Anti-GPC1 antibody or antigen-binding fragment thereof, drug conjugate thereof and use thereof

Abstract

Provided in the present application are an anti-GPC1 antibody or an antigen-binding fragment thereof, an antibody-drug conjugate and the use thereof. The anti-GPC1 antibody or the antigen-binding fragment thereof comprises complementary determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region and / or complementary determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region. The anti-GPC1 antibody or the antigen-binding fragment thereof can specifically bind to GPC1, and the anti-GPC1 antibody or the antigen-binding fragment thereof and the antibody-drug conjugate comprising the anti-GPC1 antibody or the antigen-binding fragment thereof can be used for diagnosing, preventing and / or treating a GPC1-related disease or disorder.

Description

An antibody against GPC1 or its antigen-binding fragment, a drug conjugate and its applications Technical Field

[0001] This application relates to the field of biomedical technology, specifically to an anti-GPC1 antibody or its antigen-binding fragment, a drug conjugate, and its applications. Background Technology

[0002] GPC1 (also referred to as "GPC-1" in this article), short for phosphatidylinositol polysaccharide 1, is a member of the phosphatidylinositol protein family. GPC1 consists of a core protein and three heparin sulfate chains and is anchored to the cell surface via GPI. As a growth factor co-receptor, it binds to growth factors and plays a role in tumor cell growth, invasion, and metastasis. GPC1 is mainly expressed in the central nervous system and skeletal system during embryonic development. After adulthood, GPC1 is not expressed or is expressed at low levels in normal tissues throughout the body, but it shows high expression in various tumors (such as head and neck cancer, esophageal cancer, cervical cancer, esophageal cancer, bladder cancer, etc.). This property makes GPC1 a good tumor-associated antigen (TAA). However, the development of anti-tumor drugs targeting GPC1 has been relatively slow. For example, the drug prepared by conjugating Miltuximab with radioactive elements failed in clinical development after entering Phase I clinical trials due to the large accumulation of radioactive elements in the liver (Asia Ocean J Nucl Med Biol. 2021 spring; 9(2):86–100). Other drugs in preclinical development, such as CAR-T cell therapy targeting GPC1 (WO2020154150), or the fusion of GPC1 nanobodies with immunotoxins (Mol Cancer Ther.2022 Jun 1; 21(6):960–973.), or the development of bispecific antibodies composed of GPC1 and CD3 (BMC Cancer.2020 Dec 10; 20:1214.), are still in the exploratory stage and have not yet made positive progress.

[0003] Antibody-drug conjugates (ADCs) are a new type of drug formed by linking biologically active small molecules to antibodies via chemical bonds (usually covalent bonds). By utilizing the properties of antibody drugs in vivo, they alter the distribution and pharmacokinetic properties of the loaded drug, thereby improving the overall clinical efficacy of the drug. ADCs typically consist of three components: an antibody for targeting and internalization, a linker connecting the antibody and drug, and a drug delivery system for cell-killing effects. Each component plays a crucial role in the successful clinical application of ADCs. After decades of research, the concept of ADCs has matured, and up to 15 ADC drugs have been successfully launched and benefited patients.

[0004] The development of ADC drugs using GPC1 as a target has been reported (Neoplasia (2021) 23, 939–950). This ADC was developed by Kochi University in Japan and is codenamed 01a033-T2-MMAE. However, the university's development of this ADC has only been limited to clinical efficacy studies in esophageal and pancreatic cancer, and the data indicate that the efficacy of this ADC in these two models is not very ideal.

