the human wild-type IgG1κ light chain constant region (SEQ ID NO: 17).

[0219] The variable region, constant region, and heavy chain / light chain amino acid sequences of each 2C6.9 antibody are shown in Table 1.

[0220] Codon-optimized cDNAs for the heavy and light chain amino acid sequences of the 2C6.9 humanized antibody were synthesized and ligated into the pcDNA3.4 plasmid (contracted by Nanjing Genscript Biotech Corporation). The pcDNA3.4 fragments corresponding to the heavy and light chains were co-transfected into Expi293F cells (purchased from Thermo Biosciences). The cell supernatant was purified using a Protein A affinity column (MabSelect SuRe, GE) to obtain the 2C6.9 humanized monoclonal antibody.

[0221] 6.4 Affinity Detection of Humanized Monoclonal Antibody 2C6.9 The affinity of 2C6.9-hz21 for human claudin 18.2 on the cell membrane was detected using HEK293T-claudin 18.2 cells. The specific procedure was as follows: HEK293T-claudin 18.2 cells were detached, centrifuged, and washed twice with PBS. The cells were then resuspended in PBS containing 1% BSA and seeded at 300,000 cells per well in a 50 μl volume for a total of 20 wells in a 96-well tip-bottom plate. Then, 50 μl of 2C6.9-hz21 and IMAB362 antibodies were added to each well at 11 concentrations, each diluted 3-fold starting from 1000 nM. Human IgG was used as a negative control. The reaction mixture was mixed well and incubated for 1 h at 4°C in the dark, followed by washing three times with PBS. FITC-labeled anti-human Fc secondary antibody (Biolegend, 409322) was added and incubated for 0.5 h at 4°C in the dark. After washing three times with PBS, detection was performed by flow cytometry (Beckman, Cytoflex).

[0222] As shown in Figure 2 and Table 3, the EC50 value of the binding affinity of 2C6.9-hz21 for HEK293T-claudin18.2 was lower than that of IMAB362, and the maximum fluorescence value of 2C6.9-hz21 was higher than that of IMAB362, indicating that the affinity of 2C6.9-hz21 for claudin18.2 on the cell membrane was stronger than that of IMAB362.

[0223] [Table 8]

[0224] 6.5 Measurement of affinity and specificity of humanized antibody 2C6.9. Human claudin 18.2 belongs to the tetraspanin family and has a complex structure. Therefore, a cell-based ELISA was performed to maintain the structure of claudin 18.2. The stable cell line L929-claudin 18.2 constructed in 6.1 was used for detection. Specifically, L929-claudin 18.2-adherent cells were detached by treatment with 2 mM EDTA. The cells were resuspended and collected at 2 × 10 5 The antibody was adjusted to 1 mL / mL, and 100 μL of the resuspension was seeded into a 96-well plate and incubated overnight at 37°C. The next day, the medium was removed, and the plate was washed once with PBS. 100 μL / well of 4% formaldehyde was added to the plate. After incubation at room temperature for 30 minutes, the formaldehyde was removed, followed by two washes with PBS. Next, 100 μL / well of blocking buffer (PBS containing 2% BSA) was added to the plate and incubated at 37°C for 2 hours. After removing the blocking buffer, 100 μL / well of serially diluted antibody (starting at 1 μM and diluted 4-fold for a total of 11 concentrations) was added to the corresponding wells, followed by incubation at 37°C for 2 hours. The plate was washed five times with 250 μL of PBST, with each wash lasting 2 minutes. 100 μL / well of horseradish peroxidase-conjugated anti-human IgG secondary antibody (HRP-anti-human IgG, Jackson ImmunoResearch, 109-035-003) diluted 1:10,000 in PBS (containing 2% BSA) was added to the plate. The plate was incubated at 37°C for 1 hour and washed six times with 250 μL of PBST, where the plate was left to stand for 2 minutes each time. 100 μL / well of TMB solution (Thermo, 34029) was added. The reaction was incubated at 37°C for 20 minutes and stopped with 50 μL of 2 mol / L H2SO4. OD450nm absorbance values ​​were obtained using a plate reader (MD, SpectraMax M2), and the results were subjected to curve fitting using Graphpad Prism.

[0225] The results are shown in Figure 3 and Table 4, and demonstrate that the affinity of humanized antibody 2C6.9-hz21 for human claudin 18.2 is significantly higher than that of IMAB362. In the same experiment, the affinity of humanized antibody 2C6.9-hz11 was close to that of antibody 2C6.9-hz21 (results not shown).

[0226] [Table 9]

[0227] The specificity of the candidate antibodies was detected by FACS. The specific procedure was as follows: HEK293T, HEK293T-human claudin 18.1, and HEK293T-human claudin 18.2 cells were detached and then centrifuged. After washing twice with PBS, the cells were resuspended in PBS containing 1% BSA. The candidate antibodies were added to 300,000 cells of each cell line at a final concentration of 1,000 nM, mixed well, and incubated at 4°C for 1 hour, protected from light. The cells were then washed three times with PBS, and an FITC-labeled anti-human Fc secondary antibody (BioLegend, 409322) was added, followed by incubation in the dark at 4°C for 0.5 hours. After washing three times with PBS, detection was performed by flow cytometry (Beckman, Cytoflex).

[0228] As shown in Figure 4, 2C6.9-hz21 can specifically bind to human claudin 18.2 but cannot specifically bind to human claudin 18.1.

