Antibody drug conjugates, methods of making and uses thereof

Abstract

An anti-CLDN18.2 antibody-drug conjugate and a preparation method and use thereof, the antibody-drug conjugate and a composition containing the conjugate can effectively bind to CLDN18.2 and inhibit the growth of Claudin18.2 positive tumors. Further relates to a pharmaceutical composition comprising the antibody-drug conjugate or composition, and the use of the pharmaceutical composition in the preparation of a drug for preventing and / or treating tumors.

Description

[0001] This application is based on and claims priority to CN application No. 202010410633.5, filed on May 15, 2020, the disclosure of which is incorporated herein in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine. Specifically, the present application relates to anti-Claudin 18.2 antibody-drug conjugates (ADCs) and compositions containing the same, and their uses. BACKGROUND

[0003] The treatment of gastric cancer is mainly based on surgical resection. For unresectable or recurrent gastric cancer, chemotherapy is the main treatment method, but under the existing conditions, chemotherapy can only improve symptoms and prolong survival. At present, there is no internationally recognized standard chemotherapy regimen for advanced gastric cancer. In terms of biological macromolecular drugs, in addition to Trastuzumab, Ramucirumab, Pembrolizumab, etc. approved, most other gastric cancer targeted therapy drugs have less than satisfactory results or are still in the early stages of clinical research. The core defect of existing drugs is that non-surgical treatment does not meet the clinical needs, the treatment options for advanced or recurrent gastric cancer are very limited, the prognosis is poor, and the mortality is high. As a high-incidence cancer, the demand for gastric cancer drugs is extremely large at present.

[0004] Claudin 18.2 (CLDN 18.2) is a member of the Claudin protein family. Claudin family proteins are a class of proteins that mediate tight junctions between cells. Different subtypes of Claudin proteins are expressed in different tissues and are associated with different types of cancer. Claudin 18.2 is only expressed in gastric mucosal cells in normal tissues and is not expressed in other normal tissues. Claudin 18.2 is highly expressed in 70% of primary gastric adenocarcinomas and their metastases, and is also expressed in some other cancers such as pancreatic cancer, esophageal cancer, and non-small cell lung cancer. The high specific expression of Claudin 18.2 in tumor tissues makes Claudin 18.2 a very good target for tumor immunotherapy. SUMMARY

[0005] The inventors of the present application have described antibodies 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 form part of the present application. Through a large amount of creative labor, the inventors of the present application have further invented Claudin 18.2 antibody-drug conjugates (ADCs), providing safe and effective drug options for the treatment of tumors.

[0006] Specifically, the inventors linked an antibody with high affinity and specificity to bind Claudin 18.2 to a bioactive molecule through a linker, obtaining a class of antibody-drug conjugates targeting Claudin 18.2 and compositions containing the conjugates. The conjugates or compositions have high killing activity on some tumor cells expressing Claudin 18.2. In animal in vivo tests, the conjugates can effectively inhibit the growth of Claudin 18.2-positive tumors, especially gastric cancer or gastric adenocarcinoma, and have high safety.

[0007] Antibody-drug conjugates

[0008] In one aspect, the present application provides an antibody-drug conjugate specifically binding to human CLDN18.2, which has a structure as shown in formula (I),

[0009] (D-L) γ -A

[0010] Formula (I)

[0011] wherein D is a fragment of a bioactive molecule; L is a linker;

[0012] γ is selected from an integer between 1-10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); preferably, γ is selected from an integer between 1-8 (e.g. 1, 2, 3, 4, 5, 6, 7 or 8);

[0013] A is an antibody or antigen-binding fragment thereof specifically binding to human CLDN18.2.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof specifically binding to human CLDN18.2 comprises:

[0015] (1) three heavy chain CDRs: CDR-H1, CDR-H2 and CDR-H3 contained in a VH (heavy chain variable region) as shown in SEQ ID NOs: 13, 14 or 23; and / or,

[0016] (2) three light chain CDRs: CDR-L1, CDR-L2 and CDR-L3 contained in a VL (light chain variable region) as shown in SEQ ID NO: 15 or 24;

[0017] or,

[0018] (2) three heavy chain CDRs as described in (1) or a variant thereof containing amino acid mutations compared to (1), and / or,

[0019] three light chain CDRs as described in (1) or a variant thereof containing amino acid mutations compared to (1);

[0020] 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), said amino acid mutation being a substitution, deletion or addition of one or several amino acids (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids); said antibody or antigen binding fragment thereof containing the mutation still being capable of specifically binding to CLDN18.2; preferably, to human CLDN18.2; preferably, said substitution is a conservative substitution.

[0021] In some embodiments, the CDRs are defined according to the IMGT or AbM numbering system.

[0022] In some embodiments, the antibody or antigen binding fragment thereof further comprises framework regions (FRs) from a human immunoglobulin.

[0023] In some embodiments, the antibody or antigen binding fragment thereof comprises:

[0024] (1-1) three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 of SEQ ID No: 1, CDR-H2 of SEQ ID Nos: 2 or 21, and CDR-H3 of SEQ ID No: 3; and / or,

[0025] three light chain CDRs defined according to the IMGT numbering system: CDR-L1 of SEQ ID No: 4, CDR-L2 of SEQ ID No: 5, and CDR-L3 of SEQ ID No: 6;

[0026] or,

[0027] (1-2) three heavy chain CDRs as in (1-1), or a variant thereof that contains an amino acid mutation as compared to (1-1), and / or,

[0028] three light chain CDRs as in (1-1), or a variant thereof that contains an amino acid mutation as compared to (1-1), and / or,

[0029] wherein at least one of the three heavy chain CDRs and / or three light chain CDRs contains an amino acid mutation as compared to the corresponding CDR in (1-1), the amino acid mutation being a substitution, deletion, or addition of one or several amino acids (e.g. a substitution, deletion, or addition of 1, 2, or 3 amino acids); the antibody or antigen binding fragment thereof containing the mutation still being capable of specifically binding to human CLDN18.2; preferably the substitution is a conservative substitution;

[0030] or,

[0031] (2-1) three heavy chain CDRs defined by the AbM numbering system: CDR-H1 of SEQ ID No: 7, CDR-H2 of SEQ ID Nos: 8 or 22, and CDR-H3 of SEQ ID No: 9; and / or,

[0032] three light chain CDRs defined by the AbM numbering system: CDR-L1 of SEQ ID No: 10, CDR-L2 of SEQ ID No: 11, and CDR-L3 of SEQ ID No: 12;

[0033] or,

[0034] (2-2) three heavy chain CDRs as in (2-1), or a variant thereof that contains an amino acid mutation as compared to (2-1), and / or,

[0035] three light chain CDRs as described in (2-1) or a variant thereof containing amino acid mutations compared to (2-1), (2-1) or a variant thereof containing amino acid mutations compared to (2-1), and (2-1) or a variant thereof containing amino acid mutations compared to (2-1);

[0036] wherein at least one of the three heavy chain CDRs and / or three light chain CDRs contains amino acid mutations compared to the corresponding CDRs in (2-1), the amino acid mutations being one or more amino acid substitutions, deletions, or additions (e.g. 1, 2, or 3 amino acid substitutions, deletions, or additions); the antibody or antigen binding fragment thereof containing the mutations still specifically binds to human CLDN18.2; preferably the substitutions are conservative substitutions.

[0037] In some embodiments, the antibody or antigen binding fragment thereof further comprises framework regions (FRs) from a human immunoglobulin.

[0038] In some embodiments, the antibody or antigen binding fragment thereof comprises:

[0039] (1) a VH and / or VL as described below, wherein the CDRs are defined according to the IMGT numbering system:

[0040] (1-1): a VH comprising the following 3 CDRs: CDR-H1 of SEQ ID No: 1, CDR-H2 of SEQ ID Nos: 2 or 21, and CDR-H3 of SEQ ID No: 3; and / or,

[0041] a VL comprising the following 3 CDRs: CDR-L1 of SEQ ID No: 4, CDR-L2 of SEQ ID No: 5, and CDR-L3 of SEQ ID No: 6;

[0042] or,

[0043] (1-2): at least one CDR contains mutations compared to the VH or VL described in (1-1), the mutations being one or more amino acid substitutions, deletions, or additions or any combination thereof (e.g. 1, 2, or 3 amino acid substitutions, deletions, or additions or any combination thereof); preferably the substitutions are conservative substitutions; the antibody or antigen binding fragment thereof containing the mutations still specifically binds to CLDN18.2; preferably specifically binds to human CLDN18.2;

[0044] or,

[0045] (2) a VH and / or VL as described below, wherein the CDRs are defined according to the AbM numbering system:

[0046] (2-1): a VH comprising 3 CDRs: CDR-H1 of SEQ ID No: 7, CDR-H2 of SEQ ID Nos: 8 or 22, and CDR-H3 of SEQ ID No: 9; and / or,

[0047] a VL comprising 3 CDRs: CDR-L1 of SEQ ID No: 10, CDR-L2 of SEQ ID No: 11, and CDR-L3 of SEQ ID No: 12;

[0048] or,

[0049] (2-2): at least one CDR contains a mutation compared to the VH or VL described in (2-1), the mutation being a substitution, a deletion or an addition of one or several amino acids or any combination thereof (e.g. a substitution, a deletion or an addition of 1, 2 or 3 amino acids or any combination thereof); preferably, the substitution is a conservative substitution; the antibody or antigen-binding fragment thereof containing the mutation still specifically binds to CLDN18.2; preferably, specifically binds to human CLDN18.2.

[0050] In some embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof comprises a framework region (FRs) from a human or murine immunoglobulin.

[0051] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0052] (1) a VH of SEQ ID NO: 13 or 14; and / or, a VL of SEQ ID NO: 15;

[0053] or,

[0054] (2) the VH comprised by 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 to the VH as set forth in (1); and / or, the VL comprised by 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 to the VL as set forth in (1); the antibody or antigen-binding fragment thereof containing the identical sequences still specifically binds CLDN18.2; preferably, specifically binds human CLDN18.2;

[0055] Alternatively,

[0056] (3) the VH comprised by the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) or any combination thereof as compared to the VH as set forth in (1); and / or, the VL comprised by the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) or any combination thereof as compared to the VL as set forth in (1); preferably, the substitutions are conservative substitutions. The antibody or antigen-binding fragment thereof containing the mutations still specifically binds CLDN18.2; preferably, specifically binds human CLDN18.2.

[0057] 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.

[0058] In some embodiments, the antibody or comprises:

[0059] (1) a CH (heavy chain constant region) of a human immunoglobulin or a variant thereof having 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) as compared to the wild-type sequence from which it is derived; and / or,

[0060] (2) a CL (light chain constant region) of human immunoglobulin or a variant thereof having one or more substitutions, deletions or additions of amino acids compared to the wild type sequence from which it is derived (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).

[0061] In some embodiments, the constant region is altered, e.g., mutated, to modify properties of the anti-CLDN18.2 antibody molecule (e.g., to alter one or more of the following properties: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function). Functional changes can be made by replacing at least one amino acid residue in the antibody constant region with a different residue, e.g., to change the affinity of the antibody for an effector ligand (e.g., an FcR or complement Clq), thereby altering effector function (e.g., enhancing it). In some embodiments, the constant region is altered to alter (e.g., enhance) antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular phagocytosis (ADCP).

[0062] In some embodiments, the CH is an IgG heavy chain constant region, e.g., an IgGl, IgG2, IgG3, or IgG4 heavy chain constant region.

[0063] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgGl.

[0064] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CH as set forth in SEQ ID NO: 16 or a variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4 or 5 conservative substitutions) of amino acids compared to SEQ ID NO: 16. The CH containing the mutations retains substantially the same function as SEQ ID NO: 16.

[0065] In some embodiments, the CL is selected from a light chain constant region of kappa or lambda.

[0066] In some embodiments, the CL is a kappa light chain constant region (e.g., a human kappa light chain).

[0067] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CL as set forth in SEQ ID NO: 17, or a variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids) compared to SEQ ID NO: 17. The CL containing the mutations retains substantially the same function as SEQ ID NO: 17.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CH as set forth in SEQ ID NO: 16 and / or a CL as set forth in SEQ ID NO: 17.

[0069] In some embodiments, the antibody comprises a heavy chain consisting of a VH as set forth in SEQ ID NO: 13 and a CH as set forth in SEQ ID NO: 16, and,

[0070] a light chain consisting of a VL as set forth in SEQ ID NO: 15 and a CL as set forth in SEQ ID NO: 17.

[0071] In some embodiments, the antibody comprises a heavy chain consisting of a VH as set forth in SEQ ID NO: 14 and a CH as set forth in SEQ ID NO: 16, and,

[0072] a light chain consisting of a VL as set forth in SEQ ID NO: 15 and a CL as set forth in SEQ ID NO: 17.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0074] (1) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0075] (1-1) the sequence set forth in SEQ ID NO: 18;

[0076] (1-2) a sequence having one or several (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

[0077] (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% sequence identity to the sequence set forth in SEQ ID NO: 18;

[0078] and

[0079] (2) a light chain comprising an amino acid sequence selected from the group consisting of:

[0080] (2-1) a sequence represented by SEQ ID NO: 20;

[0081] (2-2) a sequence having one or several substitutions, deletions, or additions of amino acids (e.g., 1, 2, 3, 4, or 5 substitutions, deletions, or additions of amino acids) compared to the sequence represented by SEQ ID NO: 20; or

[0082] (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% sequence identity to the sequence represented by SEQ ID NO: 20.

[0083] In some embodiments, the substitutions described in (1-2) and (2-2) above are conservative substitutions. The CH containing the mutation retains substantially the same function as SEQ ID NO: 16.

[0084] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0085] (1) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0086] (1-1) a sequence represented by SEQ ID NO: 19;

[0087] (1-2) a sequence having one or several substitutions, deletions, or additions of amino acids (e.g., 1, 2, 3, 4, or 5 substitutions, deletions, or additions of amino acids) compared to the sequence represented by SEQ ID NO: 19; or

[0088] (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% sequence identity to the sequence represented by SEQ ID NO: 19;

[0089] and

[0090] (2) a light chain comprising an amino acid sequence selected from the group consisting of:

[0091] (2-1) a sequence represented by SEQ ID NO: 20;

[0092] (2-2) a sequence having one or several substitutions, deletions, or additions of amino acids (e.g., 1, 2, 3, 4, or 5 substitutions, deletions, or additions of amino acids) compared to the sequence set forth in SEQ ID NO: 20; or

[0093] (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% sequence identity to the sequence set forth in SEQ ID NO: 20.

[0094] In some embodiments, the substitutions described in (1-2) and (2-2) above are conservative substitutions. Preferably, the antibodies or antigen-binding fragments thereof containing the mutated or identical sequences described above are still capable of specifically binding to human CLDN18.2.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of ScFv, Fab, Fab’, (Fab’)2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody, and multispecific antibody.

[0096] In some embodiments, the (D-L) γ The structure A is as shown in formula (II):

[0097] {D-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A

[0098] Formula (II)

[0099] wherein,

[0100] L1 is wherein, each R1and R2is independently hydrogen (e.g., protium or deuterium), halogen, carboxylic acid, sulfonic acid, cyano, C 1-6 alkyl, halogenated C 1-6 alkyl, cyano-substituted C 1-6 alkyl (e.g., -CH2CN), C 1-6 alkoxy, C 2-10 alkenyl, or C 2-10 alkynyl; Z1is an amino acid or a peptide consisting of 2-10 amino acids; x1and x2are each independently 0, 1, 2, 3, 4, 5, or 6; and D is connected to position 1 of L1, and L2is connected to position 2 of L1;

[0101] L2is wherein y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and L2 is attached at position 1 to L1 and at position 2 to L3;

[0102] L3 is selected from a 5-12 membered heteroaromatic ring;

[0103] L4 is wherein Z2 is selected from 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 or deuterium) and C 1-6 alkyl; Z3 is absent or selected from C 1-6 alkylene; or, R3 together with Z3 and the nitrogen atom to which they are attached form a 4-8 membered heterocyclyl; a is 0, 1, 2, 3, 4, 5, or 6, and L4 is attached at position 2 to E and at position 1 to L3;

[0104] E is wherein each R4 is independently hydrogen (e.g., protium or deuterium), b is 0, 1, 2, 3, 4, 5, or 6, and E is attached at position 2 to A and at position 1 to L4;

[0105] m1, m2, and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0106] A, D, and g are as previously described.

[0107] In some embodiments, the (D-L) γ -A structure is as shown in formula (III):

[0108] {D-[(L1’ m4 -L1-(L5) m5 -(L3) m2 -(L4) m3 -E]} γ -A

[0109] formula (III)

[0110] wherein,

[0111] L1’ is wherein R5 and R6 are each independently hydrogen (e.g., protium or deuterium) or C 1-6 alkyl; x3 is 1, 2, 3, 4, 5, or 6; and, if present, L1’ is attached at position 1 to D and at position 2 to L1;

[0112] L1 is wherein each R1and R2is independently hydrogen (e.g., protium or deuterium), halogen, carboxylic acid, sulfonic acid, cyano, C 1-6 alkyl, haloC 1-6 alkyl, cyano-substituted C 1-6 alkyl (e.g., -CH2CN), C 1-6 alkoxy, C 2-10 alkenyl, or C 2-10 alkynyl; Z1is an amino acid or a peptide consisting of 2-10 amino acids; each of x1and x2is independently 0, 1, 2, 3, 4, 5, or 6; and, L1is connected to L1’ at position 1 (when L1’ is present), or L1is connected to D at position 1 (when L1’ is not present);

[0113] L5is wherein R7is hydrogen or C 1-6 alkyl, or R7is connected to the N atom on the γ-C to form a 5-6 membered heterocyclyl; x4is 1, 2, 3, 4, 5, or 6; y1is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and L5is connected to L1at position 1 and L3at position 2;

[0114] L3is selected from a 5-12 membered heteroaromatic ring;

[0115] L4is wherein Z2is selected from C 1-6 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, and C 3-8 cycloalkylene; R3is selected from hydrogen (e.g., protium or deuterium) and C 1-6 alkyl; Z3is absent or selected from C 1-6 alkylene; or, R3is connected with Z3together with the nitrogen atom to which they are attached to form a 4-8 membered heterocyclyl; a is 0, 1, 2, 3, 4, 5, or 6, and L4is connected to E at position 2 and L3at position 1;

[0116] E is wherein each R4is independently hydrogen (e.g., protium or deuterium), b is 0, 1, 2, 3, 4, 5, or 6, and E is connected to A at position 2 and L4at position 1;

[0117] each of m1, m2, m3, and m4is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0118] A, D, and g are as previously described.