[0005] Therefore, how to develop an applicable anti-GPC1 antibody-drug conjugate and demonstrate its potential clinical application value is still in the technological gap stage. Summary of the Invention

[0006] In view of the prior art, this application provides an anti-GPC1 antibody or its antigen-binding fragment, a drug conjugate, and its applications. The anti-GPC1 antibody or its antigen-binding fragment includes complementarity-determining regions HCDR1, HCDR2, HCDR3 of the heavy chain variable region and / or complementarity-determining regions LCDR1, LCDR2, LCDR3 of the light chain variable region; the anti-GPC1 antibody or its antigen-binding fragment can specifically bind to GPC1. The anti-GPC1 antibody or its antigen-binding fragment described herein, and the antibody-drug conjugate containing the anti-GPC1 antibody or its antigen-binding fragment, can be used for the diagnosis, prevention, and / or treatment of GPC1-related diseases or conditions.

[0007] The first aspect of this application provides an anti-GPC1 antibody or its antigen-binding fragment, comprising:

[0008] (a) Heavy chain variable regions containing the following complementary determinant regions:

[0009] HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1 or 7, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1 or 7, or an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:1 or 7 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:1 or 7;

[0010] HCDR2, comprising the amino acid sequence shown in SEQ ID NO:2 or 8, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:2 or 8, or an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:2 or 8 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:2 or 8;

[0011] HCDR3, comprising the amino acid sequence shown in SEQ ID NO:3 or 9, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3 or 9, or an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:3 or 9 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:3 or 9; and / or

[0012] (b) Light chain variable regions containing the following complementary determining regions:

[0013] LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4 or 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4 or 10, or an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:4 or 10 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:4 or 10;

[0014] LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 or 11, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:5 or 11, or an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:5 or 11 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:5 or 11;

[0015] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6 or 12, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:6 or 12, or an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:6 or 12 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:6 or 12.

[0016] The substitution, deletion, or addition of one or more amino acids described in this application refers to the substitution, deletion, or addition of one or two amino acids. In some embodiments, the substitution, deletion, or addition of the one or more amino acids is a conservative modification.

[0017] The variants of the anti-GPC1 antibody or its antigen-binding fragment described in this application (including variants with at least 80% identity, variants with substitution, deletion or addition modifications) exhibit substantially the same function and effect as the anti-GPC1 antibody or its antigen-binding fragment.

[0018] In some embodiments, amino acid sequences having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 1-3 and 7-9, or amino acid sequences having one or more amino acid substitutions, deletions, or additions, contain amino acids that are predominantly or entirely located in the FR region of the heavy chain variable region. In some embodiments, amino acid sequences having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 4-6 and 10-12, or amino acid sequences having one or more amino acid substitutions, deletions, or additions, contain amino acids that are predominantly or entirely located in the FR region of the light chain variable region.

[0019] The CDRs in this document are defined according to the Kabat numbering scheme, but those skilled in the art will understand that CDRs defined using other numbering schemes (such as Chothia, IMGT) are also within the scope of protection of this application.

[0020] In some embodiments, the anti-GPC1 antibody or its antigen-binding fragment comprises:

[0021] (1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively; or

[0022] (2) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9 respectively, and LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12 respectively.

[0023] In some embodiments, the anti-GPC1 antibody or its antigen-binding fragment comprises:

[0024] (1) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:14.

[0025] (2) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:16.

[0026] (3) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:24.

[0027] (4) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:25, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:24.

[0028] (5) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:24.

[0029] (6) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:24.

[0030] (7) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:24.

[0031] (8) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:24.

[0032] (9) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:30.

[0033] (10) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:25, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:30.

[0034] (11) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:30.

[0035] (12) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:30.

[0036] (13) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:30.

[0037] (14) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:30.

[0038] (15) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:31.

[0039] (16) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:32.

[0040] (17) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:31.

[0041] (18) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:32.

[0042] (19) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:31.

[0043] (20) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:32.

[0044] (21) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:36.

[0045] (22) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:36.

[0046] (23) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:36.

[0047] (24) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:36.

[0048] (25) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:40.

[0049] (26) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:40.

[0050] (27) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:40; or

[0051] (28) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:40.

[0052] In some embodiments, the amino acids that differ from those shown in any of SEQ ID NOs: 13, 14, 15, 16, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 are predominantly or entirely located in the FR region.