[0229] 6.6 Measurement of Complement-Dependent Cytotoxicity (CDC) of Humanized Antibody 2C6.9 2C6.9 belongs to the IgG1 subtype, which can effectively activate the classical complement pathway and induce complement-dependent cytotoxicity (CDC). In our study, we used complement-rich guinea pig serum (purchased from Zhengzhou Baiji, catalog number S0001) to measure the CDC activity of 2C6.9. The specific procedure is as follows: HEK293T-claudin18.2 cells were harvested and centrifuged, and the cell density was adjusted to 5 × 10 4 Cells were seeded at 100 μl / well and incubated overnight. DMEM containing 20% ​​guinea pig serum was prepared the next day and used to dilute 2C6.9 and IMAB362. Starting at 20 μg / mL, 10 concentrations were prepared by two-fold dilutions. The original cell culture medium for HEK293T-claudin18.2 cells was removed, and 100 μL / well of the antibody dilutions were added to the corresponding wells. 10 μL / well of lysis buffer served as a positive control. The reaction was placed in a 37°C, 5% CO2 incubator and incubated for 3 hours. Afterwards, 50 μL / well of CellTiter-Glo Luminescent (CTG, purchased from Promega, product number G7573) was added for staining, followed by 30 seconds of mixing and 1 minute at room temperature. The fluorescence signal values ​​were then measured by a microplate reader (MD, SpectraMax M2), and the results were imported into Graphpad Prism for curve fitting.

[0230] As shown in Figure 5 and Table 6, the CDC activity of 2C6.9-hz21 was stronger than that of the control antibody IMAB362.

[0231] [Table 10]

[0232] 6.7 Measurement of antibody-dependent cell-mediated cytotoxicity (ADCC) of humanized antibody 2C6.9. 2C6.9 belongs to the IgG1 subtype and has relatively strong antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC activity of 2C6.9 was measured using an NK cell-mediated killing assay. The specific procedure was as follows: HEK293T-claudin18.2 cells were harvested and centrifuged, and the cell density was adjusted to 1 × 10 4 Cells were seeded onto plates at 1 × 10 cells / well and incubated overnight. The next day, the medium was removed. NK92MI-CD16a cells (Huabo Biopharm) were centrifuged, resuspended in MEMA medium, and then cultured at 1 × 10 cells / well. 6 The antibody concentrations were adjusted to 40 μg / mL, and then 50 μL / well of cells were added to the corresponding wells. 2C6.9-hz21 and IMAB362 antibodies were diluted in MEMA medium. Ten antibody concentrations were tested for HEK293T-claudin18.2 cells, starting at 40 μg / mL and diluted 5-fold. Eleven antibody concentrations were tested for NUGC-4 cells, starting at 2 mg / mL and diluted 5-fold. 50 μL / well of diluted antibody was added to the corresponding wells, and the reaction was placed in a 37°C, 5% CO2 incubator and incubated for 5.5 hours. Lysis buffer was then added to the positive control wells, and the antibody concentrations were then diluted 5-fold. The plates were then incubated for another 0.5 hours. 50 μL / well of lactate dehydrogenase (LDH) detection reagent (DOJINDO LABORATORISE, CK12) was added to the wells, and absorbance at 490 nm was measured every 10 minutes using a microplate reader (MD, SpectraMax M2). The results were imported into Graphpad Prism for curve fitting.

[0233] As shown in Figure 6 and Table 7, the ADCC activity of 2C6.9-hz21 against HEK293T-claudin18.2 was stronger than that of IMAB362.

[0234] [Table 11]

[0235] [Example 7] Preparation of ADC (2C6.9-ADC) targeting claudin 18.2 2C6.9-TL001 was prepared by conjugating TL001 obtained in Example 3 with the humanized monoclonal antibody 2C6.9. The preparation method is as follows.

[0236] (1) Conjugation: 30 mg of 2C6.9-hz21 antibody was added to 20 mM PB + 105 mM NaCl + 100 mM edetate disodium solution (pH 7.7), and the pH was adjusted to 7.7 with 2 M Tris solution. The mixture was diluted with 20 mM PB + 105 mM NaCl pH 7.7 solution (the final concentration of edetate disodium was 5 mM, and the final concentration of antibody was 15 mg / mL). Mixed evenly, 10 mM TCEP solution was added and mixed well. The resulting mixture was allowed to stand at room temperature for a while (30 or 90 minutes). Then, TL001 dissolved in dimethyl sulfoxide (molar ratio to antibody: 5:1 or 9:1) was added, mixed well, and allowed to stand at room temperature for 2 hours to obtain a conjugate sample, which was named 2C6.9-TL001.

[0237] (2) Buffer exchange: A 30 KDa 50 ml ultrafiltration tube (Millipore) was used for buffer exchange of 2C6.9-TL001, and the exchange solution was 10 mM histidine-histidine hydrochloride + 8% sucrose (pH 6.0) buffer, with an exchange ratio of 15. The sample was collected and 10% Tween-20 was added, where the final concentration of Tween-20 in the sample was 0.02% (M / V).

[0238] (3) Test: After the replacement, 2C6.9-TL001 was subjected to LC-MS molecular weight analysis under the following conditions. Chromatography measurement conditions: Liquid chromatography column: Thermo MAbPac RP 3.0*100 mm; Mobile phase A: 0.1% FA / 98% HO / 2% ACN; Mobile phase B: 0.1% FA / 2% HO / 98% ACN; Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Sample size: 1 μL.

[0239] [Table 12]

[0240] Switching valve: 0-3 min drain, 3-22 min MS, 22-30 min drain. Mass analysis conditions: Mass spectrum model: AB Sciex Triple TOF 5600+; parameters: GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 200; CE 10; m / z 600-4000; time bins to sum value 40.

[0241] The theoretical and measured molecular weights (heavy chain calculated by the major glycoform G0F) of the light and heavy chains of 2C6.9-TL001 obtained by conjugation of TL001 with 2C6.9-hz21 are shown in the table below.

[0242] [Table 13]

[0243] When the conjugation ratio between TL001 and 2C6.9-hz21 is 5:1, the light chain (LC) of antibody 2C6.9-TL001 is conjugated with 0 to 1 toxin (LC and DAR1 ratios are 57.8% and 42.2%, respectively), and the heavy chain (HC) is conjugated with 0 to 4 toxins (HC, DAR1, DAR2, DAR3, and DAR4 ratios are 22.5%, 30.3%, 25.0%, 21.9%, and 0.3%, respectively). Therefore, the toxin-antibody ratio (DAR) is 3.79, calculated as follows: DAR = light chain DAR1*2 + heavy chain (DAR1*1 + DAR2*2 + DAR3*3 + DAR4*4)*2.