[0119] In some embodiments, L1in the antibody-drug conjugate of Formula (II) or Formula (III) is wherein Z1is an amino acid or a peptide consisting of 2-5 amino acids, wherein the amino acids are selected from the group consisting of Lys, Cit, Val, D-Val, Phe, Leu, Gly, Ala and Asn; preferably Z1is selected from the group consisting of Cit, Lys, Cit-Val and Ala-Val.

[0120] In some embodiments, L1is

[0121] In some embodiments, L1is

[0122] In some embodiments, L2in the antibody-drug conjugate of formula (II) is and m1is 1.

[0123] In some embodiments, L3in the antibody-drug conjugate of formula (II) or formula (III) is a 5-6 membered heteroaromatic ring, and m2is 1.

[0124] In some embodiments, L3is a triazole, and m2is 1.

[0125] In some embodiments, L4in the antibody-drug conjugate of formula (II) or formula (III) is wherein Z2is C 1-6 alkylene, Z3is C 1-6 alkylene; and m3is 1.

[0126] In some embodiments, L4is and m3is 1.

[0127] In some embodiments, L1'in the antibody-drug conjugate of formula (III) is

[0128] In some embodiments, L5in the antibody-drug conjugate of formula (III) is wherein x4is 1, 2, 3, 4, 5 or 6; y1is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0129] In some embodiments, L5in the antibody-drug conjugate of formula (III) is

[0130] In some embodiments, D in the antibody-drug conjugate of formula (I) is

[0131] In some embodiments, D in the antibody-drug conjugate of formula (II) is

[0132] In some embodiments, D in the antibody-drug conjugate of formula (III) is

[0133] In some embodiments, D in the antibody-drug conjugate of formula (II) is -[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]- is

[0134] In some embodiments, D in the antibody-drug conjugate of formula (III) is -[(L1) m4 -L1-(L5) m5 -(L3) m2 -(L4) m3 -E]- is

[0135] In some embodiments, the antibody-drug conjugate of formula (II) has the structure:

[0136]

[0137] wherein γ is an integer selected from 1-10, e.g., γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and A is an antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2 as described above.

[0138] In some embodiments, the antibody-drug conjugate of formula (II) has the structure:

[0139]

[0140] wherein γ is an integer selected from 1-10, e.g., γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and A is 2C6.9-hz21.

[0141] In some embodiments, the antibody-drug conjugate of formula (III) has the structure:

[0142]

[0143] wherein γ is an integer selected from 1-10, e.g., γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and A is an antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2 as described above.

[0144] In some embodiments, the antibody-drug conjugate of formula (III) has the structure:

[0145]

[0146] wherein 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.

[0147] In some embodiments, the antibody-drug conjugate of formula (III) has the structure:

[0148]

[0149] wherein 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 the aforementioned antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2.

[0150] In some embodiments, the antibody-drug conjugate of formula (III) has the structure:

[0151]

[0152] wherein 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.

[0153] The present disclosure also provides a composition comprising one or more of the above antibody-drug conjugates, wherein the molar ratio (DAR value) of the biologically active molecule fragment (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)) in the composition is a fraction or an integer between 1 and 10 (e.g., a fraction or an integer between 1 and 8, for example, 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.79, 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).

[0154] The DAR value, also known as antibody drug conjugate ratio, refers to the number of (D-L) connected to each antibody on average, which can be determined and calculated by methods known in the art. For example, by LC-MS, HIC, etc. to determine the molecular weight of the antibody and (D-L) after conjugation, respectively, to calculate the proportion of light chains and heavy chains conjugated with different number of (D-L), and to calculate the DAR value by the formula DAR=(light chain DAR1*1+…+light chain DARn1*n1)*2+(heavy chain DAR1*1+…+DARn2*n2)*2; wherein DARn1 represents the proportion of light chain conjugated with n1 (D-L) in the light chain part, and DARn2 represents the proportion of heavy chain conjugated with n2 (D-L) in the heavy chain part.

[0155] In some embodiments, the composition further comprises an antibody or antigen binding fragment thereof as described above.

[0156] In some embodiments, the composition comprises 2C6.9-TL001. In some embodiments, the composition is 2C6.9-TL001.

[0157] In some embodiments, the composition comprises 2C6.9-TL002. In some embodiments, the composition is 2C6.9-TL002.

[0158] In some embodiments, the composition comprises 2C6.9-TL003. In some embodiments, the composition is 2C6.9-TL003.

[0159] The antibodies or antigen binding fragments thereof of the present application can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). Typically, derivatization (e.g., labeling) of the antibodies or antigen binding fragments thereof does not adversely affect its binding to CLDN18.2, in particular human CLDN18.2. Thus, the antibodies or antigen binding fragments thereof of the present application are also intended to include such derivatized forms.

[0160] One type of derivatized antibody (e.g., bispecific antibodies) is produced by cross-linking two or more antibodies (of the same or different types). Methods for obtaining bispecific antibodies are well known in the art, examples of which include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybrid hybridoma methods), or genetic engineering methods.

[0161] Another type of derivatized antibody is a labeled antibody. For example, the antibodies of the present application, or antigen-binding fragments thereof, can be linked to detectable labels. The detectable label described herein can be any substance that can be detected by a fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art and include but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein (FITC), fluorescein, tetramethylrhodamine (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dot, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), acridinium esters, magnetic beads (e.g., Dynal® ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for use with avidin reagents (e.g., streptavidin) modified to bind the above labels. Patents teaching the use of such labels include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). Detectable labels as described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using photodetectors to detect emitted light, enzymatic labels generally are detected by providing the enzyme with its substrate and detecting the reaction product, and calorimetric labels are detected by simple visual observation of color change markers. In certain embodiments, such labels are capable of being adapted for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In certain embodiments, detectable labels as described above can be attached to the antibodies of the present application, or antigen-binding fragments thereof, through linkers of varying lengths to reduce potential steric hindrance.

[0162] In certain embodiments, the antibodies or antigen-binding fragments thereof comprised in the antibody-drug conjugates of the present application are one or more of the foregoing derivatized antibodies or antigen-binding fragments thereof.

[0163] Therapeutic methods and pharmaceutical compositions

[0164] In another aspect, the present application also provides pharmaceutical compositions.

[0165] In some embodiments, the pharmaceutical compositions contain one or more of the above-described antibody-drug conjugates or compositions.

[0166] In some preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0167] In some preferred embodiments, the pharmaceutical composition further comprises another component having anti-tumor activity. In some embodiments, the antibody-drug conjugate or composition and the other component having anti-tumor activity are in the same formulation unit, or in different formulation units. Thus, the antibody-drug conjugate or composition of the present application and the other component having anti-tumor activity can be administered simultaneously, separately or sequentially.

[0168] In some embodiments, the other component having anti-tumor activity is a bioactive polypeptide or an active fragment thereof, or a chemotherapeutic drug. In some embodiments, the bioactive polypeptide is selected from an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody), or a cytokine (e.g., an interferon, IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, IL-21). In some embodiments, the chemotherapeutic drug is selected from one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, and nab-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, folinic acid, and 5-fluorouracil.

[0169] In another aspect, the antibody-drug conjugate or composition in the pharmaceutical composition of the present application is sufficient to:

[0170] (a) induce apoptosis of tumor cells, especially gastric cancer cells, e.g., gastric adenocarcinoma cells;

[0171] (b) inhibit proliferation of tumor cells, especially gastric cancer cells, e.g., gastric adenocarcinoma cells;

[0172] (c) induce and / or increase complement-dependent cytotoxicity activity;

[0173] (d) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0174] (e) inhibit expression and activation of CLDN18.2;

[0175] (f) inhibit CLDN18.2-mediated signaling; or

[0176] (g) any combination of (a)-(f).

[0177] In another aspect, the present application provides the use of the antibody-drug conjugate, the composition, the pharmaceutical composition for the preparation of a medicament for the prevention and / or treatment and / or adjuvant therapy of a tumor.

[0178] In some embodiments, the tumor is selected from a solid tumor, a hematological tumor, and a metastatic, refractory or recurrent lesion of a cancer.

[0179] In some embodiments, the tumor or cancer is selected from esophageal cancer, gastrointestinal cancer, gastric adenocarcinoma, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric 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 cancer, bone cancer, skin cancer, thymus cancer, cholangiocarcinoma, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia.

[0180] In some embodiments, the tumor is selected from gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer).

[0181] In some embodiments, the tumor is gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma, e.g., locally advanced unresectable or metastatic gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma.

[0182] In some embodiments, the tumor is CLDN18.2 positive, further, the tumor is HER2 negative.

[0183] In some embodiments, the tumor is HER2 negative.

[0184] In another aspect, the present application provides a method of preventing and / or treating a tumor in a subject. In another aspect, the present application provides a method of delaying progression of a tumor in a subject. In another aspect, the present application provides a method of reducing or inhibiting recurrence of a tumor in a subject. The above-mentioned methods comprise administering to a subject in need thereof an effective amount of the antibody-drug conjugate, the composition, or the pharmaceutical composition of the present application.

[0185] In some embodiments, the above-mentioned methods further comprise administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0186] In some embodiments, the second therapy can be applied separately or in conjunction with the methods described above; or, the second therapy can be applied simultaneously or sequentially with the methods described above.

[0187] In some embodiments, the second therapy is chemotherapy. In some embodiments, the drug of the chemotherapy is selected from one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, and nab-paclitaxel. In some embodiments, the drug of the chemotherapy is a combination of epirubicin, oxaliplatin, and 5-fluorouracil, or a combination of oxaliplatin, folinic acid, and 5-fluorouracil. In some embodiments, the combination of oxaliplatin, folinic acid, and 5-fluorouracil is administered in a regimen selected from FOLFOX4, FOLFOX6, or mFOLFOX6.

[0188] In some embodiments, the second therapy is immunotherapy. In some embodiments, the drug of the immunotherapy is selected from an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody), or a cytokine (e.g., an interferon, IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, IL-21).

[0189] In some embodiments, the tumor is selected from a solid tumor, a hematological tumor, and a metastatic, refractory, or recurrent lesion of a cancer.

[0190] In some embodiments, the tumor or cancer is selected from esophageal cancer, gastrointestinal cancer, gastric adenocarcinoma, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric 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 cancer, bone cancer, skin cancer, thymus cancer, cholangiocarcinoma, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia.

[0191] 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., non-small cell lung cancer).

[0192] In some embodiments, the tumor is gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma, e.g., locally advanced unresectable or metastatic gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma.

[0193] In some embodiments, the tumor is CLDN18.2 positive, further, the tumor is HER2 negative.

[0194] In some embodiments, the tumor is HER2 negative.

[0195] The antibody-drug conjugate, composition or pharmaceutical composition of the present application can be formulated into any dosage forms known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form will depend on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the antibody-drug conjugate or composition in the required amount in an appropriate solvent with one or more of the other ingredients, as described below, followed by filtered sterilization. Furthermore, sterile injection solutions can be prepared as sterile lyophilized powders for reconstitution with a suitable solvent (e.g., water for injection) prior to use. Such lyophilized powders can be prepared from an aqueous solution by the process of freeze-drying (lyophilization) to remove the water.

[0196] In addition, the antibody-drug conjugate of the present application can be present in unit dosage form in a pharmaceutical composition to facilitate administration.

[0197] The antibody-drug conjugate, composition or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracellular web, inguinal, intravesical, local (e.g., powder, salve or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In a preferred embodiment, the antibody-drug conjugate, composition or pharmaceutical composition of the present application is administered by intravenous infusion or injection.

[0198] The pharmaceutical compositions of the present application can include a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody-drug conjugate or composition of the present application. A "prophylactically effective amount" means an amount that is sufficient to prevent, hinder, or delay the onset of a disease. A "therapeutically effective amount" means an amount that is sufficient to cure or at least partially arrest the disease and its complications in an individual already suffering from a disease. The therapeutically effective amount of the antibody-drug conjugate or composition of the present application can vary according to factors such as the disease severity, the overall state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of the drug, and other therapies being administered concurrently, and the like.

[0199] In the present application, the dosage regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single dose can be administered, doses can be administered several times over a period of time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0200] A typical non-limiting range for a therapeutically or prophylactically effective amount of the antibody-drug conjugate or composition of the present application is 0.02 to 100 mg / kg, such as 0.1 to 100 mg / kg, 0.1 to 50 mg / kg, or 1 to 50 mg / kg. It is noted that dosage values can vary with the type and severity of the condition to be alleviated. It is to be expected that the specific dosage regimen for any particular patient will vary according to the patient's needs and the professional judgment of the medical practitioner and that the dosage ranges set forth herein are exemplary only. The dosages given herein are based on an adult human patient, unless otherwise noted.

[0201] In the present application, the subject can be a mammal, such as a human.

[0202] Abbreviations

[0203] Complementarity-determining region in CDR immunoglobulin variable region

[0204] FR antibody framework region: amino acid residues in an antibody variable region other than CDR residues

[0205] VH antibody heavy chain variable region

[0206] VL antibody light chain variable region

[0207] IgG immunoglobulin G

[0208] AbM AbM CDR definition is derived from Martin's related study (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), which integrates parts of both the Kabat and Chothia definitions.

[0209] Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0210] Chothia The immunoglobulin numbering system proposed by Chothia et al., which is a canonical rule for identifying the boundaries of CDR regions based on the location of structural loops (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0211] IMGT The numbering system based on the international ImMunoGeneTics information system (The international ImMunoGeneTics information (IMGT)) initiated by Lefranc et al., which can be found in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0212] mAb Monoclonal antibody

[0213] EC50 Concentration that produces 50% efficacy or binding

[0214] HRP Horseradish peroxidase

[0215] CDR-H1 Complementarity determining region 1 in an immunoglobulin heavy chain variable region

[0216] CDR-H2 Complementarity determining region 2 in an immunoglobulin heavy chain variable region

[0217] CDR-H3 Complementarity determining region 3 in an immunoglobulin heavy chain variable region

[0218] complementarity determining region 1 in a CDR-L1 immunoglobulin light chain variable region

[0219] complementarity determining region 2 in a CDR-L2 immunoglobulin light chain variable region

[0220] complementarity determining region 3 in a CDR-L3 immunoglobulin light chain variable region

[0221] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, the experimental operations of cell culture, biochemistry, nucleic acid chemistry, immunology, etc. used herein are the operations commonly used in the corresponding fields unless otherwise specified. Meanwhile, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0222] As used herein, the terms "conjugate" or "conjugate" can be interchangeable, referring to a substance obtained by linking a bioactive molecule to an antibody through a linker, and the English name is ADC (antibody-drug conjugate).

[0223] The linker can be linked to the antibody through various chemical bonds. For example, in some embodiments, the linker is linked by forming a sulfide bond with the thiol group of the antibody. In the structural formula of some specific ADC molecules (such as ADC molecules in 2C6.9-TL001, 2C6.9-TL002, or 2C6.9-TL003), -S- only represents the sulfide bond formed by the linker and the thiol group of the antibody, and does not represent that -S- is a part of the linker.

[0224] The structural formula of the ADC of the present application can be (D-L) γ -A represents, wherein, D is a fragment of a bioactive molecule; L is a linker; A is an antibody or an antigen binding fragment thereof that specifically binds to human CLDN18.2; γ is selected from an integer between 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. γ refers to the number of (D-L) linked to each antibody molecule, which is an integer. However, during the preparation of the ADC, each antibody molecule can be linked to different numbers of (D-L), so generally speaking, the product of the ADC is a composition of antibodies conjugated with different numbers of (D-L). In practice, the average number of (D-L) linked to the antibody is usually represented by DAR.

[0225] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally comprised of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be assigned to a kind, kappa (kappa) and lambda (lambda) light chains. The heavy chains can be assigned to a class that defines the isotype of an antibody, mu, delta, gamma, alpha, or epsilon, and respectively IgM, IgD, IgG, IgA, and IgE. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains do not directly participate in antibody binding to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair, VH and VL, form the antigen binding site. Assignment of amino acids to each region or domain can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883, or AbM, a related study by Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). Herein, the term "antibody" includes not only intact antibodies, but also antigen binding fragments of antibodies, unless the context clearly indicates otherwise.

[0226] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, e.g., according to the Rabat numbering system (Rabat 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 & 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 the related work of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), which integrates parts of both Rabat and Chothia definitions, as first applied in the Oxford Molecular 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, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. And, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0227] The CDRs contained by the antibodies of the application, or antigen-binding fragments thereof, can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies of the application, or antigen-binding fragments thereof, are preferably determined by the IMGT or AbM numbering system.

[0228] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in a variable region of an antibody that are not CDR residues as defined above.

[0229] As used herein, the term "antigen binding fragment" of an antibody refers to a polypeptide that is a fragment of an antibody, e.g., a fragment of a polypeptide of a full length antibody, that retains the ability to specifically bind to the same antigen bound by the full length antibody to which the fragment of the polypeptide is derived from, and / or competes with the full length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact 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 that contain at least a portion of an antibody that is sufficient to confer specific antigen binding capacity to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0230] As used herein, the term "full length antibody" means an antibody that is composed of two "full length heavy chains" or "heavy chains" and two "full length light chains" or "light chains". Wherein, a "full length heavy chain" or "heavy chain" refers to a polypeptide chain that is composed 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, a heavy chain constant region CH3 domain in the direction from N-terminus to C-terminus; and, optionally, a heavy chain constant region CH4 domain when the full length antibody is of IgE isotype. Preferably, a "full length heavy chain" is a polypeptide chain that is composed of VH, CH1, HR, CH2 and CH3 in the direction from N-terminus to C-terminus. A "full length light chain" or "light chain" is a polypeptide chain that is composed of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full length heavy chains. The full length antibody of the present application can be from a single species, e.g., human; can also be a chimeric antibody or a humanized antibody. The full length antibody of the present application contains two antigen binding sites formed by a pair of VH and VL, respectively, which specifically recognize / bind to the same antigen.