[0053] In some embodiments, the anti-GPC1 antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region;

[0054] In some embodiments, the heavy chain constant region is a heavy chain constant region of IgG, IgA, IgM, IgE or IgD or a variant thereof; the heavy chain constant region is preferably a heavy chain constant region of IgG (including IgG1, IgG2, IgG3 or IgG4) or a variant thereof; the heavy chain constant region is more preferably a heavy chain constant region of human IgG1 or a variant thereof.

[0055] In some implementations, the light chain constant region is a constant region of a human κ chain or a λ chain, or a variant thereof.

[0056] In some embodiments, the heavy chain constant region comprises a full-length heavy chain constant region or a segment thereof, the segment being selectable from the CH1 region, CH2 region, CH3 region or Fc region.

[0057] In some implementations, the Fc region is the human IgG1 Fc region.

[0058] In some embodiments, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:17, or an amino acid sequence having at least 80% identity with it; and / or

[0059] The light chain constant region contains an amino acid sequence as shown in SEQ ID NO:18 or an amino acid sequence having at least 80% identity with it.

[0060] In some embodiments, the anti-GPC1 antibody or its antigen-binding fragment comprises:

[0061] A heavy chain comprising a variable region and a constant region, the variable region comprising an amino acid sequence selected from any one of SEQ ID NOs: 33, 39, 13, 15, 23, 25, 26, 27, 28, 29, 34, 35, 37, and 38, or an amino acid sequence having at least 80% identity with such sequences, and the constant region comprising an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 80% identity with such sequences; and

[0062] The light chain comprises: a light chain variable region containing an amino acid sequence selected from any one of SEQ ID NOs: 32, 36, 14, 16, 24, 30, 31, and 40, or an amino acid sequence having at least 80% identity with such amino acid sequence; and a light chain constant region containing an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence having at least 80% identity with such amino acid sequence.

[0063] In some embodiments, the anti-GPC1 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence represented by any one of SEQ ID NOs: 19, 21, 41 and 43 or an amino acid sequence having at least 80% identity with it, and the light chain comprising an amino acid sequence represented by any one of 20, 22, 42 and 44 or an amino acid sequence having at least 80% identity with it.

[0064] In some embodiments, the anti-GPC1 antibody or its antigen-binding fragment comprises:

[0065] (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 80% identity with it, and

[0066] A light chain comprising an amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 80% identity with it;

[0067] (2) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 80% identity with it, and

[0068] Light chain comprising the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 80% identity with it;

[0069] (3) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:41 or an amino acid sequence having at least 80% identity with it, and

[0070] Light chain, comprising the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having at least 80% identity with it; or

[0071] (4) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:43 or an amino acid sequence having at least 80% identity with it, and

[0072] The light chain comprises the amino acid sequence shown in SEQ ID NO:44 or an amino acid sequence having at least 80% identity with it.

[0073] In some embodiments, the amino acids that differ from the amino acid sequences shown in any of SEQ ID NOs: 19, 20, 21, 22, 41, 42, 43 and 44 are predominantly or entirely located in the FR region or constant region (including the heavy chain constant region and the light chain constant region).

[0074] In some embodiments, the antibody is a mouse antibody, a monkey antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0075] In some specific embodiments, the antibody is a humanized antibody.

[0076] In some embodiments, the antigen-binding fragment may be selected from at least one of the following: Fab fragment, Fab' fragment, F(ab')2 fragment, Fd fragment, Fv fragment, dAb fragment, isolated CDR region, and scFv.

[0077] In some embodiments, the antigen-binding fragment may be selected from at least one of the following: Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, and scFv.

[0078] In some embodiments, the antigen-binding fragment may be selected from at least one of the Fd fragment, dAb fragment, and isolated CDR region.

[0079] In some specific embodiments, the anti-GPC1 antibody may be a monoclonal antibody.

[0080] A second aspect of this application provides a nucleic acid molecule that encodes the anti-GPC1 antibody or its antigen-binding fragment as described in the first aspect.