[0244] When the conjugation ratio between TL001 and 2C6.9-hz21 is 9:1, the antibody light chain (LC) of 2C6.9-TL001 is conjugated with 0 to 1 toxin (the proportions of LC and DAR1 are 7.5% and 92.5%, respectively), and the heavy chain (HC) is conjugated with 0 to 4 toxins (the proportions of HC, DAR1, DAR2, DAR3, and DAR4 are 2.5%, 10.2%, 9.8%, 76.4%, and 1.1%, respectively). Therefore, the toxin-antibody ratio (DAR) is 7.12.

[0245] When the feed ratio of conjugation between TL001 and 2C6.9-hz21 was 9:1, after conjugation and cation chromatography, the light chain of 2C6.9-TL001 was conjugated with 0 to 1 toxin (the ratios of LC and DAR1 were 1.5% and 24.0%, respectively), and the heavy chain was conjugated with 0 to 4 toxins (the ratios of HC, DAR1, DAR2, DAR3, and DAR4 were 0.7%, 2.1%, 14.3%, 54.5%, and 2.8%, respectively). The resulting drug-antibody ratio (DAR) was calculated to be 7.40.

[0246] The ADC molecular structure of 2C6.9-TL001 is shown below: [ka] In the formula, γ is an integer of 1 to 10, and A is 2C6.9-hz21.

[0247] The complex was subjected to SEC detection by SEC-HPLC. Chromatography conditions: Liquid chromatography column: TSKgel G3000SWxl, 300*7.8mm, 5μm; Mobile phase: 90mmol / L NaHPO, 30mmol / L NaHPO, 200mM NaCl, 5% acetonitrile; Flow rate: 0.8mL / min; Detection wavelength: 280nm; Column temperature: Room temperature; Sample chamber temperature: 8℃; Sample size: 40μL; Isocratic run: 30min.

[0248] The SEC chromatograms of 2C6.9-TL001 (DAR: 3.79) and 2C6.9-TL001 (DAR: 7.12) are shown in Figures 7 and 8, and the SEC chromatogram of 2C6.9-TL001 (DAR: 7.40) is shown in Figure 13. Based on the SEC retention time and peak area ratio, the molecular weight of the major conjugated product was approximately 150 kDa, confirming that 2C6.9-TL001 obtained by conjugation of TL001 with 2C6.9-hz21 still maintained the overall structure of the antibody.

[0249] Using the same preparation method, TL002 and TL003 prepared in Examples 4 and 5 were conjugated with humanized monoclonal antibody 2C6.9 to prepare 2C6.9-TL002 and 2C6.9-TL003. The preparation and detection methods are as described above. The DAR values ​​of the obtained 2C6.9-TL002 and 2C6.9-TL003 are 6.95 and 7.03, respectively.

[0250] The ADC molecular structure of 2C6.9-TL002 is shown below: [ka] In the formula, γ is an integer from 1 to 10, and A is 2C6.9-hz21.

[0251] The molecular structure of 2C6.9-TL003 is shown below: [ka] In the formula, γ is an integer of 1 to 10, and A is the 2C6.9 antibody.

[0252] Example 8: Measurement of affinity of 2C6.9-TL001 A cell-based ELISA was performed to measure the affinity of 2C6.9-TL001 (DAR: 7.12) for claudin 18.2 on the surface of the cell membrane. The specific procedure was as follows: L929-claudin 18.2 adherent cell line was detached with 2 mM EDTA, and 2 × 10 5The cells were resuspended to 100 cells / mL, and 100 μL of the resuspension was placed in a 96-well plate and incubated overnight at 37° C.; the next day, the medium was removed and the plate was washed once with PBS. 100 μL / well of 4% formaldehyde was added to the plate at room temperature for 30 minutes; then, after removing the formaldehyde, the plate was washed twice with PBS, and 100 μL of PBS (containing 2% BSA) was added and incubated for 2 hours; the blocking solution was removed, and the 2C6.9-TL001 to be tested was diluted 4-fold starting from 9.375 μg / mL with PBS (containing 2% BSA) to a total of 9 concentrations, and then 100 μL / well of the 2C6.9-TL001 to be tested was added to the plate and incubated at 37°C for 2 hours; the plate was washed 5 times with 250 μL of PBST, and left to stand for 2 minutes each time; PBS (2% BSA) was used to detect horseradish peroxidase (HRP)-labeled anti-human IgG secondary antibody (HRP-anti-human IgG, Jackson Immunoresearch (2C6.9-TL001) was diluted at a ratio of 1:10,000, and 100 μL of the diluted solution was added to the plate at 100 μL / well and incubated at 37°C for 1 hour. After washing six times with 250 μL of PBST, the cells were allowed to stand for 2 minutes each time. 100 μL of TMB chromogenic solution (Thermo) was added to the corresponding wells and allowed to develop for 20 minutes at 37°C. The color was terminated by adding 50 μL of 2 mol / L H2SO4. The absorbance values ​​at OD450nm were obtained using a microplate reader (MD) and imported into Graphpad Prism for curve fitting. The experimental results are shown in Figure 9, which shows the EC20 affinity of 2C6.9-TL001 (DAR: 7.12) for claudin 18.2 on the cell membrane surface. 50 The value was 39.94 ng / mL. Antibody 2C6.9-hz21 still has excellent claudin 18.2 affinity after conjugation with TL001.