[0231] As used herein, the term "Fd fragment" means an antibody fragment consisting of VH and CHI domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341 :544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CHI domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region; the term "Fab' fragment" means a fragment obtained after reduction of the disulfide bond connecting the two heavy chain fragments in an F(ab')2 fragment, consisting of one complete light chain and a Fd fragment (consisting of VH and CHI domains) of a heavy chain.

[0232] As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. An Fv fragment is generally considered to be the smallest antibody fragment that is capable of forming a complete antigen binding site. It is generally believed that the six CDRs confer the antigen binding specificity of an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding an antigen, although its affinity can be lower than that of the complete binding site.

[0233] As used herein, the term "Fc fragment" means an antibody fragment formed by disulfide bond binding of the second, third constant regions of the first heavy chain with the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

[0234] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., 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 can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4may be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present application are described by 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 the linkage of two scFv.

[0235] As used herein, the term "diabody" means that the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complement domains of 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 R.J. et al., Structure 2:1121-1123 (1994)).

[0236] Each of the above antibody fragments is capable of specifically binding to the same antigen bound by the full-length antibody from which it is derived, and / or competes with the full-length antibody for specific binding to the antigen.

[0237] As used herein, "bispecific antibody" refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody mimetic through a coupling arm, the coupling can include but not limited to chemical reaction, genetic fusion, protein fusion, polypeptide fusion and enzymatic. "Multispecific antibody" includes, for example: trispecific antibody and tetraspecific antibody, the former is an antibody with three different antigen binding specificities, and the latter is an antibody with four different antigen binding specificities.

[0238] As used herein, "antibody mimetics" refers to molecules that specifically bind to an antigen like an antibody, but do not have the structure of an antibody. They are usually artificial peptides or proteins, with a molecular weight of about 3 to 20 kDa. For example, ankyrin repeat proteins (DARPin) and fynomer. Designed ankyrin repeat proteins (DARPin) are connected to IgG antibodies, scFv-Fc antibody fragments or combinations thereof, such as CN104341529A. Anti-IL-17a fynomer is combined with anti-IL-6R antibody, such as WO2015141862A1.

[0239] In this context, the techniques for obtaining antibodies can be used to obtain antigen-binding fragments of antibodies (e.g., the antibody fragments described above) from a given antibody (e.g., the antibodies provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and screening the antigen-binding fragments of antibodies for specificity in the same manner as for the intact antibody.

[0240] As used herein, the terms "monoclonal antibody", "monoclonal", "mAb" have the same meaning and are used interchangeably, which refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, a population of identical antibody molecules except for natural mutations that can occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least 2 or more different antibodies, which usually recognize different epitopes on the antigen. In addition, the modifier "monoclonal" only indicates that the characteristics of the antibody are obtained from a population of highly homologous antibodies, and cannot be understood as requiring the antibody to be prepared by any specific method.

[0241] Monoclonal antibodies of the present application can be prepared by a variety of techniques, such as the hybridoma technique (see, e.g., Kohler et al. Nature, 256:495, 1975), recombinant DNA techniques (see, e.g., U.S. Patent Application 4,816,567), or phage library techniques (see, e.g., Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0242] For example, monoclonal antibodies can be produced as follows. Mice or other suitable host animals are first immunized with the immunogen, if necessary with the addition of an adjuvant. The immunogen or adjuvant is usually injected subcutaneously at multiple sites or intraperitoneally. The immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After the immunization, the host animal will produce lymphocytes that secrete antibodies that specifically bind to the immunogen. Alternatively, the lymphocytes can be obtained by in vitro immunization. The desired lymphocytes are collected and fused with myeloma cells using a suitable fusing agent, such as PEG, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The hybridoma cells thus produced are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells. The preferred myeloma cells should have high fusion efficiency, and the ability to grow for a prolonged period in culture. Among these, mouse myeloma lines, such as MOP-21 or MC-11 mouse tumor-derived strains (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md. USA) are preferred. There are also reports of the use of human myeloma and human-mouse heteromyeloma cell lines to produce 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). The culture medium in which the hybridoma cells are grown is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by, for example, immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), etc.For example, the affinity of the monoclonal antibodies can be determined using the method of Scatchard, Anal. Biochem. 107:220 (1980), as described by Munson et al. After the specificity, affinity, and reactivity of the antibodies produced by the hybridomas are determined, the cell lines can be subcloned by limiting dilution procedures (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996) to obtain clones which are phenotypically homogeneous. Suitable culture media can be DMEM or RPMI-1640 medium. Also, the hybridoma cells can be grown in vivo in the peritoneal cavity of the mouse. The monoclonal antibodies present in the ascitic fluid or the cell culture media can be separated from the cell culture medium by conventional immunoglobulin purification procedures.

[0243] Monoclonal antibodies can also be produced by recombinant DNA techniques. DNA molecules encoding the heavy and light chains of the monoclonal antibodies can be isolated from the hybridoma cells by PCR amplification using nucleic acid primers specific for the heavy and light chain genes. The resulting DNA molecules can be inserted into expression vectors, which are 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 suitable conditions to obtain recombinant expression of the desired antibodies.

[0244] Antibodies can be purified by 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 an antigenic epitope thereof can be immobilized on a column and the immunospecific antibodies can be purified by immunoaffinity chromatography. The purification of immunoglobulins can be found, for example, in D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0245] As used herein, the term "murine antibody" refers to the production of B cells from immunized mice, fusion of the B cells with myeloma cells, selection of the murine hybrid cells that both proliferate indefinitely and secrete antibodies, and screening, antibody production, and antibody purification. Alternatively, the production of antibodies is due to the differentiation and proliferation of B cells into plasma cells after the invasion of the antigen into the mouse. The production of antibodies is due to the differentiation and proliferation of B cells into plasma cells after the invasion of the antigen into the human, which is due to the interaction of various immune cells after the invasion of the antigen into the human, which is due to the differentiation and proliferation of B cells into plasma cells after the invasion of the antigen into the human.

[0246] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or / and the heavy chain is derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass) and the other portion of the light chain or / and the heavy chain is derived from another antibody (which can be derived from the same or a different species or belong to the same or a different antibody class or subclass), but which retains the binding activity of the target antigen (U.S. P4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :68516855 (1984)). For example, the term "chimeric antibody" can include an antibody (e.g., a human-mouse chimeric antibody) in which the variable regions of both the heavy and light chains are derived from a first antibody (e.g., a mouse antibody), and the variable regions of both the heavy and light chains are derived from a second antibody (e.g., a human antibody).

[0247] As used herein, the term "humanized antibody" refers to a non-human-derived antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human-derived antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) from a human-derived immunoglobulin (acceptor antibody). A humanized antibody typically retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance an immune response, etc. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody that has the desired property (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and / or ability to enhance an immune response).

[0248] Humanized antibodies are particularly advantageous because they can retain the desired properties of a non-human-derived donor antibody (e.g., a mouse antibody) while effectively reducing the immunogenicity of the non-human-derived donor antibody (e.g., a mouse antibody) in a human subject. However, the desired properties of a humanized antibody (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and / or ability to enhance an immune response) are typically less than those of the non-human-derived donor antibody (e.g., a mouse antibody) due to the mismatch between the CDRs of the donor antibody and the FRs of the acceptor antibody.

[0249] Thus, while researchers in the art have made significant progress in the humanization of antibodies (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 art does not provide detailed guidance on how to humanize a particular donor antibody to produce a humanized antibody that is both as humanized as possible and retains as much of the desired properties of the donor antibody as possible. The skilled artisan must engage in trial and error, exploration and tinkering, and expend significant creative labor to arrive at a humanized antibody that is both highly humanized (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% humanized) and retains the desired properties of the particular donor antibody.

[0250] In the present application, in order to make the humanized antibody retain the properties of the donor antibody as much as possible (including, for example, antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and / or the ability to enhance immune response), the framework region (FR) in the humanized antibody of the present application can contain both the amino acid residues of the human source recipient antibody and the amino acid residues of the corresponding non-human source donor antibody.

[0251] The humanized antibodies of the present application can be prepared according to the sequences of the murine monoclonal antibodies prepared above. DNA encoding the heavy and light chains can be obtained from the target murine hybridoma and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0252] To create a chimeric antibody, the murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). For example, the DNA encoding the VHand 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 regions are known in the art (see, e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encoding them can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is typically an IgGl or IgG4 constant region. For example, the DNA encoding the VLis operably linked to another DNA molecule encoding a light chain constant region, CL, to obtain a full-length light chain gene (as well as a Fab light chain gene). The sequences of human light chain constant regions are known in the art (see, e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encoding them can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is typically a kappa constant region.

[0253] To prepare humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can also be utilized that are capable of producing a complete repertoire of human antibodies upon immunization and that are incapable of 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. Transfer of the human germ-line immunoglobulin gene array VDJ and C mu into the germline of such JH- deficient mice results in the production of entirely human antibodies by the mice (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 contains human immunoglobulin genes miniloci that encode unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (see, e.g., Lonberg et al. (1994) Nature 368(6474):856-859); or the "KM mouse™" (see patent application WO 02 / 43478) which carries human heavy chain transgene and human light chain transchromosome. Other methods of 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).

[0254] As used herein, the term "degree of humanization" is a measure used to assess the number of non-human derived amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be predicted, for example, by IMGT website Domain Gap Align to compare the homology of the variable region sequence to a human V domain.

[0255] As used herein, "homologous antibody" refers to a variant of an antibody comprising heavy and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of an antibody or antigen-binding fragment thereof provided herein, and wherein the variant retains a desired functional property of an anti-CLDN18.2 antibody of the present application.

[0256] Methods for sequence alignment for comparison are well known in the art. Various programs 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 provide a detailed discussion of sequence alignment methods and homology calculations.

[0257] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half maximal effective concentration (EC50) of the interaction.

[0258] The specific binding properties between two molecules can be determined using methods well 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 kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). KD, kon, and kdis values can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., ForteBio Octet method). In addition, the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0259] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is a function of the number of matching positions shared by the sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the full length of the sequences being compared. Such a comparison can be conveniently accomplished by use of the algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443-453, as implemented in the computer program ALIGN available from DNAstar, Inc., using, for example, a program parameter of an affine gap cost of 1 for the gap open penalty and 0.5 for the gap extension penalty. Percentage 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)) as integrated 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. In addition, percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6, as incorporated in the GCG software package (available at www.gcg.com).

[0260] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence, a variant of an antibody obtained by conservative substitution of an amino acid retains the biological activity of the sequence from which it is derived, such as specifically binding to CLDN18.2. Conservative amino acid substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, for example. Conservative amino acid substitutions include substitutions of an amino acid residue for another that has similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. 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, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution is one in which the replacement amino acid residue has an atom with similar properties to the atom it is replacing. Methods of identifying amino acid conservative 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), which are incorporated herein by reference).

[0261] The nomenclature of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0262] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, physiologically and / or pharmacologically, with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength 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 to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerin), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactoserum, albumin or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like.

[0263] As used herein, the term "prevention" refers to a method implemented in order to stop or delay the occurrence of a disease or disorder or a symptom (e.g., a tumor, an infection or an autoimmune disease) in a subject.

[0264] As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present application, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether or not detectable to the eye. In addition, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0265] As used herein, the term "subject" refers to a mammal, such as a primate, e.g., a human. In certain embodiments, the subject (e.g., human) has a tumor, an infection, or an autoimmune disease, or is at risk of having such a disease.

[0266] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor, an infection, or an autoimmune disease) refers to an amount that is sufficient to prevent, arrest, or delay the onset of the disease (e.g., a tumor, an infection, or an autoimmune disease); an effective amount for treating a disease refers to an amount that is sufficient to cure, or at least partially arrest, the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and gender, the mode of administration of the drug, and other therapies that the patient may be undergoing, among other things.

[0267] As used herein, the term "immune cell" includes cells of hematopoietic origin and that 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.

[0268] As used herein, the term "immune response" refers to the action of immune cells (e.g., lymphocytes, antigen presenting cells, phagocytes, or granulocytes), and soluble macromolecules produced by immune cells or the liver (including antibodies, cytokines, and complement) that result in the selective damage, destruction of, or clearance from the body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation. In the present application, the term "antigen-specific T cell response" refers to an immune response produced by a T cell that results from the stimulation of that T cell by the antigen to which that T cell is specific. Non-limiting examples of responses produced by T cells upon antigen-specific stimulation include the proliferation of T cells and the production of cytokines (e.g., IL-2).

[0269] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region), and which vary 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), down regulation of cell surface receptors (e.g., B cell receptor), B cell activation, cytokine secretion, half-life / clearance of antibodies and antigen-antibody complexes, etc. Methods of altering the effector function of an antibody are known in the art, e.g., by introducing mutations into the Fc region.

[0270] The terms "cancer" and "tumor" are used interchangeably herein to refer to a large class 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 neighboring tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological tumors, especially hematological malignancies.

[0271] The term "hematological malignancy" includes lymphoma, leukemia, myeloma or lymphoid malignancy, as well as spleen and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphocytic leukemia. Hematological malignancies also include myelomas (e.g., multiple myeloma) and other hematological and / or B-cell or T-cell related cancers.

[0272] As used herein, the term "pharmaceutically acceptable" means moieties, molecules, or compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human as appropriate. Specific examples of materials that can serve as pharmaceutically acceptable carriers or components thereof include sugars (e.g., lactose), starches, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), gum acacia and the like.

[0273] In the present context, combination therapy includes the use of an antibody-drug conjugate, composition or pharmaceutical composition encompassed by the present application in combination with one or more second therapeutic agents (e.g., chemotherapeutic agents) or other prophylactic or therapeutic modalities (e.g., radiotherapy) of a second therapy.

[0274] Exemplary therapeutic agents for the second therapy can include chemotherapeutic agents (e.g., mitotic inhibitors), alkylating agents (e.g., nitrogen mustards), anti-metabolites (e.g., folic acid analogues), natural products (e.g., vinca alkaloids), various agents (e.g., platinum coordination complexes), hormones and antagonists (e.g., adrenocorticosteroids), immunomodulators (e.g., bropirimine (Upjohn), etc. Other anticancer therapies include other antibodies that specifically target cancer cells.

[0275] In such combination therapies, the various therapeutic agents often have different mechanisms of action that complement one another. The combination therapies can result in a synergistic effect. Combination therapies include therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells developing. Combination therapies can allow for a reduction in the dose of one or more of the agents to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies can have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition.

[0276] In the present context, "combination" includes therapies that can be administered separately, e.g., separately formulated (e.g., can be provided in separate kits), and therapies that can be administered together as a single formulation (i.e., "co-formulation"). In some embodiments, the antibody-drug conjugate, composition, or pharmaceutical composition of the present application can be administered sequentially. In other embodiments, the antibody-drug conjugate, composition, or pharmaceutical composition can be administered simultaneously. The antibody-drug conjugate of the present application can be used in combination with at least one other (active) agent in any manner.

[0277] HER2 negative means that there is no substantial amount of HER2 protein on the surface of the cell, including IHC 1+, or IHC 2+ / FISH negative, also including the range of IHC 0 to 1+ and IHC 1+ to 2+.

[0278] In the present context, the term "halogen" includes fluorine, chlorine, bromine, iodine.

[0279] In the present context, the term "C 1-6 "alkyl" denotes straight or branched chain alkyl groups containing from 1 to 6 carbon atoms, including, for example, "C 1-4 "alkyl", "C 1-3Examples of "alkyl" and the like include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, i-hexyl, 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, 1,2-dimethylpropyl, and the like.

[0280] In the present text, the term "C 1-6 "Alkylene" means a divalent group obtained by removing two hydrogen atoms from a straight-chain or branched alkane containing 1 to 6 carbon atoms, including, for example, "C 1-4 "Alkylene", "C 1-3 "Alkylene" and the like. Examples thereof include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene, or 1,6-hexylene, and the like.

[0281] In the present text, the term "C 2-10 "Alkenyl" means a straight-chain or branched alkenyl group having at least one double bond and 2 to 10 carbon atoms, including, for example, "C 2-6 "Alkenyl", "C 2-4 "Alkenyl" and the like. Examples thereof include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-buten- dienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, and the like.

[0282] In the present text, the term "C 2-10 "Alkenylene" means a divalent group obtained by removing two hydrogen atoms from an alkene containing 2 to 10 carbon atoms. Including, for example, "C 2-8 "Alkenylene", "C 4-6 "Alkenylene" and the like. Examples thereof include, but are not limited to, 1,5-pentenylene, 1,5-pent-2-enylene, 1,6-hexenylene, and the like.

[0283] In the present text, the term "C 2-10 "Alkynyl" means a straight-chain or branched alkynyl group having at least one triple bond and 2 to 10 carbon atoms, including, for example, "C 2-6 "Alkynyl", "C 2-4 "Alkynyl" and the like. Examples thereof include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, and the like.

[0284] In the present text, the term "C 2-10 Alkynylene" refers to a divalent group obtained by the loss of two hydrogen atoms from an alkyne containing 2-10 carbon atoms. Examples include "C 2-8 Alkynylene", "C 4-6 Alkynylene", and the like. Examples include, but are not limited to, 1,5-pentynylene, 1,5-pent-2-ynylene, 1,6-hexynylene, and the like.