[0081] A third aspect of this application provides a recombinant vector comprising the nucleic acid molecule of claim 11.

[0082] The fourth aspect of this application provides a host cell comprising the nucleic acid molecule of claim 11 or the recombinant vector of claim 12.

[0083] The fifth aspect of this application provides a method for preparing an anti-GPC1 antibody or an antigen-binding fragment thereof, comprising culturing the host cells described in the fourth aspect, and isolating and / or purifying the antibody or antigen-binding fragment expressed by the cells.

[0084] In some embodiments, the antibody or its antigen-binding fragment is separated and / or purified by affinity chromatography.

[0085] The sixth aspect of this application provides an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, comprising the anti-GPC1 antibody or its antigen-binding fragment as described in the first aspect and a drug; wherein the drug is selected from: cytotoxic agents, radiolabels, fluorophores, chromophores, imaging agents, immunomodulators, protein degrading agents, angiogenesis inhibitors, cell proliferation inhibitors, apoptosis-promoting agents, cell lysins, and any combination thereof.

[0086] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate, or its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, has a structure represented by general formula (I):

[0087] in:

[0088] Bp is the anti-GPC1 antibody or its antigen-binding fragment;

[0089] L stands for connector;

[0090] D represents the drug unit;

[0091] p is any integer between 1 and 20.

[0092] In some implementations, p is any integer between 3 and 8.

[0093] In some embodiments, the connector is a breakable connector.

[0094] In some embodiments, the linker is an enzyme-responsive breakable linker.

[0095] In some embodiments, the linker comprises peptide units that can be cleaved by proteases.

[0096] In some embodiments, the linker comprises peptide units that can be cleaved by lysosomal enzymes.

[0097] In some embodiments, the peptide unit is selected from -valine-citrulline-(-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-(SEQ ID NO:48)), and -glycine-proline-arginine-asparagine-leucine-valine-citrulline-(-Gly-Pro-Arg-Asn-Leu-Val-Cit-(SEQ ID NO:47)).

[0098] In some embodiments, the linker is maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxy (MC-VC-PAB).

[0099] In some embodiments, the pharmaceutical unit is selected from immunomodulators, protein degraders, and cytotoxic agents.

[0100] In some embodiments, the pharmaceutical unit includes: amanitins, anthracyclines, auristatins, baccatins, calicheamicins, camptothecins, cemadotins, colchicine, colcimids, comprbetastatins, cryptophycins, discormolides, duocarmycins, docetaxel, doxorubicin, and echinomycins. Elutherobins, epothilones, estramustines, lexitropsins, maytansines, maytansinoids, methotrexate, netropsins, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), puromycins, rhizoxins, SN-38, taxanes, tubulolysins, vincaalkaloids, tetrahydroisoquinoline alkaloids or their derivatives, protein degrading agents or their derivatives.

[0101] In some embodiments, the pharmaceutical unit includes maytansine alkaloids or derivatives thereof, camptothecin or derivatives thereof, auristatin or derivatives thereof, tetrahydroisoquinoline alkaloids or derivatives thereof, BTK protein degraders or derivatives thereof, GSPT1 protein degraders or derivatives thereof, and CRBN protein degraders or derivatives thereof.

[0102] In some embodiments, the pharmaceutical unit includes CC885, DX8951, MMAE, DM1, DM4, Dxd, SN38, Trabectedin (ET743), Lurbinectedin, and CC-90009.

[0103] In some embodiments, the antibody-drug conjugate has the following structure:

[0104] in,

[0105] Anti-GPC1 is the anti-GPC1 antibody or its antigen-binding fragment;

[0106] L1 is the connection segment between P and Anti-GPC1;

[0107] P is a peptide unit that can be cleaved by proteases, selected from -valine-citrulline-(-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-(SEQ ID NO:48)), and -glycine-proline-arginine-asparagine-leucine-valine-citrulline-(-Gly-Pro-Arg-Asn-Leu-Val-Cit-(SEQ ID NO:47)).