[0253] Example 9: Detection of the killing activity of 2C6.9-ADC against tumor cell lines with high claudin 18.2 expression. The killing activity of 2C6.9-TL001 against cell lines with high claudin 18.2 expression was detected using HEK293T-claudin 18.2 and HEK293T-claudin 18.1 cells. The specific experimental procedure was as follows: On the day before the experiment, the cells were diluted with DMEM + 10% FBS, and the suspension was added at 1 × 10 cells per well in 100 μL. 4 The next day, the ADC molecules were diluted 4-fold with DMEM basic medium, starting at 150 μg / mL (DAR: 7.12) or 262.5 μg / mL (DAR: 3.79), to a total of 11 concentrations. 100 μL of the dilutions were added to the corresponding wells at a final serum concentration of 5%. The cells were then incubated at 37°C in a 5% CO2 incubator for 48 hours. CCK8 (Rhinogen) was then added (20 μL / well), and the cells were placed in a 5% CO2 incubator at 37°C for 0.5 to 2.5 hours. OD450nm absorbance values ​​were obtained every 30 minutes using a microplate reader (MD), and the values ​​were imported into Graphpad Prism for curve fitting.

[0254] As shown in Figures 10A, 10B, and 10C, 2C6.9-TL001 can effectively kill HEK293T-claudin18.2 cells. With a DAR value of 7.12, its EC 50 The value was 473 ng / mL; the DAR value was 3.79, and the EC 50 The EC value of 2C6.9-TL001 (DAR: 7.12) for killing HEK293T-claudin 18 was 794.1 ng / mL. 50 is 39 The killing activity of 2C6.9-TL001 (DAR: 7.12) against claudin 18.2 cells was significantly stronger (approximately 8-fold difference) than that against claudin 18.1 cells, indicating that the killing effect of 2C6.9-TL001 is specific to claudin 18.2.

[0255] The killing activity of 2C6.9-TL002 and 2C6.9-TL003 against HEK293T-claudin18.2 and HEK293T-claudin18.1 cells was detected using the same experimental method as above. The experimental results shown in Figures 10D and 10E show that 2C6.9-TL002 and 2C6.9-TL003 can effectively kill HEK293T-claudin18.2 cells and inhibit EC 50 The EC values ​​of 2C6.9-TL002 and 2C6.9-TL003 for killing HEK293T-claudin18.1 were 628.9 ng / mL and 540.2 ng / mL, respectively; and 50 The values ​​were 30,590 ng / mL and 8,258 ng / mL, respectively. The killing activity of 2C6.9-TL002 and 2C6.9-TL003 against HEK293T-claudin18.2 cells was significantly stronger than that against claudin18.1, indicating that the killing effect was specific to claudin18.2.

[0256] Example 10: Detection of the killing activity of 2C6.9-ADC against endogenously expressed claudin 18.2 cell lines. The gastric cancer cell line NUGC-4 was selected to detect the killing activity of 2C6.9-TL001 against endogenously expressed claudin 18.2 cells. The specific experimental procedure was as follows: On the day before the experiment, the cells were diluted with RPMI 1640 + 10% FBS, and the suspension was added at 1 x 10 cells per well in 100 μL. 4The next day, 2C6.9-TL001 was diluted 3-fold in RPMI 1640 medium, starting at 1000 μg / mL (DAR 7.12) or 1750 μg / mL (DAR 3.79), to a total of 11 concentrations. 100 μL of the dilutions were added to the corresponding wells at a final serum concentration of 5%. The cells were then incubated at 37°C in a 5% CO2 incubator for 72 hours. 20 μL of CCK8 (Rhinogen) was then added to the wells at a final serum concentration of 5%. The cells were then incubated at 37°C in a 5% CO2 incubator for 0.5–2.5 hours. OD450nm absorbance readings were obtained every 30 minutes using a microplate reader (MD). The values ​​were imported into Graphpad Prism for curve fitting. The experimental results are shown in Figure 11A and Figure 11B; 2C6.9-TL001 can effectively kill NUGC-4 cells; its EC 50 The EC value is 2.383 μg / mL; when the DAR value is 3.79, the EC 50 The value is 10.01 μg / mL.

[0257] The killing activity of 2C6.9-TL002 and 2C6.9-TL003 against NUGC-4 cells was detected by the same experimental method as above. The initial concentration of ADC was 500 μg / mL, and it was diluted 4 times to a total of 11 concentrations. As shown in Figure 11C, 2C6.9-TL002 and 2C6.9-TL003 could effectively kill NUGC-4 cells, and EC 50 The values ​​are 54.92 μg / mL and 123.94 μg / mL, respectively.

[0258] [Example 11] Detection of 2C6.9-hz21 antibody internalization NUGC-4 was selected to detect the internalization activity of antibody 2C6.9. The specific experimental steps were as follows: NUGC-4 cells were digested with trypsin, counted, and resuspended in PBS (containing 1% BSA) at a cell density of 3 × 10 6The 2C6.9-hz21 antibody was added to 100 μL of resuspended cells at a final concentration of 100 μg / mL, and isotype human IgG was set as a negative control and incubated on ice for 1 hour. After incubation, the cells were washed three times with pre-cooled PBS and resuspended in NUGC-4 cell culture medium (1640 + 10% PBS). The cells were resuspended in 0.5% FBS and divided into two portions: one portion was incubated at 37°C for 4 hours in a cell incubator (endocytosis group), and the other portion was incubated on ice for 4 hours (affinity group). After incubation, the cells were washed three times with pre-cooled PBS and resuspended in 50 μL of PBS (containing 1% BSA). Anti-human fluorescent secondary antibody (Biolegend) was added and incubated at 4°C for 30 minutes. After incubation, the cells were washed three times with pre-cooled PBS and detected by flow cytometry (Beckman). The antibody internalization ratio was calculated according to the following formula: endocytosis (%) = [1-(MFI 37℃抗体群 -MFI 37℃対照群 ) / (MFI 氷上の抗体群 -MFI 氷上の対照群 )] × 100%. The experimental results showed that the 4-hour internalization ratio of 2C6.9-hz21 in NUGC-4 cells was 37.79%, indicating that the 2C6.9-hz21 complex has the potential to internalize the drug into cells and kill tumor cells.