[0285] In the present text, the term "C 1-6 Alkoxy" refers to a radical having the structure C 1-6 alkyl-O-structure, wherein C 1-6 alkyl is as defined above. Specific examples include, but are not limited to, methoxy, ethoxy, propyloxy, isopropyloxy, n-propyloxy, isopropyloxy, n-butyloxy, isobutyloxy, t-butyloxy, pentyloxy, hexyloxy, and the like.

[0286] In the present text, the term "5-12 membered heteroaryl" refers to an aromatic cyclic radical containing 5-12 ring members, and wherein at least one ring member is a heteroatom selected from N, O, and S. Specific examples include, but are not limited to, 5-10 membered heteroaryl, 5-10 membered nitrogen-containing heteroaryl, 5-6 membered oxygen-containing heteroaryl, and the like, for example, furanyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, 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, 1,2,4,5-tetrazinyl, and the like.

[0287] The application is not limited to the particular methodology, protocols, cell lines, vectors, or reagents described herein as these can vary. In addition, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application.

[0288] As used herein, and unless otherwise indicated, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. In addition, the term "or" as used herein means "and / or" unless otherwise indicated. Furthermore, to the extent that any definition or usage of a term herein is inconsistent with or contrary to the definition or usage of that term as found in the patent statutes, regulations, rules, or decisions of any jurisdiction having a bearing on the interpretation of this disclosure, the definition or usage of that term as found in the patent statutes, regulations, rules, or decisions of that jurisdiction shall control. BRIEF DESCRIPTION OF DRAWINGS

[0289] Figure 1AFlow cytometry detection of HEK293T-Claudin 18.2 monoclonal stable cell line is shown.

[0290] Figure 1B Flow cytometry detection of L929-Claudin 18.2 monoclonal stable cell line is shown.

[0291] Figure 1C Flow cytometry detection of KATOIII-Claudin 18.2 monoclonal stable cell line is shown.

[0292] Figure 1D Flow cytometry detection of NCI-N87-Claudin 18.2 monoclonal stable cell line is shown.

[0293] Figure 1E Western blot detection of HEK293T-Claudin 18.1 monoclonal stable cell line is shown.

[0294] Figure 2 2C6.9-hz21, IMAB362 antibody affinity detection (flow cytometry) is shown.

[0295] Figure 3 2C6.9-hz21, IMAB362 antibody affinity detection (L929-Claudin 18.2 cells) is shown.

[0296] Figure 4 Specificity of 2C6.9-hz21 antibody detection by flow cytometry is shown.

[0297] Figure 5 2C6.9-hz21, IMAB362 CDC killing activity assay on HEK293T-Claudin 18.2 cells is shown.

[0298] Figure 6 2C6.9-hz21, IMAB362 antibody ADCC activity detection (HEK293T-Claudin 18.2 cells) is shown.

[0299] Figure 7 HPLC-SEC detection profile of 2C6.9-TL001 (DAR: 3.79) is shown.

[0300] Figure 8 HPLC-SEC detection profile of 2C6.9-TL001 (DAR: 7.12) is shown.

[0301] Figure 9The results of affinity detection of 2C6.9-TL001 (DAR: 7.12) to cell membrane surface Claudin 18.2 are shown.

[0302] Figure 10A The results of killing activity detection of 2C6.9-TL001 (DAR: 7.12) to HEK293T-Claudin 18.2 cells are shown.

[0303] Figure 10B The results of killing activity detection of 2C6.9-TL001 (DAR: 3.79) to HEK293T-Claudin 18.2 cells are shown.

[0304] Figure 10C The results of killing activity detection of 2C6.9-TL001 (DAR: 7.12) to HEK293T-Claudin 18.1 cells are shown.

[0305] Figure 10D The results of killing activity detection of 2C6.9-TL002, 2C6.9-TL003 to HEK293T-Claudin 18.2 cells are shown.

[0306] Figure 10E The results of killing activity detection of 2C6.9-TL002, 2C6.9-TL003 to HEK293T-Claudin 18.1 cells are shown.

[0307] Figure 11A The results of killing activity detection of 2C6.9-TL001 (DAR: 7.12) to NUGC-4 cells are shown.

[0308] Figure 11B The results of killing activity detection of 2C6.9-TL001 (DAR: 3.79) to NUGC-4 cells are shown.

[0309] Figure 11C The results of killing activity detection of 2C6.9-TL002, 2C6.9-TL003 to NUGC-4 cells are shown.

[0310] Figure 12A The changes of tumor volume of each group of mice in the Balb / c Nude mouse subcutaneous NCI-N87-Claudin 18.2 cell xenograft model are shown (*: P<0.05; ****: P<0.0001).

[0311] Figure 12B The changes of body weight of each group of mice in the Balb / c Nude mouse subcutaneous NCI-N87-Claudin 18.2 cell xenograft model are shown.

[0312] Figure 12C The results show the in vivo efficacy comparison between 2C6.9-TL001 and 2C6.9 monoclonal antibody plus chemotherapy in the CDX model at 11 days (****: P<0.0001).

[0313] Figure 12D The results show the in vivo efficacy comparison between 2C6.9-TL001 and 2C6.9 monoclonal antibody + chemotherapy in the CDX model at 21 days (****: P<0.0001).

[0314] Figure 12E The changes in tumor volume in mice of different groups with different DAR values ​​2C6.9-TL001 at 21 days in the CDX(NUGC-4) model are shown (****: P<0.001).

[0315] Figure 12F This shows the changes in body weight of mice in different groups with different DAR values ​​2C6.9-TL001 in the CDX(NUGC-4) model over 21 days.

[0316] Figure 12G Subcutaneous tissue of Balb / c Nude mice was shown. Changes in tumor volume in mice of different groups over 17 days in the GA0006 PDX model of gastric cancer (****: P<0.0001).

[0317] Figure 12H Subcutaneous tissue of Balb / c Nude mice was shown. Changes in body weight of mice in different groups over 17 days in the GA0006 PDX model of gastric cancer.

[0318] Figure 12I Subcutaneous tissue of Balb / c Nude mice was shown. Changes in tumor volume in mice of different groups over 24 days in the GA0006 PDX model of gastric cancer (****: P<0.0001).

[0319] Figure 12J Subcutaneous tissue of Balb / c Nude mice was shown. Changes in body weight of mice in different groups over 24 days in the GA0006 PDX model of gastric cancer.

[0320] Figure 12K The changes in tumor volume in each group of mice in the subcutaneous NCI-N87-Claudin18.2 cell xenograft model of Balb / c Nude mice are shown (***: P<0.001; ****: P<0.0001).

[0321] Figure 12LThe change in the body weight of each group of mice in the subcutaneous NCI-N87-Claudin18.2 cell xenograft model of Balb / c Nude mice is shown.

[0322] Figure 12M The change in the tumor volume of each group of mice in the subcutaneous HEK293T-Claudin18.2 cell xenograft model of Balb / c Nude mice is shown (**: P<0.01; ****: P<0.0001).

[0323] Figure 12N The change in the body weight of each group of mice in the subcutaneous HEK293T-Claudin18.2 cell xenograft model of Balb / c Nude mice is shown.

[0324] Figure 12O The change in the tumor volume of each group of mice in the subcutaneous NUGC-4 cell xenograft model of Balb / c Nude mice is shown (****: P<0.0001).

[0325] Figure 12P The change in the body weight of each group of mice in the subcutaneous NUGC-4 cell xenograft model of Balb / c Nude mice is shown.

[0326] Figure 13 The HPLC-SEC detection spectrum of 2C6.9-TL001 (DAR: 7.40) is shown. DETAILED DESCRIPTION

[0327] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not noted in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not noted for the manufacturer are all conventional products that can be obtained by purchase on the market.

[0328] Example 1 Synthesis of biologically active molecule (TOXIN-1)

[0329]

[0330] Methanesulfonyl chloride (462 mg, 12.77 mmol, purity ~ 70%) was added dropwise to a solution of belotecan hydrochloride (3 g, 6.38 mmol) and triethylamine (2.58 g, 25.54 mmol) in dichloromethane (40 mL) at room temperature for 2 h. The reaction mixture was filtered, and the filter cake was washed with dichloromethane (3 mL) three times 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).

[0331] Structural characterization data are as follows:

[0332] 1 H NMR (400 MHz, DMSO-d6) δ 8.32 (d, J = 8.4 Hz, 1H), 8.20 (dd, J = 8.4, 1.2 Hz, 1H), 7.93 - 7.84 (m, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.35 (s, 1H), 6.56 (s, 1H), 5.44 (d, J = 9.2 Hz, 4H), 3.98 (p, J = 6.7 Hz, 1H), 3.50 (t, J = 8.0 Hz, 2H), 3.42 - 3.35 (m, 2H), 3.00 (s, 3H), 1.93 - 1.82 (m, 2H), 1.15 (d, J = 6.7 Hz, 6H), 0.88 (t, J = 7.3 Hz, 3H).

[0333] ESI-MS (m / z): 512.2 [M+H] + .

[0334] [α] D 20 = +28.19° (c = 0.101 g / 100 mL, CH3CN).

[0335] Example 2. Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-4)

[0336]

[0337] Step 1. Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate (Compound 3-2)

[0338] Step two: synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (compound 3-3) + .

[0339] Step two: synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (compound 3-3)

[0340] 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 reaction was stirred at room temperature for 4 h, diluted with water, extracted with ethyl acetate (20 mL x 2), and the aqueous phase was adjusted to pH = 3 with 1N hydrochloric acid and 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, and the solvent was removed by evaporation under reduced pressure to obtain 120 mg of the title compound.

[0341] Step three: synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (compound 3-4)

[0342] Compound 3-3 (20 mg, 0.085 mmol) was dissolved in dichloromethane (4 mL) at room temperature, and meta-chloroperoxybenzoic acid (22 mg, 0.127 mmol) was added. After addition, the reaction was stirred at room temperature overnight, and purified by preparative liquid chromatography to obtain 20 mg of the title compound. ESI-MS (m / z): 269.1 [M+H] + .

[0343] Example Three (Synthesis of (4-((S)-2-(4-aminobutyl)-3-5-(4-((6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamido)methyl)-1H-1,2,3-triazol-1-yl)- 4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanoylamido) benzyl)((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)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)

[0344]

[0345] Step One: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propargyl)hex-5- ynamide (Compound 3-5)

[0346] Propargylamine (189 mg, 3.4 mmol) and compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL) at 25 °C, and N,N-diisopropylethylamine (738 mg, 5.67 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.63 g, 4.25 mmol) were added successively, and the reaction was stirred for 2 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by 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] + .

[0347] Step Two: Synthesis of (4-((S)-3-5-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)- 4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanoylamido)benzyl) ((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)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)

[0348] Under nitrogen protection at 25°C, TOXIN-1 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL), cooled to 0°C, and a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added. Then, a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at 0°C for 20 min, and the reaction solution was purged with nitrogen for 20 min. A solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonazo-6-azatridodecanoamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanoamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added. The mixture was stirred at 0 °C for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (HPLC) to give 500 mg of the title compound. ESI-MS (m / z): 1597.5 [M+H] + .

[0349] Step 3: Synthesis of ((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzazido[1,2-b]quinoline-4-yl)(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentadecanoamide)benzyl)carbonate (compound 33-2)

[0350] Compound 33-1 (80 mg, 0.05 mmol) and fragment 3-5 (23 mg, 0.075 mmol) were dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL) at room temperature. Cuprous bromide (11 mg, 0.08 mmol) was added, and the mixture was stirred for 1 h. The mixture was purified by preparative high-performance liquid chromatography (HPLC) to give 30 mg of the title compound. ESI-MS (m / z): 815.9 [(M-273) / 2+H] + .

[0351] Step four: synthesis of (4-((S)-2-(4-aminobutyl)-3-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanoylamino)benzyl)((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)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)

[0352] Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) and trifluoroacetic acid (0.2 mL) was added. The reaction was stirred at room temperature for 30 min. Purification by preparative HPLC gave 20.0 mg of the title compound as a trifluoroacetate salt. Its structure was characterized as follows:

[0353] 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.10 (s, 2H), 8.38 (t, J = 5.56 Hz, 1H), 8.32 (d, J = 8.40 Hz, 1H), 8.22 - 8.20 (m, 2H), 8.09 (t, J = 5.68 Hz, 1H), 7.91 - 7.87 (m, 2H), 7.82 - 7.78 (m, 1H), 7.69 (brs, 3H), 7.61 (d, J = 8.56 Hz, 2H), 7.32 (d, J = 8.56 Hz, 2H), 7.06 (s, 1H), 5.56 (d, J = 16.96 Hz, 1H), 5.51 (d, J = 16.96 Hz, 1H), 5.47 (d, J = 19.28 Hz, 1H), 5.42 (d, J = 19.28 Hz, 1H), 5.14 (d, J = 12.20 Hz, 1H), 5.07 (d, J = 12.16 Hz, 1H), 4.48 (t, J = 5.24 Hz, 2H), 4.46 - 4.43 (m, 1H), 4.29 (d, J = 5.60 Hz, 2H), 4.08 - 3.95 (m, 5H), 3.79 (t, J = 5.28 Hz, 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 = 7.08 Hz, 2H), 2.29 (t, J = 7.36 Hz, 2H), 2.23 - 2.13 (m, 2H), 1.82 (p, J = 7.24 Hz, 2H), 1.78 - 1.63 (m, 2H), 1.61 - 1.49 (m, 2H), 1.42 - 1.27 (m, 2H), 1.15 (d, J = 6.80 Hz, 3H), 1.13 (d, J = 6.76 Hz, 3H), 0.90 (t, J = 7.32 Hz, 3H).

[0354] ESI-MS (m / z): 816.0 [M / 2+H] + .

[0355] [α] D 20 = -19.55° (c = 1.000 g / 100 mL, CH3CN).

[0356] Example 4. Synthesis of (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-((S)-2-((S)-3- methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamino)methyl)- 1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)butanoyl- amino)butanoylamino)propionamido)benzyl)methan-1,2-diylbis(methylcarbamate) trifluoroacetate (TL002)

[0357]

[0358] Step 1. Synthesis of (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

[0359] (S)-4,11-diethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline- 3,14(4H,12H)-dione (150 mg, 0.37 mmol) was dissolved in dichloromethane (15 mL), diisopropylethylamine (96.81 mg, 0.74 mmol) was added, then a solution of bis(4- nitrophenyl) carbonate (127.92 mg, 0.41 mmol) in dichloromethane (15 mL) was added, and the reaction was stirred at 25 °C for 3 h. The reaction was concentrated to give 207 mg of the title compound. It was used directly in the next step.

[0360] ESI-MS (m / z): 558.1 [M+H] + .

[0361] 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]indolizino[1,2-b]quinolin-9-yl)ethan-1,2-diylbis(methyl carbamate)

[0362] (S)-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,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 mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated, and the residue was purified by column chromatography on silica gel (eluent: dichloromethane / methanol = 9 / 1) to give the title compound 207 mg.

[0363] ESI-MS (m / z): 607.3 [M+H] + .

[0364] Step three: Synthesis of (S)-N-methyl-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14- tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-9-yl)(2- (methylamino)ethyl)carbamate trifluoroacetate

[0365] (S)-tert-butyl (4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-lH- pyrano[3',4':6,7]indolizino[l,2-b]quinolin-9-yl)ethane-l,2-diylbis(methylcarbamate) (207 mg, 0.19 mmol) was dissolved in dichloromethane (8 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated to give the title compound 200 mg. It was used directly in the next step.

[0366] ESI-MS (m / z): 507.2 [M+H] + .

[0367] Step four: Synthesis of (4-((S)-2-((S)-2-(4-(l-(26-azido-3,6,9,12,15,18,21,24- octaoxa-hexacosyl)piperidin-4-yl)butanamido)-3-methylbutanamido)propanamido)benzyl)((S)- 4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2- b]quinolin-9-yl)ethane-l,2-diylbis(methylcarbamate)

[0368] (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)-3-methylbutamido)propamido)benzyl)(4-nitrophenyl)carbonate (150 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (3 mL), and 1-hydroxybenzotriazole (34.03 mg, 0.25 mmol) was added. The compound was reacted with diisopropylethylamine (48.82 mg, 0.37 mmol) and (S)-N-methyl-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]inzazo[1,2-b]quinoline-9-yl)(2-(methylamino)ethyl)carbamate trifluoroacetate (102.11 mg, 0.12 mmol), and reacted at 25 °C for 16 h. The reaction solution was purified by preparative high performance liquid chromatography, and the preparative solution was freeze-dried to give 44 mg of the title compound.

[0369] ESI-MS (m / z): 1400.7 [M+H] + .

[0370] Column: Waters XBridge Prep C18 OBD 19mm×150mm×5.0μm

[0371] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0372] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0.00 10 90 28 3.00 10 90 28 19.00 90 10 28

[0373] Step 5: Synthesis of ((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]indazazino[1,2-b]quinoline-9-yl)(4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)butamido)propamido)benzyl)ethane-1,2-diylbis(methylcarbamate)trifluoroacetate

[0374] (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)-3-methylbutamido)propamido)benzyl)((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]inzazin[ [1,2-b]quinoline-9-yl)ethane-1,2-diylbis(methylcarbamate) (44 mg, 0.30 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-propynyl)hexanoamide (14.09 mg, 0.045 mmol) were dissolved in dimethyl sulfoxide (3 mL) and water (0.75 mL), and cuprous bromide (8.65 mg, 0.06 mmol) was added. The reaction mixture was reacted at 25 °C for 1 h. The reaction solution was purified by preparative high-performance liquid chromatography (HPLC), and the preparative solution was lyophilized and dissolved in dichloromethane (2 mL). Trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at 25 °C for 0.5 h. The preparative solution was purified by preparative HPLC and lyophilized to give 16 mg of the title compound.

[0375] ESI-MS (m / z): 853.6 [M / 2+H] + .