[0108] L2 is the connection segment between P and D;

[0109] D is selected from MMAE, DX-895, and CC885;

[0110] n is any integer between 3 and 8.

[0111] In some embodiments, L1 is selected from MC- (maleimide-hexanoyl-), MeO2S-Pym-, NHS-, Mal-PEG8-, DBCO-, and Hydrazide-. In some specific embodiments, L1 is MC-.

[0112] In some embodiments, P is -valine-citrulline-(-VC-).

[0113] In some implementations, L2 is empty or selected from -PAB-, -PAB-DMEDA-, and -NMEDA-. In some specific implementations, L2 is -PAB-. In some implementations, L1-P-L2 is MC-VC-PAB-.

[0114] In some embodiments, the DAR value of the antibody-drug conjugate is an integer or decimal of 2-8, 2.5-8, 3-8, 3-7, 3-6.5, 3-6, 3-5.5, 3-5, or 3-4.8.

[0115] In some specific embodiments, the antibody-drug conjugate is selected from the following structures:

[0116] The seventh aspect of this application provides the use of GPC1 protein as a diagnostic marker for squamous cell carcinoma of the lung in the preparation of diagnostic reagents or kits for squamous cell carcinoma of the lung.

[0117] The eighth aspect of this application provides the use of antibody-drug conjugates targeting GPC1 (e.g., human GPC1) in the preparation of medicaments for the diagnosis, prevention and / or treatment of squamous cell carcinoma of the lung.

[0118] In some embodiments, this application relates to antibody-drug conjugates targeting GPC1 for the diagnosis, prevention, and / or treatment of squamous cell carcinoma of the lung. Alternatively, this application relates to a method for the prevention and / or treatment of squamous cell carcinoma of the lung, comprising administering an antibody-drug conjugate targeting GPC1 to a subject in need.

[0119] The ninth aspect of this application provides the use of the anti-GPC1 antibody or its antigen-binding fragment as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant vector as described in the third aspect, the host cell as described in the fourth aspect, the anti-GPC1 antibody or its antigen-binding fragment prepared in the fifth aspect, or the antibody-drug conjugate or solvate as described in the sixth aspect, or their tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, in the preparation of medicaments for the diagnosis, prevention, and / or treatment of GPC1-related diseases or conditions.

[0120] In some embodiments, this application relates to the anti-GPC1 antibody or its antigen-binding fragment as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant vector as described in the third aspect, the host cell as described in the fourth aspect, the anti-GPC1 antibody or its antigen-binding fragment prepared in the fifth aspect, or the antibody-drug conjugate or solvate as described in the sixth aspect, or in the form of tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, for the diagnosis, prevention, and / or treatment of GPC1-related diseases or conditions. Alternatively, this application relates to a method for the prevention and / or treatment of GPC1-related diseases or conditions, comprising administering to a subject in need the anti-GPC1 antibody or its antigen-binding fragment as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant vector as described in the third aspect, the host cell as described in the fourth aspect, the anti-GPC1 antibody or its antigen-binding fragment prepared in the fifth aspect, or the antibody-drug conjugate or solvate as described in the sixth aspect, or in the form of tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof. Alternatively, this application provides the use of the anti-GPC1 antibody or its antigen-binding fragment as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant vector as described in the third aspect, the host cell as described in the fourth aspect, the anti-GPC1 antibody or its antigen-binding fragment prepared in the fifth aspect, or the antibody-drug conjugate or solvate as described in the sixth aspect, or their tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof for the diagnosis, prevention, and / or treatment of GPC1-related diseases or conditions.

[0121] In some implementations, the disease or ailment is cancer.

[0122] In some implementations, the cancer is a solid tumor.

[0123] In some embodiments, the cancer is squamous cell carcinoma of the lung, esophageal cancer, pancreatic cancer, cervical cancer, bladder cancer, colorectal cancer, liver cancer, glioma, lung cancer, head and neck cancer, thyroid cancer, endometrial cancer, breast cancer, or ovarian cancer.