[0259] Example 12: In vivo efficacy of 2C6.9-ADC was evaluated using cancer cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models to evaluate the antitumor effects of ADC molecules. 12.1 NCI-N87-claudin18.2+Balb / c nude mouse CDX model NCI-N87-claudin18.2 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested and resuspended in PBS, and then 5x10 cells were transferred to female Balb / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). 6The cells were subcutaneously inoculated into mice at a dose of 1000 cells / mouse (suspended in 0.1 mL of PBS) to establish a subcutaneous tumor model. The average tumor volume was 70–100 mm. 3 When tumor volume reached 100 μg / kg, the mice were randomly divided into groups (7 mice / group). The day of grouping was recorded as day 0, and the groups were divided into the following groups: human IgG1 isotype control antibody (negative control) group (abbreviated as IgG1), 2C6.9-TL001 (DAR: 7.12) 1 mg / kg group and 3 mg / kg group, and 2C6.9-TL002 (DAR: 6.95) 1 mg / kg group and 3 mg / kg group. All samples were injected via the tail vein twice a week, a total of 6 times.

[0260] After administration, the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 (wherein a represents the longest diameter of the tumor and b represents the shortest diameter of the tumor.) Mortality was observed and recorded daily.

[0261] The tumor growth inhibition rate (TGI) (%) was calculated using the following formula to evaluate the antitumor effect: TGI (%) = [1-(V Tend -V Tstart ) / (V Cend -V Cstart )]*100% where V Tend : Mean tumor volume at the end of the experiment in the treatment group V Tstart : Mean tumor volume at the start of treatment in the treatment group V Cend : Mean tumor volume at the end of the experiment in the negative control group V Cstart : Mean tumor volume at the start of administration in the negative control group.

[0262] The relative tumor growth rate T / C (%) was calculated using the following formula to evaluate the antitumor effect: T / C (%) (tumor volume) = (T t / T0) / (C t / C0) × 100% where, T0: mean tumor volume of the treatment group at the initial stage (i.e., P0), T t : Average tumor volume of the treatment group at each measurement C0: Average tumor volume of the negative control group at the initial stage (i.e., P0) C t : Mean tumor volume of the negative control group at each measurement.

[0263] The experimental results are shown in Table 8 and Figures 12A and 12B. 2C6.9-TL001 (DAR: 7.12) demonstrated significant dose-dependent tumor growth inhibition in the NCI-N87-Claudin18.2 gastric cancer xenograft model. Compared with the negative control group, after six doses (day 21), the tumor inhibition rate (TGI) of the 2C6.9-TL001 1 mg / kg group was up to 96.03%, with four mice showing partial tumor regression; while in the 3 mg / kg group, the TGI reached 133.50%, with three mice showing partial tumor regression and four mice showing complete tumor regression. The TGI of the 2C6.9-TL002 3 mg / kg group was 40.11%, with no significant antitumor effect in the 1 mg / kg group. During the observation period, no significant weight loss was observed in any of the treatment groups, and the treatment was well tolerated by the animals.

[0264] [Table 14]

[0265] 12.2 Comparison of the in vivo efficacy of 2C6.9-TL001 and 2C6.9 monoclonal antibody plus chemotherapy in the CDX model. An NCI-N87-claudin18.2 subcutaneously implanted tumor model was established according to Example 12.1 and divided into groups. The day of grouping was recorded as day 0. The groups were a human IgG1 isotype control antibody (negative control) group (abbreviated as IgG1), a paclitaxel group (albumin-bound), a 2C6.9 mAb combined with paclitaxel group, and a 2C6.9-TL001 (DAR: 7.12) group. All samples were injected via the tail vein twice a week for a total of three weeks. The dosages are shown in Table 9.

[0266] The experimental results are shown in Table 9 and Figure 12C. Compared with the negative control group, all three treatment groups, especially the 2C6.9-TL001 (DAR: 7.12) group, can significantly inhibit tumor growth after 11 days of drug administration. The tumor inhibition rate (TGI) of the 2C6.9-TL001 (DAR: 7.12) group was up to 121.68%, and 6 out of 7 mice showed partial tumor regression.

[0267] [Table 15]

[0268] After 21 days of treatment, all three treatment groups were able to significantly inhibit tumor growth compared to the negative control group, with the 2C6.9-TL001 (DAR: 7.12) group being the most significant. The tumor inhibition rate (TGI) of the 2C6.9-TL001 (DAR: 7.12) group was up to 125.73%, and five of the seven mice showed complete tumor regression, and two mice showed partial tumor regression. The experimental results are shown in Table 10 and Figure 12D.

[0269] [Table 16]

[0270] 12.3 Comparison of the in vivo efficacy of 2C6.9-TL001 with different DAR values ​​in the CDX model. NUGC-4 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, resuspended in PBS, and then inoculated into female Balb / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) at 5 × 10 6 A subcutaneous tumor model was established by subcutaneous inoculation of 1000 cells / mice (suspended in 0.1 mL of PBS). The average tumor volume was approximately 70-100 mm. 3When tumor volume reached 1000 mg / kg, the mice were randomly divided into groups (7 mice per group). The day of grouping was recorded as day 0, and the groups were divided into three groups: human IgG1 isotype control antibody (negative control) group (abbreviated as IgG1), 2C6.9-TL001 (DAR: 3.79) 5.25 mg / kg group, 2C6.9-TL001 (DAR: 3.79) 17.5 mg / kg group, and 2C6.9-TL001 (DAR: 7.12) 3 mg / kg group. The mice were divided into two groups: a 10 mg / kg group (2C6.9-TL001 group) and a 10 mg / kg group (2C6.9-TL001 (DAR: 7.12) group). (ADC drugs with different DAR values ​​were administered at the same toxin load and designed dose.) All samples were injected via the tail vein twice a week for a total of 3 weeks. The doses are shown in Table 11.