[0376] Column: Waters SunFire Prep C18 ODS 5μm 19x50mm

[0377] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0378] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0.00 10 90 28

[0379] 4.00 10 90 28 20.00 90 10 28

[0380] Example 5 (4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-((methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butamido)butamido)propamido)benzyl)2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]inzazo[1,2-b]quinoline-11-yl)ethyl isopropyl carbamate trifluoroacetate (TL003)

[0381]

[0382] Step one: synthesis of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1- oxopropyl-2-yl)-3-methylbutanamide

[0383] ((9H-fluoren-9-yl)methyl)((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1- oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate (2.00 g, 3.88 mmol) was dissolved in N,N-dimethylformamide (12 mL) at room temperature, piperidine (3 mL) was added, and the reaction was stirred at 25 °C for 2 h. The reaction was poured into water, and a solid was precipitated, which was suction filtered and purified by preparative high performance liquid chromatography. The fractions were lyophilized to give the title compound 810 mg.

[0384] ESI-MS (m / z): 294.0 [M+H] + .

[0385] Column: Waters SunFire Prep C18 ODS 8 pm 45x450mm

[0386] Mobile phase A: acetonitrile; mobile phase B: water

[0387] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0.00 7 93 60 7.00 7 93 60 50.00 50 50 60

[0388] Step two: synthesis of tert-butyl 4-(4-((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1- oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)amino)-4-oxobutyl)piperidine-1- carboxylate

[0389] (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3- methylbutanamide (810 mg, 2.76 mmol), 4-(1-(tert-butoxycarbonyl)piperidin-4-yl)butanoic acid and 2-ethoxy-1-ethoxy carbonyl-1,2-dihydroquinoline were dissolved in dichloromethane (8 mL) and methanol (8 mL) at room temperature, and the reaction was stirred at 45 °C for 2 h. The reaction was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) to give the title compound 1.31 g.

[0390] ESI-MS (m / z): 546.8 [M+H] + .

[0391] Step three: synthesis of (S)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2- yl)-3-methyl-2-(4-(piperidin-4-yl)butanamido)butanamide trifluoroacetate

[0392] tert-Butyl 4-(4-((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)amino)-4-oxobutyl)piperidine-1-carboxylate (1.3 g, 2.14 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (5 mL) was added, and the reaction was stirred at 25 °C for 2 h. The reaction 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 reaction was stirred at 25 °C for 2 h. The mixture was filtered, the filter cake was washed with acetonitrile, and the filtrate was concentrated under reduced pressure to give the title compound 900 mg.

[0393] ESI-MS (m / z): 446.9 [M+H] + .

[0394] Step four: Synthesis of (S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24- octaoxa-hexacosyl)piperidin-4-yl)butanamido)-N-((S)-1-((4- (hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutanamido

[0395] (S)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methyl-2-(4-(piperidin-4-yl)butanamido)butanamide trifluoroacetate (487 mg, 0.78 mmol) and 26-azido-3,6,9,12,15,18,21,24- octa-oxa-hexacosyl 4-methylbenzenesulfonate (619 mg, 0.94 mmol) were dissolved in acetonitrile (20 mL), potassium carbonate (655 mg, 4.69 mmol) was added, and the reaction was stirred at 16 °C for 6 h. The reaction was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluent: dichloromethane / methanol = 8 / 1) to give the title compound 586 mg.

[0396] ESI-MS (m / z): 868.5 [M+H] + .

[0397] Step five: Synthesis of (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24- octa-oxa-hexacosyl)piperidin-4-yl)butanamido)-3-methylbutanamido)propanamido)benzyl)(4-nitrophenyl) carbonate

[0398] (S)-2-(4-(l-(26-azido-3,6,9,12,15,18,21,24-oxa-hexacosyl)piperidin-4-yl)butanamido)-N- ((S)-l-((4-(hydroxymethyl)phenyl)amino)-l-oxopropan-2-yl)-3-methylbutanamide (585 mg, 0.64 mmol) was dissolved in dichloromethane (30 mL) at room temperature, added N,N- diisopropylethylamine (334.31 mg, 2.56 mmol), then added dropwise di(p-nitrophenyl) carbonate (602.35 mg, 1.92 mmol) in dichloromethane (30 mL), and reacted at 25 °C for 6 h. The reaction solution was concentrated under reduced pressure, the residue was added to methyl tert-butyl ether, and filtered under suction to obtain 760 mg of the title compound.

[0399] ESI-MS (m / z): 1033.4 [M+H] + .

[0400] Step six: Synthesis of (4-((S)-2-((S)-2-(4-(l-(26-azido-3,6,9,12,15,18,21,24-oxa- hexacosyl)piperidin-4-yl)butanamido)-3-methylbutanamido)propanamido)benzyl) 2-((S)-4- ethyl-4-hydroxy-3, 14-dioxo-3, 4, 12, 14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin- 11-yl)ethyl isopropylcarbamate

[0401] (4-((S)-2-((S)-2-(4-(l-(26-azido-3,6,9,12,15,18,21,24-oxa-hexacosyl)piperidin-4-yl)butanamido)- 3-methylbutanamido)propanamido)benzyl)(4-nitrophenyl) carbonate (150 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (3 mL) at room temperature, added 1- hydroxybenzotriazole (34.03 mg, 0.25 mmol) and N,N-diisopropylethylamine (48.82 mg, 0.37 mmol), then added (S)-4-ethyl-4-hydroxy-l l-(2-(isopropylamino)ethyl)-l,12-dihydro-14H- pyrano[3',4':6,7]indolizino[l,2-b]quinoline-3,14(4H)-dione hydrochloride (59.79 mg, 0.12 mmol), and the reaction solution was stirred at room temperature overnight. The reaction solution was purified by preparative high-performance liquid chromatography, and the preparation was freeze-dried to obtain 64 mg of the title compound.

[0402] ESI-MS (m / z): 1327.6 [M+H] + .

[0403] Step seven: synthesis of (4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24-octaoxa-hexacosyl)piperidin-4-yl)butyramido)butyramido)propanamido)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)ethyl isopropylcarbamate trifluoroacetate

[0404] At room temperature, (4-((S)-2-((S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxa-hexacosyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propanamido)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)ethyl isopropylcarbamate (64 mg, 0.046 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hexanamide (21.63 mg, 0.069 mmol) were dissolved in dimethyl sulfoxide (4 mL) and water (1 mL), copper bromide (13.27 mg, 0.092 mmol) was added, and the reaction was allowed to proceed at 25 °C for 1 h. The reaction solution was purified by preparative high performance liquid chromatography, and the preparation was freeze-dried to obtain 35 mg of the title compound. ESI-MS (m / z): 1632.8 [M+H] + .

[0405] Column: Waters SunFire Prep C18 OBD 5 µm 19x150mm

[0406] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0407] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0408] Example six: preparation of monoclonal antibodies targeting human Claudin 18.2

[0409] The monoclonal antibodies targeting human Claudin 18.2 in the present application are humanized monoclonal antibodies, including 2C6.9-hz11 and 2C6.9-hz21, the CDR, variable region sequence and constant region sequence information of which are shown in Table 1:

[0410] Table 1: sequence information summary

[0411]

[0412]

[0413] 6.1 Construction and identification of human Claudin 18.2 and human Claudin 18.1 overexpression cell lines

[0414] 6.1.1 Construction of human Claudin 18.2 and human Claudin 18.1 overexpression cell lines

[0415] To verify the specificity and function of human Claudin 18.2 antibody, the complete coding sequence of human Claudin 18.2 (Gene ID: NM_001002026.2, synthesized by Nanjing Kingsriver Biotechnology Co., Ltd.) and the complete coding sequence of human Claudin 18.1 (Gene ID: NM_016369.3, synthesized by Nanjing Kingsriver Biotechnology Co., Ltd.) were cloned into the lentiviral vector pLVX-IRES-puro, and the virus was prepared by the literature described (Mohammadi Z et al., Mol Biotechnol. 2015 Sep;57(9):793-800.) lentivirus packaging system. After obtaining the virus, HEK293T, L929, KATOIII and NCI-N87 cells were infected, respectively, and through puromycin screening and single clone selection, single clone HEK293T-Claudin 18.1, HEK293T-Claudin 18.2, L929-Claudin 18.2, KATOIII-Claudin 18.2, NCI-N87-Claudin 18.2 stable cell lines were obtained. The human Claudin 18.2 and human Claudin 18.1 plasmids were transfected into BaF / 3 cells (DSMZ, Cat# ACC300) by 4D-Nucleofector X Transfection Kit (Lonza, Cat# V4XC-3012). After 48 hours of transfection, 1.25 mg / mL hygromycin (Thermo Fisher Sci. Cat# 10687010) was added for screening, and after 12 days of screening, single clones were sorted to obtain single clone BaF / 3-Claud18.1 and BaF / 3-Claud18.2 cell lines.

[0416] 6.1.2 Detection of human Claudin 18.2 and human Claudin 18.1 overexpression cell lines

[0417] HEK293T-Claudin 18.1 was identified by Western Blot (detection antibody: Proteintech, 66167-1-Ig), and other cell lines were identified by flow cytometry (flow cytometer: Beckman, CytoFlex; detection antibody IMAB362, sequence from patent: CN 101312989B). As shown in FIG. 6.2, the flow cytometry results show that HEK293T-Claudin 18.2, L929-Claudin 18.2, KATOIII-Claudin 18.2, and NCI-N87-Claudin 18.2 all obtained monoclonal cell lines with high positive rate (close to 100%) and good uniformity, which can be used for subsequent experiments. Western Blot results (FIG. 6.3) show that three HEK293T-Claudin 18.1 stable cell lines all overexpress human Claudin 18.1, and HEK293T-Claudin 18.1-1C2 has higher expression, which can be used for subsequent experiments. Figure 1A-1D Figure 1E

[0418] 6.2 Preparation of anti-human Claudin 18.2 murine monoclonal antibody

[0419] The present application uses DNA immunization and cell immunization to immunize wild-type mice to obtain anti-human Claudin 18.2 murine monoclonal antibody. Each Balb / c mouse is injected with 100 μg of plasmid containing the complete coding sequence of human Claudin 18.2 through the tail vein injection method. After the fourth and sixth immunization, the serum titer is detected by flow cytometry analysis. Select the mouse with higher titer for 3-5 days of booster immunization with BaF / 3-Claudin 18.2 overexpression cell line before fusion. Use the standard fusion process to fuse the mouse spleen cells and Sp2 / 0 (ATCC, Cat# CRL-1581) mouse myeloma cell line by PEG fusion method, and then screen by HAT pressure selection. After 10-14 days, perform flow cytometry screening.

[0420] The supernatant of 6000 hybridoma clones was screened by flow cytometry (purchased from Sartorius, model: iQue Screener Plus) to obtain 43 positive hybridoma clones that can recognize HEK293T-Claudin 18.2 cell line for subcloning. Select HEK293T-Claudin 18.2 and HEK293T-Claudin 18.1 cell lines for further screening by flow cytometry to obtain 14 positive clones that only bind to human Claudin 18.2 but not to human Claudin 18.1. Finally, subclone and sort by limiting dilution method to obtain monoclonal cells.​​

[0421] Human gastric cancer cell line NUGC4 (purchased from JCRB Cell Bank, Japan, Cat# JCRB0834) endogenously expresses Claudin 18.2 protein, which is widely used to detect the binding of antibodies to endogenous Claudin 18.2 and the development of functional detection methods. The candidate clones were detected using NUGC4 cells, and finally 7 subclones were selected as candidate clones. After further affinity detection, 2C6.9M was selected for variable region amplification and humanization.

[0422] In order to detect the antibody subtype of the candidate hybridoma clone, Pierce Rapid Isotyping Kit (Thermo Fisher Sci. Cat# 26179) was used to identify 2C6.9M. The identification results showed that the heavy chain was IgG1 subtype and the light chain was Kappa subtype.

[0423] Hybridoma cells were cultured to about 8000, the cells were lysed and the first strand cDNA was synthesized using cDNA reverse transcription kit (Thermo Fisher Sci. Cat# 18080-200). The VH and VK (VLKappa) genes were amplified from cDNA by PCR using primers, and the PCR products were purified by DNA purification kit (Qiagen, Cat# 28104) and connected to TOPO vector (Thermo Fisher Sci. Cat# K457540). About 12 clones were selected for sequencing from each ligation reaction. The sequences were analyzed by Vector NTI 11.5 (Thermo Fisher Sci.) and Sequencer 5.4.6 (Genecodes). The variable region sequences and CDR sequences of murine antibody 2C6.9M are shown in Table 2.

[0424] Table 2: 2C6.9 variable region and CDR amino acid sequences (SEQ ID NO:)

[0425]

[0426] 6.3 Humanization of murine antibody 2C6.9M

[0427] The murine antibody 2C6.9M was humanized by CDR grafting antibody humanization method. Briefly, the humanization involved the following steps: comparing the amino acid sequence of the murine monoclonal antibody with the amino acid sequence of the human embryonic antibody, finding the sequence with high homology and better physicochemical properties, as the human embryonic framework sequence; analyzing the HLA-DR affinity, selecting the human embryonic framework sequence with low affinity; and grafting the six CDRs of the murine antibody onto the selected heavy chain and light chain framework sequences, respectively.

[0428] Specifically, the CDR regions of the heavy chain and light chain of the mouse-derived antibody 2C6.9M were respectively grafted onto the FR framework of the corresponding humanized template. The heavy chain and light chain humanized templates of 2C6.9M were human germline gene sequences IGHV4-59*01 (see IMGT Accession No. AB019438) and IGKV4-1*01 (see IMGT Accession No. Z00023), respectively.

[0429] Further, by using computer simulation technology, the variable region and the framework amino acid sequences around it were analyzed by molecular docking to investigate the spatial stereochemical binding mode. By calculating the electrostatic force, van der Waals force, hydrophobicity and entropy, the key amino acids in the amino acid sequence of the mouse-derived antibody that can interact with Claudin 18.2 and maintain the spatial framework were analyzed, and these mouse-derived amino acids were retained in the grafted antibody. That is, a series of back mutations were performed on the FR region amino acid residues of the above-mentioned humanized template, so that the humanized antibody can retain the antigen binding ability of the mouse-derived antibody as much as possible.

[0430] The variable region sequence of the mouse-derived antibody 2C6.9M is shown in SEQ ID NO: 23, SEQ ID NO: 24, and the CDR sequence is shown in SEQ ID NO: 1 to SEQ ID NO: 12. In order to avoid the occurrence of isomers without affecting the affinity, the 2C6.9 CDR-H2 amino acid sequence is modified, and the modified sequence is shown in SEQ ID NO: 21, SEQ ID NO: 22. Finally, two humanized antibodies were constructed, which were named 2C6.9-hz11 and 2C6.9-hz21, respectively. The heavy chain constant region of each antibody is a human wild-type IgG1 heavy chain constant region (SEQ ID NO: 16), and the light chain constant region of the antibody is a human wild-type IgG1 kappa light chain constant region (SEQ ID NO: 17).

[0431] The variable region, constant region and heavy / light chain amino acid sequences of 2C6.9 antibodies are shown in Table 1.

[0432] The heavy chain and light chain amino acid sequences of the humanized antibodies of 2C6.9 were synthesized into cDNA by codon optimization and connected to the plasmid pcDNA3.4 (commissioned to Nanjing Kingsway Biological Technology Co., Ltd.). The corresponding pcDNA3.4 of the heavy chain and light chain was transfected into Expi293F cells (purchased from Thermo) at the same time, and the humanized monoclonal antibodies of 2C6.9 were obtained by purifying the supernatant of the cultured cells through protein A (MabSelect SuRe, GE) affinity chromatography column.

[0433] 6.4 Affinity detection of humanized monoclonal antibodies of 2C6.9

[0434] 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 steps are as follows: Digest HEK293T-Claudin18.2 cells, centrifuge and resuspend, wash twice with PBS; resuspend cells in PBS (containing 1% BSA), seed cells at 300,000 cells / well into 96-well conical plates, 50 μl per well, for 20 wells; add 50 μL of 2C6.9-hz21 and IMAB362 antibodies to each well, starting at 1000 nM, and serially dilute 3-fold for a total of 11 concentration points; use Human IgG as a negative control; mix well and incubate at 4°C in the dark for 1 hour; wash 3 times with PBS, add FITC-labeled anti-human Fc secondary antibody (brand: BioLegend, catalog number: 409322), and incubate at 4°C in the dark for 0.5 hours; wash 3 times with PBS, and perform flow cytometry (Beckman, model: Cytoflex) for detection.

[0435] Experimental results are as follows Figure 2 As shown in Table 3, the EC50 value of the binding affinity of 2C6.9-hz21 to HEK293T-Claudin 18.2 detected by flow cytometry was less than that of IMAB362, while the maximum fluorescence signal value was greater than that of IMAB362, indicating that 2C6.9-hz21 has a better affinity for Claudin 18.2 on the cell membrane than IMAB362.

[0436] Table 3: Flow cytometry affinity assay of humanized antibody 2C6.9-hz21 binding to HEK293T-Claudin 18.2

[0437] Antibody name EC50(nM) Max MFI 2C6.9-hz21 5.767 165866.3 IMAB362 8.217 156715.7

[0438] 6.5 Determination of affinity and specificity of humanized monoclonal antibodies against 2C6.9

[0439] Human Claudin 18.2 is a four-transmembrane protein with a relatively complex structure. To ensure the integrity of the Claudin 18.2 antigen molecule, a cell-based ELISA method was used for detection. The stable L929-Claudin 18.2 cell line constructed in section 6.1 was selected for detection. The specific experimental steps are as follows: The adherent L929-Claudin 18.2 cell line was digested with 2 mM EDTA and resuspended to a concentration of 2 × 10⁻⁶ mcg. 5Cells / mL, 100 μL per well, were seeded into 96-well plates and incubated overnight at 37°C. The next day, the culture medium was removed, the plates were washed once with PBS, and 100 μL of 4% formaldehyde was added to each well for fixation at room temperature for 30 minutes. After removing the formaldehyde, the plates were washed twice with PBS, and 100 μL of PBS (containing 2% BSA) was added for blocking at 37°C for 2 hours. The blocking buffer was removed, and the antibody was diluted with PBS (containing 2% BSA) at a starting concentration of 1 μM, followed by 4-fold dilutions, resulting in 11 concentration spots, at 100 μL per well, and incubated at 37°C for 2 hours. The plates were washed 5 times with 250 μL of PBST, with each wash incubated for 2 minutes. Horseradish peroxidase (HRP)-labeled anti-human IgG secondary antibody (HRP-anti-Human IgG, Jackson ImmunoResearch, 109-035-003) was diluted 1:10000 with PBS (containing 2% BSA), at 1:10000, at 100 μL per well, and incubated at 37°C for 1 hour. 250 μL Wash 6 times with PBST, allowing it to stand for 2 minutes each time; add 100 μL of TMB chromogenic solution (Thermo, 34029) to the corresponding wells and incubate at 37°C for 20 minutes; add 50 μL of 2 mol / L H2SO4 to stop the reaction, and read the value at 450 nm using a microplate reader (MD, SpectraMax M2). Import the results into Graphpad Prism for curve fitting.