[0124] In some specific embodiments, the cancer is squamous cell carcinoma of the lung or esophageal cancer.

[0125] The binding affinity and endocytosis efficiency of the antibody portion of an ADC drug directly affect its in vivo efficacy. This application employs multiple immunoassay methods and incorporates antibody endocytosis efficiency as a key screening indicator to obtain candidate antibodies with both high affinity and high endocytosis efficiency. Furthermore, the antibody developed in this application exhibits cross-recognition of human and monkey antigens, while showing no cross-recognition with homologous antigens such as GPC2, GPC3, and GPC4, significantly simplifying preclinical research.

[0126] Regarding the linker-drug delivery system, this application employs different types of drug delivery systems, such as vcMMAE, Exatecan, and the molecular glue CC885, as well as combinations of these with different types of linkers, including novel linkers with extracellular / intracellular release mechanisms. The resulting ADC demonstrated excellent efficacy and acceptable preclinical toxicity in both in vitro and in vivo pharmacodynamic models.

[0127] Regarding indications, this application is the first to investigate the expression of GPC1 protein in lung squamous cell carcinoma using immunohistochemistry (IHC), and preliminarily establishes the epidemiological characteristics of GPC1 expression in lung squamous cell carcinoma. Simultaneously, by comparing IHC staining of tumor sections from lung squamous cell carcinoma and esophageal cancer PDX models, it was confirmed that the expression rate and protein level of GPC1 in lung squamous cell carcinoma PDX sections were both higher than those in esophageal cancer PDX sections. This application is the first to propose and preclinically demonstrate the potential of anti-GPC1 antibody-drug conjugates for the treatment of lung squamous cell carcinoma.

[0128] This application prepared the full-length extracellular protein of GPC1, the N-terminal extracellular protein of GPC1, the C-terminal extracellular protein of GPC1, and HEK293 cells expressing human GPC1 protein. Mice were then subjected to monoimmunization, cross-immunization, or cross-immunization with proteins. After obtaining sufficient immunogenicity, mouse spleens were harvested for hybridoma cell preparation. A phage display library was established using hybridoma cells, and the phage library was panned using antigens. Finally, the endocytosis capacity of candidate antibodies with strong binding affinity was tested. Through comprehensive evaluation of the antibody binding affinity and endocytosis capacity, as well as the in vitro and in vivo efficacy and PK stability of the ADC prepared from the chimeric antibody, candidate antibodies were ultimately selected for humanization.

[0129] Candidate ADC drugs were prepared by conjugating humanized antibodies with various linkers and drug-carrying agents. The clinical application prospects of the candidate ADC drugs were determined by evaluating their in vitro stability, rat PK stability, cellular efficacy, tumor model efficacy, and preliminary toxicology.

[0130] The beneficial effects of this application are as follows:

[0131] 1. The anti-GPC1 antibody or its antigen-binding fragment obtained in this application has the characteristics of high binding force and high endocytosis, and exhibits human-monkey cross-species antigens, and does not bind to GPC2, GPC3, and GPC5 antigens of the same family;

[0132] 2. The ADC drug prepared in this application possesses excellent cellular efficacy (inhibition of tumor cell proliferation) and in vivo efficacy in CDX models (exhibiting good inhibitory activity against tumor growth in vivo). At safe doses, the inhibition rate against CDX model tumors with moderate antigen expression can reach 90% or more;

[0133] 3. The ADC drug prepared in this application can reach a non-severe toxicity dose of 5 mpk in monkey toxicology, demonstrating good safety;

[0134] 4. This application is the first to propose that the prepared anti-GPC1-ADC drug has good potential application value in the treatment of squamous cell carcinoma of the lung. Attached Figure Description

[0135] Figure 1-1 shows the results of the antibody's specific recognition test of the homologous protein huGPC2-his in Example 4.

[0136] Figures 1-2 show the results of the antibody's specific recognition test of the homologous protein huGPC3-his in Example 4.