[0271] The experimental results are shown in Table 11 and Figures 12E and 12F. At the same toxin load, the efficacy of 2C6.9-TL001 with a high DAR value (7.12) was essentially equivalent to that of 2C6.9-TL001 with a low DAR value (3.79) after 21 days of drug administration. No significant weight loss was observed in any treatment group during the observation period, and the treatment was well tolerated by the animals.

[0272] [Table 17]

[0273] 12.4 HuPrime® Gastric Cancer GA0006 + Balb / c Nude Mouse PDX Model In the HuPrime® gastric cancer xenograft model GA0006 (Crown Bioscience (Taicang) Inc.; a high-claudin18.2-expressing gastric tumor derived from a 57-year-old female patient), tumor tissues were harvested from tumor-bearing mice, cut into small pieces with a diameter of 3 × 3 × 3 mm, and inoculated subcutaneously into the right anterior scapula of Balb / c nude mice. The average tumor volume was approximately 150–250 mm. 3When tumor size reached 100 mg / kg, the mice were randomly divided into three groups (7 mice per group) based on tumor size, and the day of grouping was recorded as day 0. There were three groups: a human IgG1 homotypic control antibody (negative control) group (IgG1 10 mg / kg), a 2C6.9-TL001-DAR7.12 3 mg / kg group, and a 10 mg / kg group. All samples were injected into the tail vein twice a week for a total of five times. After drug administration, the tumor volume and body weight of the mice were observed and measured periodically using the method described in Example 12.1.

[0274] The experimental results in Table 12 and Figures 12G and 12H show that 2C6.9-TL001-DAR7.12 has a significant dose-dependent inhibitory effect on tumor growth in the GA0006 gastric cancer PDX model. Compared with the negative control group, the tumor inhibition rate (TGI) of the 3 mg / kg group was up to 94.72% after five doses (day 17), and tumors in four mice partially disappeared; the TGI of the 10 mg / kg group was as high as 124.49%, and tumors in all seven mice disappeared, indicating that 2C6.9-TL001 can effectively inhibit tumor growth. No significant weight loss was observed in any treatment group during the observation period, and the treatment was well tolerated by the animals.

[0275] [Table 18]

[0276] The experimental results are shown in Table 13 and Figures 12I and 12J. On day 24 post-administration, 2C6.9-TL001-DAR7.12 demonstrated significant and dose-dependent inhibition of tumor growth in the GA0006 gastric cancer PDX model. Compared with the negative control group, the tumor inhibition rate (TGI) of the 3 mg / kg group was up to 103.76%, and tumors in six mice partially regressed. The TGI of the 10 mg / kg group reached 113.70%, and tumors in all seven mice regressed; this indicates that 2C6.9-TL001 can effectively inhibit tumor growth. No significant weight loss was observed in any treatment group during the observation period, and the treatment was well tolerated by the animals.

[0277] [Table 19]

[0278] 12.5 Evaluation of In Vivo Efficacy for NCI-N87-Claudin18.2 in Balb / c Nude Mouse CDX Model The NCI-N87-Claudin18.2 subcutaneously implanted tumor model was established according to the method in Example 12.1. The mean tumor volume was approximately 140 mm 3 When tumor volume reached 100 μg / kg, the mice were randomly assigned to groups (8 mice / group). The day of group assignment was recorded as day 0, and the groups were assigned to a human IgG1 isotype control antibody (negative control) group (abbreviated as IgG1), 2C6.9-TL001 (DAR: 7.40) 0.3 mg / kg group, 1 mg / kg group, and 3 mg / kg group, respectively. All samples were injected into the tail vein twice a week for a total of 6 doses, and the doses are shown in Table 14.

[0279] The results are shown in Table 14 and Figures 12K and 12L. When the observation period was extended to 31 days after the drug withdrawal, the tumor inhibition rate (TGI) of the 2C6.9-TL001 0.3 mg / kg group was 34.04%, while the TGI of the 1 mg / kg group and the 3 mg / kg group was up to 122.57% (partial tumor regression was observed in all mice) and 184.22% (complete tumor regression was observed in 4 mice, and partial tumor regression was observed in 4 mice), respectively, compared with the negative control group. No significant weight loss was observed in any treatment group during the observation period, and the treatment was well tolerated by the animals.

[0280] [Table 20]

[0281] 12.6 Evaluation of in vivo efficacy of HEK293T-claudin18.2 in Balb / c nude mouse CDX model. HEK293T-claudin18.2 cells (human embryonic kidney cells) were cultured in DMEM medium containing 3 μg / mL puromycin and 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, resuspended in PBS, and then inoculated into female Balb / c nude mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) at 1 × 10 7 A subcutaneous tumor model was established by subcutaneous inoculation of 1000 cells / mice (suspended in 0.1 mL of PBS). The average tumor volume was approximately 120–140 mm. 3 When tumor volume reached 100 μg / kg, the mice were randomly assigned to groups (8 mice / group). The day of group assignment was recorded as day 0, and the groups were assigned to a human IgG1 isotype control antibody (negative control) group (referred to as IgG1), 2C6.9-TL001 (DAR: 7.40) 0.3 mg / kg group, 1 mg / kg group, and 3 mg / kg group, respectively. All samples were injected into the tail vein twice a week for a total of 6 doses, and the doses are shown in Table 15.

[0282] The results are shown in Table 15 and Figures 12M and 12N. Compared with the negative control group, 2C6.9-TL001 (DAR: 7.40) showed significant and dose-dependent inhibition of tumor growth after six doses (21 days after the first dose) in the HEK293T-claudin18.2 human embryonic kidney cell xenograft tumor model. The tumor inhibition rate (TGI) of the 2C6.9-TL001 0.3 mg / kg group was 70.99% (complete tumor regression was observed in one mouse), while the TGI of the 1 mg / kg and 3 mg / kg groups was 94.98% (partial tumor regression was observed in two mice) and 182.81% (partial tumor regression was observed in two mice and complete tumor regression was observed in six mice), respectively.