[0440] Experimental results are as follows Figure 3 As shown in Table 4, the affinity of humanized antibody 2C6.9-hz21 for human Claudin 18.2 is significantly better than that for IMAB362. The same experiment showed that the affinity of humanized antibody 2C6.9-hz11 for antibody 2C6.9-hz21 was close (results omitted).

[0441] Table 4: Affinity assay of humanized antibody 2C6.9-hz21 binding to L929-Claudin 18.2 cells using ELISA

[0442] Antibody name EC50(nM) Max signal value (OD450) 2C6.9-hz21 0.12 0.92 IMAB362 0.15 0.53

[0443] The application also detects the specificity of the candidate antibody by flow cytometry. The specific steps are as follows: digest HEK293T, HEK293T-human Claudin 18.1, HEK293T-human Claudin 18.2 cells, centrifuge and resuspend, wash twice with PBS; resuspend the cells with PBS (containing 1% BSA), take 300,000 cells of each cell, add the candidate antibody at a final concentration of 1000 nM, mix well, react at 4°C for 1 hour; wash 3 times with PBS, add FITC-labeled anti-human Fc secondary antibody (brand: BioLegend, item number: 409322), incubate at 4°C for 0.5 hours; wash 3 times with PBS, and detect by flow cytometry (flow cytometer brand: Beckman, model: Cytoflex).

[0444] The experimental results are shown in Figure 4 As shown in the table, 2C6.9-hz21 can specifically bind to human Claudin 18.2, but not to human Claudin 18.1.

[0445] 6.6 Complement-dependent cytotoxicity (CDC) detection of humanized antibody of 2C6.9

[0446] 2C6.9 is an IgG1 subtype, which can effectively activate the complement classical pathway and exert complement-dependent cytotoxicity (CDC) function. To determine the CDC effect of antibody 2C6.9, the application uses guinea pig serum (purchased from Zhengzhou Baiji, item number: S0001) rich in complement as the material for the experiment. The specific experimental steps are as follows: take HEK293T-Claudin 18.2 cells, adjust the cell density after centrifugation, plate 5×10 4 / well overnight; the next day, prepare DMEM+20% guinea pig serum medium, dilute 2C6.9-hz21 and IMAB362 antibody with this medium, start with 20 μg / mL, dilute 2 times, 10 concentration points; remove the original culture medium of HEK293T-Claudin 18.2 cells, add the diluted antibody in the previous step to the corresponding wells, 100 μL / well; set the positive control group for complete killing, that is, add 10 μl / well of lysis buffer; after incubation at 37°C in a 5% CO2 incubator for 3 hours, add CellTiter-Glo Luminescent (CTG, purchased from Promega, item number: G7573) staining solution, 50 μl / well, mix well for 30 seconds, place at room temperature for 1 minute, then measure the fluorescence signal value with a microplate reader (MD, SpectraMax M2), and import the results into Graphpad Prism for curve fitting.

[0447] The experimental results are shown inFigure 5 As shown in Table 6, the CDC activity of 2C6.9-hz21 is superior to that of the control antibody IMAB362.

[0448] Table 6: Detection of CDC activity of anti-Claudin 18.2 humanized antibody 2C6.9-hz21

[0449] Antibody name EC50 value (ng / mL) 2C6.9-hz21 1363 IMAB362 3317

[0450] 6.7 Antibody-dependent cellular cytotoxicity (ADCC) activity assay of humanized antibody of 2C6.9

[0451] 2C6.9 is of IgG1 subtype, and has strong antibody-dependent cell-mediated cytotoxicity (ADCC) activity. To detect the ADCC activity of 2C6.9-hz21, the present application uses the method of NK cell killing for detection. The specific experimental steps are as follows: HEK293T-Claudin 18.2 cells are taken, centrifuged, and the cell density is adjusted to 1 x 105cells / well for overnight plating; the next day, the culture medium in the plate is removed, NK92MI-CD16a cells (Huabio) are centrifuged, resuspended with empty MEMA medium, and the cell density is adjusted to 1 x 105cells / mL, 50 μl / well is added to the corresponding well; 2C6.9-hz21 and IMAB362 antibodies are diluted with empty MEMA medium, HEK293T-Claudin 18.2 cells are started at 40 μg / mL, 5-fold dilution, 10 concentration points; NUGC-4 cells are started at 2 mg / mL, 5-fold dilution, 11 concentration points; the diluted antibodies are added to the corresponding wells at 50 μl / well, and are placed in a 37°C, 5% CO2 cell incubator for incubation for 5.5 hours; then lysis buffer is added to the positive control wells, and after incubation for 0.5 hours, lactate dehydrogenase (LDH) detection reagent (Dongren Chemical, CK12) is added at 50 μl / well, and the enzyme marker (MD, SpectraMax M2) is read at 490 nm every 10 minutes, and the results are imported into Graphpad Prism for curve fitting. 4 cells / well for overnight plating; the next day, the culture medium in the plate is removed, NK92MI-CD16a cells (Huabio) are centrifuged, resuspended with empty MEMA medium, and the cell density is adjusted to 1 x 105cells / mL, 50 μl / well is added to the corresponding well; 2C6.9-hz21 and IMAB362 antibodies are diluted with empty MEMA medium, HEK293T-Claudin 18.2 cells are started at 40 μg / mL, 5-fold dilution, 10 concentration points; NUGC-4 cells are started at 2 mg / mL, 5-fold dilution, 11 concentration points; the diluted antibodies are added to the corresponding wells at 50 μl / well, and are placed in a 37°C, 5% CO2 cell incubator for incubation for 5.5 hours; then lysis buffer is added to the positive control wells, and after incubation for 0.5 hours, lactate dehydrogenase (LDH) detection reagent (Dongren Chemical, CK12) is added at 50 μl / well, and the enzyme marker (MD, SpectraMax M2) is read at 490 nm every 10 minutes, and the results are imported into Graphpad Prism for curve fitting. 6 cells / well for overnight plating; the next day, the culture medium in the plate is removed, NK92MI-CD16a cells (Huabio) are centrifuged, resuspended with empty MEMA medium, and the cell density is adjusted to 1 x 105cells / mL, 50 μl / well is added to the corresponding well; 2C6.9-hz21 and IMAB362 antibodies are diluted with empty MEMA medium, HEK293T-Claudin 18.2 cells are started at 40 μg / mL, 5-fold dilution, 10 concentration points; NUGC-4 cells are started at 2 mg / mL, 5-fold dilution, 11 concentration points; the diluted antibodies are added to the corresponding wells at 50 μl / well, and are placed in a 37°C, 5% CO2 cell incubator for incubation for 5.5 hours; then lysis buffer is added to the positive control wells, and after incubation for 0.5 hours, lactate dehydrogenase (LDH) detection reagent (Dongren Chemical, CK12) is added at 50 μl / well, and the enzyme marker (MD, SpectraMax M2) is read at 490 nm every 10 minutes, and the results are imported into Graphpad Prism for curve fitting.

[0452] The experimental results are shown in Table 7 and Figure 2. Figure 6 As shown in Table 7 and Figure 2, the ADCC cell killing activity of antibody 2C6.9-hz21 on HEK293T-Claudin 18.2 is superior to that of IMAB362.

[0453] Table 7: ADCC activity assay of anti-Claudin 18.2 humanized antibody 2C6.9-hz21

[0454] Antibody name EC50 value (ng / mL) Max killing rate (%) 2C6.9-hz21 67.67 30 IMAB362 57.66 20

[0455] Example Seven Preparation of a Claudin 18.2 targeting ADC (2C6.9-ADC)

[0456] The 2C6.9-TL001 was prepared by conjugating the TL001 prepared in Example Three with the humanized monoclonal antibody 2C6.9. The preparation method is as follows:

[0457] (1) Conjugation: 30 mg of 2C6.9-hz21 antibody was taken, 20 mM PB + 105 mM NaCl + 100 mM disodium edetate solution, pH 7.7 was added, the pH was adjusted to 7.7 with 2M Tris solution, then diluted with 20 mM PB + 105 mM NaCl pH 7.7 solution (the final concentration of disodium edetate was 5 mM, and the final concentration of antibody was 15 mg / mL), and mixed well; then, 10 mM TCEP solution was added and mixed well, and left to stand at room temperature for a certain period of time (30 or 90 minutes); TL001 dissolved in dimethyl sulfoxide was added to the above solution system (molar ratio of antibody: 5:1 or 9:1), mixed well, and left to stand at room temperature for 2 hours to obtain the conjugated sample, which was named 2C6.9-TL001.

[0458] (2) Buffer replacement: the 2C6.9-TL001 was subjected to buffer replacement with a 30KDa 50ml ultrafiltration tube (Millipore), and the replacement liquid was 10 mM histidine-histidine hydrochloride + 8% sucrose (pH 6.0) buffer, and the replacement ratio was 15 times; the sample was collected, and 10% Tween-20 was added to make the final concentration of Tween-20 in the sample 0.02% (M / V).

[0459] (3) Detection:

[0460] The 2C6.9-TL001 after replacement was subjected to LC-MS molecular weight analysis, and the conditions were as follows:

[0461] Chromatographic determination conditions:

[0462] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0463] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN;

[0464] Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0465] Flow rate: 0.25 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 1 μl;

[0466] Time (min) 2 20 22 25 26 30 Mobile phase A (%) 75 60 5 5 75 75 Mobile phase B (%) 25 40 95 95 25 25

[0467] Switching valve: 0-3 min to waste, 3-22 min to MS, 22-30 min to waste

[0468] Mass spectrometry measurement conditions:

[0469] Mass spectrometry model: AB Sciex Triple TOF 5600+;

[0470] Parameters: GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 200; CE 10; m / z 600-4000; Time bins to sum 40.

[0471] The theoretical molecular weight and the actual molecular weight of the light chain and the heavy chain of 2C6.9-TL001 obtained after coupling TL001 with 2C6.9-hz21 are shown in the following table (the heavy chain is calculated based on the main glycoform G0F):

[0472]

[0473] When the coupling feed ratio of TL001 and 2C6.9-hz21 is 5:1, the antibody light chain (LC) in 2C6.9-TL001 is coupled with 0-1 toxins (the LC, DAR1 ratio is 57.8%, 42.2% respectively), and the heavy chain (HC) is coupled with 0-4 toxins (the HC, DAR1, DAR2, DAR3, DAR4 ratio is 22.5%, 30.3%, 25.0%, 21.9%, 0.3% respectively), thus the coupling ratio (DAR) of the antibody and the toxin is 3.79, and the calculation formula is: DAR = light chain DAR1*2 + heavy chain (DAR1*1 + DAR2*2 + DAR3*3 + DAR4*4)*2.

[0474] When the coupling feed ratio of TL001 and 2C6.9-hz21 is 9:1, the antibody light chain in 2C6.9-TL001 is coupled with 0-1 toxins (the LC, DAR1 ratio is 7.5%, 92.5% respectively), and the heavy chain is coupled with 0-4 toxins (the HC, DAR1, DAR2, DAR3, DAR4 ratio is 2.5%, 10.2%, 9.8%, 76.4%, 1.1% respectively), thus the coupling ratio (DAR) of the antibody and the toxin is 7.12.

[0475] When TL001 was coupled with 2C6.9-hz21 at a coupling feed ratio of 9:1, after coupling and cation chromatography, the antibody light chain in 2C6.9-TL001 was coupled with 0-1 toxins (LC, DAR1 ratios were 1.5%, 24.0%, respectively), and the heavy chain was coupled with 0-4 toxins (HC, DAR1, DAR2, DAR3, DAR4 ratios were 0.7%, 2.1%, 14.3%, 54.5%, 2.8%, respectively), and the calculated antibody-toxin coupling ratio (DAR) of 2C6.9-TL001 was 7.40.

[0476] The molecular structure of the ADC in 2C6.9-TL001 is as follows:

[0477]

[0478] wherein γ is an integer of 1-10, and A is 2C6.9-hz21.

[0479] SEC detection was performed on the conjugate by SEC-HPLC.

[0480] Chromatographic conditions:

[0481] Liquid chromatography column: TSKgel G3000SWxl, 300*7.8mm, 5μm;

[0482] Mobile phase: 90mmol / L Na2HPO4, 30mmol / L NaH2PO4, 200mM NaCl, 5% acetonitrile;

[0483] Flow rate: 0.8ml / min; detection wavelength: 280nm; column temperature: room temperature; sample chamber temperature: 8°C;

[0484] Injection volume: 40μl; isocratic run: 30min.

[0485] The SEC chromatogram of 2C6.9-TL001 (DAR: 3.79) and 2C6.9-TL001 (DAR: 7.12) is shown in FIG. 1, and the SEC chromatogram of 2C6.9-TL001 (DAR: 7.40) is shown in FIG. 2. Figure 7-8 Figure 13 According to the SEC retention time and peak area ratio, it was confirmed that the main coupling product had a molecular weight of about 150kD, i.e., 2C6.9-TL001 obtained by coupling TL001 with 2C6.9-hz21, which still maintained the intact structure of the antibody.

[0486] ​TL002, TL003 prepared in Example Four and Example Five were coupled with humanized monoclonal antibody 2C6.9 by using the same preparation method, to prepare 2C6.9-TL002, 2C6.9-TL003, the preparation and detection method is as described above, and the DAR value of 2C6.9-TL002, 2C6.9-TL003 is 6.95, 7.03 respectively.

[0487] The ADC molecule structure in 2C6.9-TL002 is as follows:

[0488]

[0489] wherein γ is an integer of 1-10, and A is 2C6.9-hz21.

[0490] The ADC molecule structure in 2C6.9-TL003 is as follows:

[0491]

[0492] wherein γ is an integer of 1-10, and A is 2C6.9 antibody.

[0493] Affinity detection of 2C6.9-TL001 in Example Eight

[0494] The present application adopts cell ELISA method to detect the affinity of 2C6.9-TL001 (DAR: 7.12) to cell membrane surface Claudin 18.2. The specific experimental steps are as follows: the adherent growth L929-Claudin 18.2 cell line is digested with 2 mM EDTA, resuspended to 2*10^5 cells / mL, 100 μL per well is plated in a 96-well plate, and incubated at 37°C overnight; the next day, remove the culture medium, wash once with PBS, add 100 μL of 4% formaldehyde to each well and fix at room temperature for 30 minutes, remove the formaldehyde, wash twice with PBS, and add 100 μL of PBS containing 2% BSA to each well and incubate for 2 hours; remove the blocking solution, dilute the 2C6.9-TL001 to be tested with PBS (containing 2% BSA), start with 9.375 μg / mL, dilute 4 times, 9 concentration points, 100 μL per well, and incubate at 37°C for 2 hours; wash 5 times with 250 μL of PBST, and stand for 2 minutes each time; dilute the horseradish peroxidase (HRP) labeled anti-human IgG secondary antibody (HRP-anti-Human IgG, Jackson ImmunoResearch) with PBS (2% BSA) to 1:10000, 100 μL per well, and incubate at 37°C for 1 hour; wash 6 times with 250 μL of PBST, and stand for 2 minutes each time; add 100 μL of TMB color developing liquid (Thermo) to the corresponding wells, and develop at 37°C for 20 minutes; add 50 μL of 2 mol / L H2SO4 to stop the reaction, and read at 450 nm on an enzyme label instrument (MD) and import into Graphpad Prism for curve fitting. The experimental results are shown in Figure 9 The EC50 value of 2C6.9-TL001 (DAR: 7.12) to cell membrane surface Claudin 18.2 affinity is 39.94 ng / mL. After conjugation with TL001, the antibody 2C6.9-hz21 still has excellent affinity to Claudin 18.2. 50 The EC50 value of 2C6.9-TL001 (DAR: 7.12) to cell membrane surface Claudin 18.2 affinity is 39.94 ng / mL. After conjugation with TL001, the antibody 2C6.9-hz21 still has excellent affinity to Claudin 18.2.

[0495] Example Nine Detection of the killing activity of 2C6.9-ADC on Claudin 18.2 highly expressed tumor cell lines

[0496] The present application selects HEK293T-Claudin 18.2, HEK293T-Claudin 18.1 cells to detect the killing activity of 2C6.9-TL001 on Claudin 18.2 high expression cell lines. The specific experimental steps are as follows: the day before the experiment, dilute the cells with DMEM+10%FBS, 100ul / well, 1*10^4 cells per well, and plate overnight; the next day, dilute the ADC molecules with DMEM base medium, 150ug / mL (DAR:7.12) or 262.5ug / mL (DAR:3.79) as the starting point, 4-fold dilution, 11 concentration points, add to the corresponding wells, 100ul per well, and the final serum concentration is 5%; incubate in a 37℃, 5%CO2 incubator for 48 hours; add CCK8 (Rhinogen), 20ul / well, incubate in a 37℃, 5%CO2 incubator for 0.5-2.5 hours, take readings every half hour with an enzyme marker (MD) at 450nm and import Graphpad Prism for curve fitting.