[0137] Figures 1-3 show the results of the antibody's specific recognition test of the homologous protein huGPC5-his in Example 4.

[0138] Figures 2-1 to 2-8 show the results of cross-testing of antibodies against target antigens in humans and monkeys in Example 4.

[0139] Figure 3 shows the endocytosis efficiency test results after the antibody binds to the antigen in Example 5.

[0140] Figure 4 shows the FACS detection results of the expression level of the lung squamous cell carcinoma cell line GPC1 in Example 8.

[0141] Figure 5 shows the FACS detection results of the expression level of the esophageal cancer cell line GPC1 in Example 8.

[0142] Figure 6 shows the rat PK property test results of ADC in Example 9.

[0143] Figure 7 shows the tumor inhibition effect of ADC on the NCI-H1703 CDX model of lung squamous cell carcinoma in Example 10.

[0144] Figure 8 shows the tumor inhibition effect of ADC on the SK-MES-1CDX model of squamous cell carcinoma of the lung in Example 10.

[0145] Figure 9 shows the tumor inhibition effect of ADC on the KYSE-520CDX esophageal cancer model in Example 10.

[0146] Figure 10 shows the tumor inhibition effect of ADC on the TE8 CDX model of esophageal cancer in Example 10.

[0147] Figure 11 shows the results of the pretoxicology test on the effect of ADC at a dose of 5 mpk on the body weight of cynomolgus monkeys in Example 11.

[0148] Figure 12 shows the effect of ADC at a dose of 5 mpk on the main hematological parameters of cynomolgus monkeys in Example 11.

[0149] Figure 13 shows the effect of ADC at a dose of 5 mpk on the main blood biochemical indicators of cynomolgus monkeys in Example 11.

[0150] Figure 14 shows an example of IHC staining results for lung squamous cell carcinoma tumor tissue sections in Example 12.

[0151] Figure 15 shows example results of PDX sections of lung squamous cell carcinoma and esophageal cancer in Example 13. Detailed Implementation

[0152] Terminology Definition

[0153] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Inc., New York, USA (2012); Abbas et al., Cellular and Molecular Immunology, Elsevier Science Health Science div (2009); He Wei et al., Medical Immunology (2nd ed.), People's Medical Publishing House, 2010.

[0154] Where appropriate, unless otherwise stated, procedures involving the use of commercially available kits and reagents are generally performed in accordance with the manufacturer’s defined protocols and conditions.

[0155] Unless otherwise stated, the singular forms “a,” “single,” and “the” include the plural referents.

[0156] Unless otherwise stated, the terms “comprise”, “comprises”, and “comprising” or their equivalents (contain, contain, containing, include, include, including) used herein are open-ended expressions, meaning that they may cover other unspecified elements, components, and steps in addition to those listed.

[0157] Unless otherwise stated, the terms “optional” or “optionally” as used herein mean that the object or event it modifies exists or does not exist, or occurs or does not occur. Unless the context clearly indicates otherwise, the word “or” in this document is intended to include “and”.

[0158] As used herein, “about” means within the acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may, in accordance with art practice, mean within one or more standard deviations. Alternatively, “about” may mean a range of up to 20% (i.e., ±20%), such as fluctuations within a given specific numerical range of ±10%, ±5%, ±2%, ±1%, or ±0.5%. Furthermore, particularly for biological systems or methods, the term may mean up to an order of magnitude or up to five times a certain value. When a specific value is given in this application or claim, unless otherwise stated, “about” should be understood as meaning within the acceptable range of error for that specific value. In this document, unless otherwise stated, the values ​​of various measurements, step parameters, or conditions are implicitly modified by “about”.

[0159] In this application, unless the context clearly specifies otherwise, singular terms encompass plural references, and vice versa.