[0283] [Table 21]

[0284] 12.7 Evaluation of in vivo efficacy of NUGC-4 in Balb / c nude mouse CDX model. NUGC-4 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, resuspended in PBS, and then inoculated into female Balb / c nude mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) at 5 × 10 6 A subcutaneous tumor model was established by subcutaneous inoculation of 1000 cells / mice (suspended in 0.1 mL of PBS). The average tumor volume was approximately 80 mm. 3 When tumor volume reached 100 μg / kg, the mice were randomly assigned to groups (8 mice / group). The day of group assignment was recorded as day 0, and the groups were assigned to a human IgG1 isotype control antibody (negative control) group (referred to as IgG1), a 2C6.9-TL001 (DAR: 7.40) 3 mg / kg group, and a 10 mg / kg group, respectively. All samples were injected into the tail vein twice a week for a total of 6 doses, and the doses are shown in Table 16.

[0285] The results are shown in Table 16 and Figures 12O and 12P. Compared with the negative control group, 2C6.9-TL001 (DAR: 7.40) showed significant and dose-dependent inhibition of tumor growth after six doses (21 days after the first dose) in the NUGC-4 gastric cancer xenograft tumor model. The 2C6.9-TL001 3 mg / kg group showed a tumor inhibition rate (TGI) of 56.62%, while the TGI of the 10 mg / kg group was 90.28% (partial tumor regression was observed in two mice).

[0286] [Table 22]

[0287] In conclusion, 2C6.9-TL001 can effectively inhibit the growth of claudin-18.2-positive tumors in either CDX or PDX models in a dose-dependent manner and is safe for use.

[0288] Although embodiments of the present invention have been described in detail, it will be understood that those skilled in the art, guided by the teachings of all the disclosures, can make modifications and changes to the details, and all such modifications will fall within the scope of the present invention, which is given by the claims appended hereto and their equivalents.

Claims

1. The structure is of formula (I): (D-L) γ -A formula (I) An antibody-drug conjugate (ADC) as shown in wherein D is a fragment of a biologically active molecule; L is a linker; γ is an integer from 1 to 10; A is an antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2, and the antibody or antigen-binding fragment thereof (1) The following VH and VL, with CDRs defined according to the IMGT numbering system: a VH comprising the following three CDRs: CDR-H1 having the sequence of SEQ ID NO: 1, CDR-H2 having the sequence of SEQ ID NO: 2 or 21, and CDR-H3 having the sequence of SEQ ID NO: 3; and a VL comprising the following three CDRs: CDR-L1 having the sequence of SEQ ID NO: 4, CDR-L2 having the sequence of SEQ ID NO: 5, and CDR-L3 having the sequence of SEQ ID NO: 6; or (2) The following VH and VL, with CDRs defined according to the AbM numbering system: a VH comprising the following three CDRs: CDR-H1 having the sequence of SEQ ID NO: 7, CDR-H2 having the sequence of SEQ ID NO: 8 or 22, and CDR-H3 having the sequence of SEQ ID NO: 9; and a VL comprising the following three CDRs: CDR-L1 having the sequence of SEQ ID NO: 10, CDR-L2 having the sequence of SEQ ID NO: 11, and CDR-L3 having the sequence of SEQ ID NO: 12; An ADC comprising:

2. The ADC is (1) The antibody or antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO: 13 or 14 and a VL set forth in SEQ ID NO: 15; or (2) The VH contained in the antibody or its antigen-binding fragment has at least 90% identity compared to the VH described in (1), and / or the VL contained in the antibody or its antigen-binding fragment has at least 90% identity compared to the VL described in (1); 2. The ADC of claim 1, wherein:

3. The antibody (1) A heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 13 and a CH of the sequence set forth in SEQ ID NO: 16, and a light chain comprising a VL of the sequence set forth in SEQ ID NO: 15 and a CL of the sequence set forth in SEQ ID NO: 17; or (2) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 14 and a CH of the sequence set forth in SEQ ID NO: 16, and a light chain comprising a VL of the sequence set forth in SEQ ID NO: 15 and a CL of the sequence set forth in SEQ ID NO: 17; 3. The ADC of claim 1 or 2, wherein:

4. the antibody or antigen-binding fragment thereof (1) A heavy chain comprising an amino acid sequence selected from the group consisting of: (1-1) the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 19; or (1-3) a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 19; and (2) a light chain comprising an amino acid sequence selected from the group consisting of: (2-1) the sequence set forth in SEQ ID NO: 20; or (2-3) a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 20; The ADC of any one of claims 1 to 3, comprising:

5. The antibody or antigen-binding fragment thereof may be an ScFv, Fab, Fab', (Fab') ２ 5. The ADC of any one of claims 1 to 4, which is selected from an Fv fragment, a disulfide-linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

6. Formula (II): {D-[L １ -(L ２ ) ｍ１ -(L ３ ) ｍ２ -(L ４ ) ｍ３ -E]} γ -A Formula (II) having the structure shown in During the ceremony, L １ teeth 【Chemical 1】 where R １ and R ２ are each independently hydrogen, halogen, a carboxylic acid group, a sulfonic acid group, cyano, C １－６ Alkyl, halogenated C １－６ Alkyl, cyano-substituted C １－６ Alkyl, C １－６ Alkoxy, C ２－１０ alkenyl, or C ２－１０ alkynyl; Z １ is an amino acid or a peptide consisting of 2 to 10 amino acids; x １ is 0, 1, 2, 3, 4, 5 or 6, and x ２ is 0, 1, 2, 3 or 4; L １ is connected to D in the first place and to L in the second place. ２ connected to; L ２ teeth 【Chemistry 2】 where y １ is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; L ２ is ranked 1st １ Connected to L in second place ３ connected to; L ３ is selected from 5- to 12-membered aromatic heterocycles; L ４ teeth 【Chemistry 3】 where Z ２ is C １－６ Alkylene, C ２－１０ Alkenylene, C ２－１０ a quinylene, and C ３－８ cycloalkylene; R ３ is hydrogen and C １－６ alkyl; Z ３ does not exist or C １－６ alkylene; or R ３ and Z ３ together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclylene; α is 0, 1, 2, 3, 4, 5, or 6; and L ４ is connected to E at the second position and to L at the first position. ３ connected to; E is 【Chemistry 4】 and E is connected to A at the 2nd position and to L at the 1st position. ４ connected to; m １ , m ２ and m ３ are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is as defined in any one of claims 1 to 5; D and γ are as defined in claim 1. The ADC according to any one of claims 1 to 5.