[0497] The experimental results are shown in Figure 10A , Figure 10B and Figure 10C , the 2C6.9-TL001 molecule can effectively kill HEK293T-Claudin 18.2 cells, when the DAR value is 7.12, the EC 50 value is 473ng / mL; when the DAR value is 3.79, the EC 50 value is 794.1ng / mL. At the same time, the killing activity EC50 value of 2C6.9-TL001 (DAR:7.12) molecule on HEK293T-Claudin 18.1 is 3900ng / mL; the killing activity of 2C6.9-TL001 (DAR:7.12) molecule on Claudin 18.2 cells is obviously higher than that on Claudin 18.1 cells (about 8 times difference), indicating that this killing effect is specific to Claudin 18.2.

[0498] The same experimental method as described above is used to detect the killing activity of 2C6.9-TL002 and 2C6.9-TL003 on HEK293T-Claudin 18.2 and HEK293T-Claudin 18.1 cells. The experimental results are shown in Figure 10D , Figure 10E , 2C6.9-TL002 and 2C6.9-TL003 can effectively kill HEK293T-Claudin 18.2 cells, and the EC 50The EC50 values of 2C6.9-TL002 and 2C6.9-TL003 against HEK293T-Claudin 18.1 killing activity were 628.9 ng / mL and 540.2 ng / mL, respectively. The killing activity of 2C6.9-TL002 and 2C6.9-TL003 against HEK293T-Claudin 18.2 cells was significantly higher than that against Claudin 18.1 cells, indicating that the killing effect of 2C6.9-TL002 and 2C6.9-TL003 was specific to Claudin 18.2.

[0499] Example 10 Detection of killing activity of 2C6.9-antibody-drug conjugate on endogenous Claudin 18.2 cell line

[0500] The present application selects gastric cancer cell line NUGC-4 to detect the killing activity of 2C6.9-TL001 on endogenous Claudin 18.2 cells. The specific experimental steps are as follows: the day before the experiment, dilute the cells with RPMI 1640+10% FBS, 100 μL / well, 1*10^4 cells per well, and plate overnight; the next day, dilute 2C6.9-TL001 molecules with RPMI 1640 medium, starting with 1000 μg / mL (DAR 7.12) or 1750 μg / mL (DAR: 3.79), 3-fold dilution, 11 concentration points, add to the corresponding wells, 100 μL per well, and the final serum concentration is 5%; incubate in a 37°C, 5% CO2 incubator for 72 hours; add CCK8 (Rhinogen), 20 μl / well, incubate in a 37°C, 5% CO2 incubator for 0.5-2.5 hours, read every half hour on a microplate reader (MD) at 450 nm and import into Graphpad Prism for curve fitting. The experimental results are shown in Figure 11A and 11B As shown, 2C6.9-TL001 molecules can effectively kill NUGC-4 cells, and when the DAR value is 7.12, the EC 50 value is 2.383 μg / mL; when the DAR value is 3.79, the EC 50 value is 10.01 μg / mL.

[0501] The killing activity of 2C6.9-TL002 and 2C6.9-TL003 on NUGC-4 cells was detected by a similar experimental method as described above, and the starting concentration of ADC was 500 μg / mL, 4-fold dilution, 11 concentration points. The experimental results are shown in Figure 11C As shown, 2C6.9-TL002 and 2C6.9-TL003 can effectively kill NUGC-4 cells, and the EC 50 values are 54.92 μg / mL and 123.94 μg / mL, respectively.

[0502] Example 11 2C6.9-hz21 antibody internalization detection

[0503] The present application selects NUGC-4 cells to detect the internalization activity of 2C6.9 antibody. The specific experimental steps are as follows: take NUGC-4 cells, trypsinize and count, resuspend the cells with PBS (containing 1% BSA), adjust the cell density to 3x10 6 / mL; take 100 μL of resuspended cells, add 2C6.9-hz21 antibody at a final concentration of 100 μg / mL, use isotype Human IgG as negative control, and incubate on ice for 1 hour; after incubation, wash 3 times with pre-cooled PBS, resuspend the cells with NUGC-4 cell culture medium (1640+10% FBS) and divide the cells into two parts, one part is incubated at 37°C for 4 hours (endocytosis group), and the other part continues to be incubated on ice for 4 hours (affinity group); after incubation, wash 3 times with pre-cooled PBS, resuspend the cells with 50 uL PBS (containing 1% BSA), add anti-human fluorescent secondary antibody (BioLegend), and incubate at 4°C for half an hour; after incubation, wash 3 times with pre-cooled PBS, and detect by flow cytometry (Beckman). According to the formula: endocytosis (%) = [1-(MFI 37℃抗体组 -MFI 37℃对照组 ) / (MFI 冰上抗体组 –MFI 冰上对照组 )]x100%, the internalization rate of the antibody is calculated. The experimental results show that the internalization rate of 2C6.9-hz21 on NUGC-4 cells for 4 hours is 37.79%. It shows that the conjugate after conjugation of 2C6.9-hz21 with drugs has the potential to internalize drugs into cells and kill tumor cells.

[0504] Example 12 2C6.9-antibody-drug conjugate in vivo efficacy detection

[0505] The present application uses Cancer cell line-derived xenograft (CDX) model and patient-derived xenograft (PDX) model to evaluate the anti-tumor effect of ADC molecules.

[0506] 12.1 NCI-N87-Claudin18.2+Balb / c Nude mouse CDX model NCI-N87-Claudin18.2 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37°C, 5% CO2. Exponential growth phase cells were collected, resuspended with PBS, and inoculated into the right flank of Balb / c nude mice at a dose of 5x10 6Cells (suspended in 0.1 ml PBS) were subcutaneously injected into female Balb / cNude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) to establish a subcutaneous xenograft tumor model. The tumors were allowed to grow to an average volume of 70–100 mm². 3 Animals were randomly assigned to groups of seven based on tumor volume. The day of group assignment was designated Day 0. The groups were: Human IgG1 isotype control antibody (negative control) group (IgG1), 2C6.9-TL001 (DAR: 7.12) 1 mg / kg and 3 mg / kg groups, and 2C6.9-TL002 (DAR: 6.95) 1 mg / kg and 3 mg / kg groups. All samples were administered via tail vein injection twice weekly for a total of six administrations.

[0507] After administration, the tumor diameter was measured twice a week using calipers, and the tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[0508] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the efficacy of tumor suppression:

[0509] TGI(%) = [1-(V T末 -V T始 ) / (V C末 -V C始 )]*100%

[0510] Where V T末 Mean tumor volume at the end of the experiment in the treatment group

[0511] V T始 Mean tumor volume at the start of treatment in the treatment group

[0512] V C末 Mean tumor volume at the end of the experiment in the negative control group

[0513] V C始 Mean tumor volume at the start of drug administration in the negative control group

[0514] The relative tumor proliferation rate (T / C%) was calculated using the following formula to evaluate the efficacy of tumor suppression:

[0515] T / C (%) (tumor volume) = (T t / T0) / (C t / C0)×100%

[0516] Where, T0: the average tumor volume of the treatment group at the initial stage (i.e., P0).

[0517] T tAverage tumor volume of the treatment group at each measurement

[0518] C0: Average tumor volume of the negative control group at the initial (i.e. P0) time

[0519] C t Average tumor volume of the negative control group at each measurement.

[0520] The experimental results are shown in Table 8 and Figure 12A , 12B 2C6.9-TL001 (DAR: 7.12) had a significant inhibitory effect on the growth of NCI-N87-Claudin18.2 gastric cancer xenograft tumor model, and was dose-dependent. After 6 doses (Day 21), the tumor inhibition rate (TGI) of the 2C6.9-TL001 1 mg / kg group was as high as 96.03%, and 4 mice had partial tumor regression; while the TGI of the 3 mg / kg group reached 133.50%, 3 mice had partial tumor regression, and 4 mice had complete tumor regression. The TGI of the 2C6.9-TL002 3 mg / kg group was 40.11%, and the 1 mg / kg group had no significant tumor inhibition effect. All mice in the treatment groups had no significant weight loss during the observation period, and the animals had good tolerance.

[0521] Table 8: NCI-N87-Claudin18.2+Balb / c Nude Mouse CDX Model

[0522]

[0523] Note: TGI: Tumor Growth Inhibition Rate; T / C: Relative Tumor Proliferation Rate; PR: Partial Tumor Regression; CR: Complete Tumor Regression. P value is the comparison result with IgG1 group; N / A: Not applicable; ns: P>0.05, no statistical difference.

[0524] 12.2 2C6.9-TL001 and 2C6.9 Monoclonal Antibody + Chemotherapy CDX Model In Vivo Efficacy Comparison

[0525] The NCI-N87-Claudin18.2 subcutaneous xenograft tumor model was established and grouped according to the method in Experimental Example 12.1. The day of grouping was recorded as Day 0, and the groups were Human IgG1 isotype control antibody (negative control) group (referred to as IgG1), paclitaxel group (albumin-bound type), 2C6.9 monoclonal antibody combined with paclitaxel group, and 2C6.9-TL001 (DAR: 7.12) group. All samples were injected intravenously, twice a week, for a total of 3 weeks of administration, and the doses are shown in Table 9.

[0526] The experimental results are shown in Table 9 and Figure 12CAs shown, after 11 days of administration, compared with the negative control group, the tumor growth of the three administration groups was significantly inhibited, and the 2C6.9-TL001 (DAR: 7.12) group was the most significant. The tumor inhibition rate (TGI) of the 2C6.9-TL001 (DAR: 7.12) group was as high as 121.68%, and the tumors of 6 out of 7 mice partially regressed.

[0527] Table 9: Comparison of in vivo efficacy of 2C6.9-TL001 and 2C6.9 mAb + chemotherapy in CDX model

[0528]

[0529] Note: TGI: tumor growth inhibition rate; T / C: relative tumor proliferation rate; PR: partial regression of tumor; CR: complete regression of tumor. P value is the comparison result with the IgG1 group; N / A: not applicable; ns: P > 0.05, no statistical difference.

[0530] After 21 days of administration, compared with the negative control group, the tumor growth of the three administration groups was significantly inhibited, and the 2C6.9-TL001 (DAR: 7.12) group was the most significant. The tumor inhibition rate (TGI) of the 2C6.9-TL001 (DAR: 7.12) group was as high as 125.73%, and the tumors of 5 out of 7 mice completely regressed, and the tumors of 2 mice partially regressed. The experimental results are shown in Table 10 and Figure 12D .

[0531] Table 10: Comparison of in vivo efficacy of 2C6.9-TL001 and 2C6.9 mAb + chemotherapy in CDX model

[0532]

[0533] Note: TGI: tumor growth inhibition rate; T / C: relative tumor proliferation rate; PR: partial regression of tumor; CR: complete regression of tumor. P value is the comparison result with the IgG1 group; N / A: not applicable.

[0534] 12.3 Comparison of in vivo efficacy of 2C6.9-TL001 with different DAR values in CDX model

[0535] NUGC-4 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37℃, 5% CO2. Exponential growth phase cells were collected, resuspended with PBS, and inoculated subcutaneously into female Balb / c Nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) at a cell amount of 5x10 6 / mouse (suspended in 0.1 ml PBS) to establish a subcutaneous tumor model. When the average tumor volume reached 70-100 mm 3When the tumor volume was randomly grouped, 7 in each group. The day of grouping was recorded as Day 0 (Day 0), and the group was Human IgG1 isotype control antibody (negative control) group (referred to as IgG1), 2C6.9-TL001 (DAR: 3.79) 5.25 mg / kg group, 2C6.9-TL001 (DAR: 3.79) 17.5 mg / kg group, 2C6.9-TL001 (DAR: 7.12) 3 mg / kg group, 2C6.9-TL001 (DAR: 7.12) 10 mg / kg group (different DAR value ADC drugs were designed according to the same toxin load). All samples were injected into the tail vein, twice a week, for a total of 3 weeks of drug administration, and the dose is shown in Table 11.

[0536] The experimental results are shown in Table 11 and Figure 12E 、 12F Under the condition of the same toxin load, after 21 days of administration, the efficacy of 2C6.9-TL001 high DAR value (7.12) and low DAR value (3.79) was basically the same. All treatment groups of mice had no obvious weight loss during the observation period, and the animal tolerance was good.

[0537] Table 11: Comparison of in vivo efficacy of 2C6.9-TL001 CDX model with different DAR values

[0538]

[0539] Note: TGI: Tumor growth inhibition rate; T / C: Relative tumor proliferation rate; PR: Partial regression of tumor; CR: Complete regression of tumor. P value is the comparison result with IgG1 group; N / A: Not applicable.

[0540] 12.4 Gastric cancer GA0006 + Balb / c nude mouse PDX model

[0541] From Gastric cancer xenograft model GA0006 (Zhongmei Guanke Biotechnology (Taicang) Co., Ltd., gastric tumor with high expression of Claudin 18.2 derived from a 57-year-old female patient) tumor-bearing mice were collected tumor tissues, cut into tumor blocks with a diameter of 3x3x3mm and inoculated subcutaneously in the right anterior scapular region of Balb / c nude mice. When the average tumor volume of tumor-bearing mice reached about 150-250mm 3Day 0). The groups were 3 groups: Human IgG1 isotype control antibody (negative control) group (IgG1 10 mg / kg), 2C6.9-TL001-DAR7.12 3 mg / kg group and 10 mg / kg group. All samples were injected via tail vein, twice a week, for a total of 5 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly, and the measurement method is described in Experimental Example 12.1.

[0542] The experimental results are shown in Table 12 and Figure 12G 、 12H Table 12: Tumor growth inhibition of 2C6.9-TL001-DAR7.12 in gastric cancer GA0006 + Balb / c Nude mice PDX model

[0543] Table 12: Gastric cancer GA0006 + Balb / c Nude mice PDX model

[0544]

[0545] Note: TGI: Tumor growth inhibition rate; T / C: Relative tumor proliferation rate; PR: Partial tumor regression; CR: Complete tumor regression. P value is the comparison result with IgG1 group; N / A: Not applicable.

[0546] The experimental results are shown in Table 13 and Figure 12I 、 12J Table 13: Tumor growth inhibition of 2C6.9-TL001-DAR7.12 in gastric cancer GA0006 + Balb / c Nude mice PDX model

[0547] Table 13: Gastric cancer GA0006 + Balb / c Nude mice PDX model

[0548]

[0549] Note: TGI: Tumor growth inhibition rate; T / C: Relative tumor proliferation rate; PR: Partial tumor regression; CR: Complete tumor regression. P value is the comparison result with IgG1 group; N / A: Not applicable.

[0550] 12.5 In vivo efficacy evaluation of NCI-N87-Claudin18.2+ Balb / c Nude mice CDX model

[0551] The NCI-N87-Claudin18.2 subcutaneous transplantation tumor model was established according to the method in Experimental Example 12.1. When the average tumor volume reached 140mm 3 left and right, randomly grouped according to tumor volume, 8 mice per group. The grouping day was recorded as Day 0 (Day 0), and the groups were Human IgG1 isotype control antibody (negative control) group (referred to as IgG1), 2C6.9-TL001 (DAR: 7.40) 0.3mg / kg group, 1mg / kg group and 3mg / kg group. All samples were injected intravenously through the tail vein, twice a week, for a total of 6 doses, and the doses are shown in Table 14.

[0552] The experimental results are shown in Table 14 and Figure 12K and 12L After 6 doses, the drug was stopped and the observation was extended to Day 31 (31 days after the first dose). Compared with the negative control group, the tumor inhibition rate (TGI) of the 2C6.9-TL001 0.3mg / kg group was 34.04%; while the TGI of the 1mg / kg group and the 3mg / kg group was as high as 122.57% (all mice had partial tumor regression) and 184.22% (4 mice had complete tumor regression and 4 mice had partial tumor regression), respectively. All treated mice had no significant weight loss during the observation period, and the animals had good tolerance.

[0553] Table 14: In vivo efficacy evaluation of NCI-N87-Claudin18.2+ Balb / c Nude mice CDX model

[0554]

[0555] Note: TGI: Tumor growth inhibition rate; T / C: Relative tumor proliferation rate; PR: Partial tumor regression; CR: Complete tumor regression. P value is the comparison result with IgG1 group; N / A: Not applicable.

[0556] 12.6 In vivo efficacy evaluation of HEK293T-Claudin18.2+ Balb / c Nude mice CDX model

[0557] HEK293T-Claudin18.2 cells (human embryonic kidney cells) were cultured in DMEM medium containing 3 pg / mL puromycin and 10% fetal bovine serum (FBS) at 37°C, 5% CO2. Exponentially growing cells were collected, resuspended in PBS, and subcutaneously inoculated into female Balb / c Nude mice (Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd.) at a cell amount of 1 x 106 cells (suspended in 0.1 ml PBS) to establish a subcutaneous xenograft model. When the average tumor volume reached 120-140 mm3, the mice were randomly divided into groups according to the tumor volume, with 8 mice in each group. The day of grouping was recorded as Day 0, and the groups were as follows: 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. All samples were injected intravenously via the tail vein, twice a week, for a total of 6 doses, and the doses are shown in Table 15. 7 3 The day of grouping was recorded as Day 0, and the groups were as follows: 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. All samples were injected intravenously via the tail vein, twice a week, for a total of 6 doses, and the doses are shown in Table 15.

[0558] The results are shown in Table 15 and Figure 12M 12N As shown in Table 15 and

[0559] Table 15: In vivo efficacy evaluation of HEK293T-Claudin18.2+Balb / c Nude mouse CDX model

[0560]

[0561]

[0562] Note: TGI: tumor growth inhibition rate; T / C: relative tumor proliferation rate; PR: partial tumor regression; CR: complete tumor regression. P value is the comparison result with the IgG1 group; N / A: not applicable.