7. Formula (III): {D-[(L １ ') ｍ４ -L １ -(L ５ ) ｍ５ -(L ３ ) ｍ２ -(L ４ ) ｍ３ -E]} γ -A Formula (III) having the structure shown in During the ceremony, L １ 'teeth 【Chemistry 5】 where R ５ and R ６ are each independently hydrogen or C １－６ is alkyl; x ３ is 1, 2, 3, 4, 5 or 6; L １ ', if it exists, connects with D at the first position and with L at the second position. １ Connect with; L １ teeth 【Chemistry 6】 where R １ and R ２ are each independently hydrogen, halogen, a carboxylic acid group, a sulfonic acid group, cyano, C １－６ Alkyl, halogenated C １－６ Alkyl, cyano-substituted C １－６ Alkyl, C １－６ Alkoxy, C ２－１０ alkenyl, or C ２－１０ alkynyl; Z １ is an amino acid or a peptide consisting of 2 to 10 amino acids; x １ is 0, 1, 2, 3, 4, 5 or 6, and x ２ is 0, 1, 2, 3 or 4, and L １ is ranked 1st １ ' (L １ ' exists), or connected to D at the first position (L １ ' is not present); L １ is in second place ５ connected to; L ５ teeth 【Chemistry 7】 where R ７ is hydrogen or C １－６ alkyl or R ７ is connected to the N atom on the γ-C to form a 5- to 6-membered heterocyclylene; x ４ is 1, 2, 3, 4, 5 or 6; y １ is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; L ５ is ranked 1st １ Connected to L in second place ３ connected to; L ３ is selected from 5- to 12-membered aromatic heterocycles; L ４ teeth 【Chemistry 8】 where Z ２ is C １－６ Alkylene, C ２－１０ Alkenylene, C ２－１０ Alkynylene, and C ３－８ cycloalkylene; R ３ is hydrogen and C １－６ alkyl; Z ３ does not exist or C １－６ alkylene; or R ３ and Z ３ together with the nitrogen atom to which they are attached form a 4- to 8-membered heterocyclylene; α is 0, 1, 2, 3, 4, 5, or 6; and L ４ is connected to E at the second position and to L at the first position. ３ connected to; E is 【Chemistry 9】 and And E is connected to A at the second place and L at the first place. ４ connected to; m ２ , m ３ , m ４ and m ５ are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is as defined in any one of claims 1 to 5; D and γ are as defined in claim 1. The ADC according to any one of claims 1 to 5.

8. L １ but 【Chemistry 10】 wherein Z １ is an amino acid or a peptide consisting of 2 to 5 amino acids, wherein the amino acids are selected from Lys, Cit, Val, D-Val, Phe, Leu, Gly, Ala, and Asn.

9. L ２ but 【Chemistry 13】 and m １ The ADC of claim 6 or 8, wherein

10. L ３ is a 5- to 6-membered aromatic heterocycle, and m ２ is 1, The ADC according to any one of claims 6 to 9.

11. L ４ but 【Chemistry 14】 and Z ２ is C １－６ alkylene, and Z ３ is C １－６ alkylene; and m ３ The ADC of any one of claims 6 to 10, wherein

12. L １ 'but 【Chemistry 16】 12. The ADC of any one of claims 7, 8, 10 and 11, wherein

13. L ５ but 【Chemistry 17】 where x ４ is 1, 2, 3, 4, 5 or 6; y １ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; the ADC of any one of claims 7, 8, 10 to 12.

14. D is 【Chemistry 19】 【Chemistry 20】 or 【Chemical 21】 is; The ADC according to any one of claims 1 to 13.

15. D-[(L １ ') ｍ４ -L １ - (L ５ ) ｍ５ - (L ３ ) ｍ２ - ( L ４ ) ｍ３ -E]- is 【Chemistry 24】 or 【Chemistry 25】 8. The ADC of claim 7, wherein:

16. ADC, 【Chemical Formula 26】 is selected from where γ is an integer from 1 to 10. The ADC according to any one of claims 1 to 15.

17. The ADC of any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof bears a label.

18. 18. The ADC of any one of claims 1 to 17, wherein γ is an integer from 1 to 8.

19. A composition comprising the ADC of any one of claims 1 to 18, wherein the molar ratio (DAR value) of the fragment of the biologically active molecule to the antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 is a decimal or integer number from 1 to 10.

20. A pharmaceutical composition comprising the ADC of any one of claims 1 to 18, or the composition of claim 19, and a pharmaceutically acceptable carrier and / or excipient.

21. Use of the ADC of any one of claims 1 to 18, the composition of claim 19 or the pharmaceutical composition of claim 20 in the preparation of a medicament for the prevention and / or treatment and / or adjuvant treatment of a tumor, comprising: The medicament may further comprise an additional antitumor active ingredient.

22. 22. The use according to claim 21, wherein the tumor is selected from a solid tumor, a hematological tumor, or a metastatic, refractory, or recurrent lesion of cancer.

23. An effective amount of the antibody-drug conjugate (ADC) of any one of claims 1 to 18, the composition of claim 19, or the pharmaceutical composition of claim 20. A pharmaceutical composition for preventing and / or treating a tumor and / or delaying tumor progression and / or reducing or inhibiting tumor recurrence in a subject.

24. 24. The pharmaceutical composition of claim 23 for combination with a second therapy, comprising: wherein the second therapy is selected from surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; wherein said second therapy is applied separately, in combination, simultaneously, or sequentially with said pharmaceutical composition.

Images