[0563] 12.7 NUGC-4+Balb / c Nude mouse CDX model in vivo efficacy evaluation

[0564] ​​NUGC-4 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37°C in 5% CO2. Exponentially growing cells were collected, resuspended in PBS, and subcutaneously inoculated into female Balb / c Nude mice (Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd.) at a cell amount of 5x10 6 / per mouse (suspended in 0.1 ml PBS) to establish a subcutaneous xenograft model. When the average tumor volume reached 80 mm 3 around, the mice were randomly divided into groups according to the tumor volume, with 8 mice in each group. The day of grouping was recorded as Day 0, and the groups were as follows: 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. All samples were injected intravenously via the tail vein, twice a week, for a total of 6 doses, and the doses are shown in Table 16.

[0565] The experimental results are shown in Table 16 and Figure 12O and 12P Compared with the negative control group, 2C6.9-TL001 (DAR: 7.40) had a significant inhibitory effect on the growth of NUGC-4 gastric cancer xenograft models in a dose-dependent manner after 6 doses (21 days after the first dose). The tumor inhibition rate (TGI) of the 2C6.9-TL001 3 mg / kg group was 56.62%, and the TGI of the 10 mg / kg group was 90.28% (tumor partially regressed in 2 mice).

[0566] Table 16: In vivo efficacy evaluation of NUGC-4 + Balb / c Nude mouse CDX model

[0567]

[0568] Note: TGI: tumor growth inhibition rate; T / C: relative tumor proliferation rate; PR: partial tumor regression; CR: complete tumor regression. P value is the comparison result with the IgG1 group; N / A: not applicable.

[0569] In summary, whether it is a CDX model or a PDX model, 2C6.9-TL001 in the present application can effectively inhibit the growth of Claudin18.2-positive tumors in a dose-dependent manner, and has good safety.

[0570] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed herein, and these changes are within the scope of protection of the present application. The scope of protection of the present application is given by the appended claims and any equivalents thereof.

Claims

1. An antibody-drug conjugate, the structure of which is shown in formula (I), (D-L) γ -A Formula (I) in, D represents the bioactive molecular fragment; L represents the linker; γ is selected from an integer between 1 and 10; A is an antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2, wherein the antibody or antigen-binding fragment thereof comprises: (1) The following VH and VL, where CDR is defined according to the IMGT numbering system: A VH containing the following three CDRs: CDR-H1 with sequence SEQ ID No: 1, CDR-H2 with sequence SEQ ID No: 2 or 21, and CDR-H3 with sequence SEQ ID No: 3; and, A VL containing the following three CDRs: CDR-L1 with sequence SEQ ID No: 4, CDR-L2 with sequence SEQ ID No: 5, and CDR-L3 with sequence SEQ ID No: 6; or, (2) The following VH and VL, where CDR is defined according to the AbM numbering system: A VH containing the following three CDRs: CDR-H1 with sequence SEQ ID No: 7, CDR-H2 with sequence SEQ ID Nos: 8 or 22, and CDR-H3 with sequence SEQ ID No: 9; and, A VL containing the following three CDRs: CDR-L1 with sequence SEQ ID No: 10, CDR-L2 with sequence SEQ ID No: 11, and CDR-L3 with sequence SEQ ID No:

12.

2. The antibody-drug conjugate according to claim 1, wherein: γ is selected from an integer between 1 and 8.

3. The antibody-drug conjugate according to claim 2, wherein: γ can be 1, 2, 3, 4, 5, 6, 7 or 8.

4. The antibody-drug conjugate according to claim 1, wherein: The antibody or its antigen-binding fragment comprises VH as shown in SEQ ID NOs: 13 or 14; and / or, the antibody or its antigen-binding fragment comprises VL as shown in SEQ ID NO:

15.

5. The antibody-drug conjugate according to claim 1, wherein, The antibody comprises: (1) CH of human immunoglobulin or a variant thereof, said variant having at most 10 amino acid substitutions, deletions, or additions compared to the wild-type sequence from which it is derived; and / or, (2) CL of human immunoglobulin or a variant thereof, wherein the variant has a substitution, deletion or addition of up to 10 amino acids compared with the wild-type sequence from which it is derived.

6. The antibody-drug conjugate according to claim 5, wherein, The antibody comprises: (1) CH of human immunoglobulin or a variant thereof, said variant having at most 5 amino acid substitutions, deletions, or additions compared to the wild-type sequence from which it is derived; and / or, (2) CL of human immunoglobulin or a variant thereof, wherein the variant has a substitution, deletion or addition of up to 5 amino acids compared to the wild-type sequence from which it is derived.

7. The antibody-drug conjugate according to claim 6, wherein, The antibody comprises: (1) CH of human immunoglobulin or a variant thereof, said variant having one, two, three, four or five amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived; and / or, (2) CL of human immunoglobulin or a variant thereof, wherein the variant has one, two, three, four or five amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived.

8. The antibody-drug conjugate according to claim 5, wherein, CH is the constant region of the IgG heavy chain.

9. The antibody-drug conjugate according to claim 8, wherein, The CH is the constant region of the IgG1, IgG2, IgG3, or IgG4 heavy chain.

10. The antibody-drug conjugate according to claim 5, wherein, The antibody contains the heavy chain constant region of human IgG1.

11. The antibody-drug conjugate according to claim 5, wherein, The antibody comprises CH as shown in SEQ ID NO: 16 or a variant thereof, the variant having a conservative substitution of up to 10 amino acids compared to SEQ ID NO:

16.

12. The antibody-drug conjugate according to claim 11, wherein, The antibody comprises CH as shown in SEQ ID NO: 16 or a variant thereof, the variant having a conservative substitution of up to 5 amino acids compared to SEQ ID NO:

16.

13. The antibody-drug conjugate according to claim 12, wherein, The antibody comprises CH as shown in SEQ ID NO: 16 or a variant thereof, the variant having a conserved substitution of 1, 2, 3, 4 or 5 amino acids compared to SEQ ID NO:

16.

14. The antibody-drug conjugate according to claim 5, wherein, The CL is the constant region of the κ light chain.

15. The antibody-drug conjugate according to claim 5, wherein, The antibody comprises CL or a variant thereof as shown in SEQ ID NO: 17, the variant having a conservative substitution of up to 10 amino acids compared to SEQ ID NO:

17.

16. The antibody-drug conjugate according to claim 15, wherein, The antibody comprises CL or a variant thereof as shown in SEQ ID NO: 17, the variant having a conservative substitution of up to 5 amino acids compared to SEQ ID NO:

17.

17. The antibody-drug conjugate according to claim 16, wherein, The antibody comprises CL or a variant thereof as shown in SEQ ID NO: 17, the variant having a conserved substitution of 1, 2, 3, 4 or 5 amino acids compared to SEQ ID NO:

17.

18. The antibody-drug conjugate according to claim 5, wherein, The antibody comprises CH as shown in SEQ ID NO: 16 and / or CL as shown in SEQ ID NO:

17.

19. The antibody-drug conjugate according to claim 1, wherein, The antibody is: (1) The heavy chain comprising VH of the sequence shown in SEQ ID NO: 13 and CH of the sequence shown in SEQ ID NO: 16, and the light chain comprising VL of the sequence shown in SEQ ID NO: 15 and CL of the sequence shown in SEQ ID NO: 17; or, (2) The heavy chain comprising VH of the sequence shown in SEQ ID NO: 14 and CH of the sequence shown in SEQ ID NO: 16, and the light chain comprising VL of the sequence shown in SEQ ID NO: 15 and CL of the sequence shown in SEQ ID NO:

17.

20. The antibody-drug conjugate according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: (1) Heavy chain, which contains an amino acid sequence selected from the following: The sequence shown in SEQ ID NO: 18 or SEQ ID NO: 19; and (2) Light chain, which contains an amino acid sequence selected from the following: The sequence shown in SEQ ID NO:

20.

21. The antibody-drug conjugate according to claim 1, wherein, The antibody or its antigen-binding fragment is selected from ScFv, Fab, Fab', F(ab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

22. The antibody-drug conjugate according to any one of claims 1-21, having the structure shown in formula (II), {D-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A Equation (II) in, L1 is Each of R1 and R2 is independently hydrogen, halogen, carboxylic acid, sulfonic acid, cyano, or C. 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 2-10 alkenyl or C 2-10 Alkyne group; Z1 is an amino acid or a peptide composed of 2-10 amino acids; x1 and x2 are each independently 0, 1, 2, 3, 4, 5 or 6; and L1 is connected to D at position 1 and L2 at position 2. L2 is Where y1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and L2 is connected to L1 at position 1 and L2 is connected to L3 at position 2. L3 is selected from 5-12 heterocyclic aromatic rings; L4 is Among them, Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 etyne and C 3-8 Cycloalkylene; R3 is selected from hydrogen and C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene; or, R3 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position and to L3 at the 1 position; E is In this case, each R4 is independently hydrogen, β is 0, 1, 2, 3, 4, 5 or 6, and E is connected to A at position 2 and to L4 at position 1. m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; A is as defined in any one of claims 1-21; D and γ are as defined in claim 1.

23. The antibody-drug conjugate according to any one of claims 1-21, having the structure shown in formula (III): {D-[(L1') m4 -L1-(L5) m5 -(L3) m2 -(L4) m3 -E]} γ -A Equation (III) in, L1' is R5 and R6 are each independently hydrogen or C. 1-6 Alkyl group; x3 is 1, 2, 3, 4, 5 or 6; and if L1' is present, it is connected to D at position 1 and to L1 at position 2; L1 is Each of R1 and R2 is independently hydrogen, halogen, carboxylic acid, sulfonic acid, cyano, or C. 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 2-10 alkenyl or C 2-10 Alkyne group; Z1 is an amino acid or a peptide consisting of 2-10 amino acids; x1 and x2 are each independently 0, 1, 2, 3, 4, 5 or 6; and when L1' is present, L1 is connected to L1' at position 1, and when L1' is absent, L1 is connected to D at position 1; L1 is connected to L5 at position 2. L5 is Wherein, R7 is hydrogen or C 1-6 Alkyl group, or R7 connected to the N atom on its γ-C to form a 5-6 membered heterocyclic group; 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 position 1 and to L3 at position 2. L3 is selected from 5-12 heterocyclic aromatic rings; L4 is Z2 is selected from C. 1-6 Alkylene, C 2-10 imidene group, C 2-10 etyne and C 3-8 Cycloalkylene; R3 is selected from hydrogen and C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene; or, R3 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position and to L3 at the 1 position; E is In this case, each R4 is independently hydrogen, β is 0, 1, 2, 3, 4, 5 or 6, and E is connected to A at position 2 and to L4 at position 1. m5, m2, m3 and m4 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; A is as defined in any one of claims 1-21; D and γ are as defined in claim 1.

24. The antibody-drug conjugate according to claim 22, wherein, L1 is Z1 is an amino acid or a peptide composed of 2-5 amino acids, wherein the amino acid is selected from Lys, Cit, Val, D-Val, Phe, Leu, Gly, Ala and Asn.

25. The antibody-drug conjugate according to claim 24, wherein, Z1 is selected from Cit, Lys, Cit-Val, and Ala-Val.

26. The antibody-drug conjugate according to claim 25, wherein, L1 is 27. The antibody-drug conjugate according to claim 25, wherein, L1 is 28. The antibody-drug conjugate according to claim 22, wherein, L2 is And m1 is 1.

29. The antibody-drug conjugate according to claim 22, wherein, L3 is a 5-6 membered heteroaryl ring, and m2 is 1.

30. The antibody-drug conjugate according to claim 29, wherein, L3 is triazole, and m2 is 1.

31. The antibody-drug conjugate according to claim 22, wherein, L4 is Z2 is C 1-6 Alkylene, Z3 is C 1-6 Alkylene, and m3 is 1.

32. The antibody-drug conjugate according to claim 31, wherein, L4 is And m3 is 1.

33. The antibody-drug conjugate according to claim 23, wherein, L1' is 34. The antibody-drug conjugate according to claim 23, wherein, L5 is Where x4 is 1, 2, 3, 4, 5 or 6; y1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

35. The antibody-drug conjugate according to claim 34, wherein, L5 is 36. The antibody-drug conjugate according to any one of claims 1-21, wherein, D is 37. The antibody-drug conjugate according to claim 36, wherein, D is 38. The antibody-drug conjugate according to claim 22, wherein, In equation (II), D-[L1-(L2)] m1 -(L3) m2 -(L4) m3 -E]-for 39. The antibody-drug conjugate according to claim 23, wherein, In equation (III), D-[(L1') m4 -L1-(L5) m5 -(L3) m2 -(L4) m3 -E]-for 40. The antibody-drug conjugate according to any one of claims 1-21, wherein the antibody-drug conjugate is selected from: in, γ is selected from integers between 1 and 10.

41. The antibody-drug conjugate according to claim 40, wherein γ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

42. The antibody-drug conjugate according to claim 41, wherein, The antibody or its antigen-binding fragment is labeled.

43. The antibody-drug conjugate according to claim 42, wherein, The antibody or its antigen-binding fragment carries a detectable marker.

44. The antibody-drug conjugate of claim 43, wherein the detectable label is an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, or biotin.

45. A composition comprising the antibody-drug conjugate according to any one of claims 1-44, wherein the molar ratio (DAR value) of the bioactive molecular fragment in the composition to the antibody or its antigen-binding fragment that specifically binds to CLDN18.2 is a decimal or integer between 1 and 10.

46. ​​The composition according to claim 45, wherein the molar ratio (DAR value) of the bioactive molecular fragment in the composition to the antibody or its antigen-binding fragment that specifically binds to CLDN18.2 is a decimal or an integer between 3 and 8.

47. The composition according to claim 45, wherein the molar ratio (DAR value) of the bioactive molecular fragment in the composition to the antibody or its antigen-binding fragment specifically binding to CLDN18.2 is 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.7, 9, 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.

48. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1-44, or a composition according to any one of claims 45-46, and a pharmaceutically acceptable carrier and / or excipient.

49. Use of the antibody-drug conjugate of any one of claims 1-44, the composition of any one of claims 45-46, or the pharmaceutical composition of claim 48 in the preparation of a medicament for the prevention and / or treatment and / or adjuvant treatment of tumors or cancer, optionally, the medicament further comprising additional antitumor active ingredients.

50. The use according to claim 49, wherein, The antibody-drug conjugate, composition, or pharmaceutical composition is administered separately, simultaneously, or sequentially with other antitumor active ingredients.

51. The use according to claim 50, wherein, The other antitumor active ingredient is a bioactive polypeptide or its active fragment, or a chemotherapy drug.

52. The use according to claim 51, wherein, The bioactive polypeptide is selected from immune checkpoint inhibitors or cytokines.

53. The use according to claim 52, wherein, The immune checkpoint inhibitor is a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, or a LAG-3 antibody; the cytokines are interferon, IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, or IL-21.

54. The use according to claim 51, wherein, The chemotherapy drug is selected from one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, leucovorin, paclitaxel, and albumin-bound paclitaxel.

55. The use according to claim 49, wherein the tumor is selected from solid tumors, hematologic malignancies, and metastatic, refractory, or recurrent lesions of cancer.

56. The use according to claim 49, wherein the tumor or cancer is selected from esophageal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer, liver cancer, gastric 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 cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma, sarcoma, glioblastoma, and leukemia.

57. The use according to claim 49, wherein the tumor or cancer is gastrointestinal cancer.

58. The use according to claim 56, wherein the tumor or cancer is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, pancreatic cancer, and lung cancer.

59. The use according to claim 58, wherein the tumor or cancer is gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma.

60. The use according to claim 59, wherein the tumor or cancer is locally advanced unresectable or metastatic gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma.

61. The use according to any one of claims 56, 58-60, wherein the gastric cancer is gastric adenocarcinoma.

62. The use according to claim 56 or 58, wherein the lung cancer is non-small cell lung cancer.

63. The use according to claim 56, wherein the myeloma is multiple myeloma.

64. The use according to claim 49, wherein the tumor is CLDN18.2 positive.

65. The use according to claim 49, wherein the tumor is HER2 negative.

66. The antibody-drug conjugate according to claim 1, wherein the conjugate is Where γ is selected from an integer between 1 and 10, and A is an antibody that specifically binds to human CLDN18.

2.

67. An antibody-drug conjugate, wherein the conjugate is Where A is an antibody containing VH and VL; (i) The VH comprises CDR-H1 having the sequence SEQ ID No:1, CDR-H2 having the sequence SEQ ID No:2 or SEQ ID No:3, and CDR-H3 having the sequence SEQ ID No:3; and the VL comprises CDR-L1 having the sequence SEQ ID No:4, CDR-L2 having the sequence SEQ ID No:5, and CDR-L3 having the sequence SEQ ID No:6; or (ii) The VH comprises CDR-H1 having the sequence SEQ ID No:7, CDR-H2 having the sequence SEQ ID No:8 or 22, and CDR-H3 having the sequence SEQ ID No:9; and the VL comprises CDR-L1 having the sequence SEQ ID No:10, CDR-L2 having the sequence SEQ ID No:11, and CDR-L3 having the sequence SEQ ID No:12; and Where γ is an integer selected from 1 to 10.

68. The antibody-drug conjugate of claim 67, wherein A comprises VH having the sequence of SEQ ID NO:14; and VL having the sequence of SEQ ID NO:15, wherein γ is an integer selected from 1 to 10.

69. An antibody-drug conjugate, wherein the conjugate is Wherein A comprises a heavy chain of the sequence shown in SEQ ID NO: 19 and a light chain of the sequence shown in SEQ ID NO: 20, wherein A is linked by one or more thiol groups to form a conjugate, and γ is selected from an integer from 1 to 10.

70. A pharmaceutical composition comprising one or more conjugates as described in any one of claims 67-69, and one or more pharmaceutical excipients.

71. The pharmaceutical composition according to claim 70, having a DAR value of 3 to 8.

72. The pharmaceutical composition according to claim 70, having a DAR value of about 3.

8.

73. The pharmaceutical composition according to claim 70, having a DAR value of about 7.

1.

74. The pharmaceutical composition according to claim 70, having a DAR value of about 7.4.

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