[0160] Unless otherwise stated, the term "treatment" as used herein means that, after administration, it can inhibit, suppress, reduce, improve, alleviate, relieve, or eliminate a disease or its related symptoms; it can delay, slow, stop, or terminate the progression of a disease or its related symptoms; or it can prevent, control, or reduce the recurrence of a disease or its related symptoms. For the prevention and treatment of disease, the effective therapeutic or preventative dose can be determined by a clinician based on the subject's individual condition, disease severity, sex, age, weight, route of administration, etc., using routine methods or experience.

[0161] Unless otherwise stated, the term "subject" as used herein encompasses any vertebrate, such as mammals, including humans, non-human primates, sheep, dogs, cats, horses, cattle, chickens, pigs, rats, etc. Preferably, the subject in this application is a human.

[0162] In this application, the term "antibody" refers to a polypeptide encoded by an immunoglobulin gene and capable of binding an antigen. The term "complementarity-determining region" can refer to a portion of the variable region of an antibody that confers antigen-binding specificity, and can refer to an amino acid sequence found in the highly variable region of the heavy or light chain of an immunoglobulin. The heavy chain may include three complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3); and the light chain may include three complementarity-determining regions (CDRs) (LCDR1, LCDR2, and LCDR3). The CDRs can provide contact residues that play an important role in the binding of the antibody to its antigen or antigenic epitope. In this document, antibodies may include monoclonal antibodies, polyclonal antibodies, neutralizing antibodies, antagonistic antibodies, anti-idiotypic antibodies, trehalosyl-free antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies), or nanobodies.

[0163] In this application, the term "variable" generally refers to the fact that certain portions of the sequence of the variable domain of an antibody vary strongly, resulting in the binding and specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs). More conserved portions within the variable domain are referred to as frames (FRs). In the art, antibody CDRs can be defined by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th Edition, NIH, Bethesda, MD (1991)), the Chothia definition rule based on the location of the structural loop region (see, Al-Lazikani et al., JMol Biol 273:927-48, 1997), and the KABAT definition rule based on the concepts of IMGT ontology and the IMGT Scientific diagram rules.

[0164] In this application, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with human antibodies. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance immune responses.

[0165] The term "chimeric antibody" refers to an antibody whose variable region sequence originates from one species and whose constant region sequence originates from another species, such as an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody.

[0166] In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that individual antibodies within the cluster are identical, except for a small number of possible natural mutations. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody formulations (which usually have different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of antibodies through any particular method. For example, the monoclonal antibodies used in this application can be prepared in hybridoma cells or through recombinant DNA methods.

[0167] In this application, the terms "fully human antibody," "fully human antibody," or "completely human antibody," also known as "fully human monoclonal antibody," refer to antibodies whose variable and constant regions are both human-derived, thus eliminating immunogenicity and toxicity. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The antibody or ligand described in this application can be a fully human monoclonal antibody. Related technologies for the preparation of fully human antibodies include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology, etc. The terms "full-length antibody," "intact antibody," and "all antibody" are used interchangeably herein to refer to antibodies that have a structure substantially similar to naturally occurring antibodies and contain a heavy chain including an Fc region. For example, when used to refer to IgG molecules, a "full-length antibody" is an antibody containing two heavy chains and two light chains.

[0168] In this application, the term "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain, which, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. J Allergy Clin Immunol, 2010, 125:S41-52, incorporated herein by reference in its entirety. The Fc region may be a naturally occurring Fc region or an Fc region modified in accordance with the manner described in the art or elsewhere in this disclosure.

[0169] In this application, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise stated, as used herein, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope). The affinity of a molecule X for its partner Y can be expressed using the dissociation equilibrium constant (K0). D The kinetic components affecting the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) techniques (e.g., Or biological layer interferometry (e.g., FORTEBIO).

[0170] In this application, the term "K" d (sec) -1 This refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as K. off value.

[0171] In this application, the term "k" a (M) -1 -×sec -1 (k) refers to the binding rate constant of a specific antibody-antigen interaction. This value is also known as k. on value.

[0172] In this application, the term "K" D "(M), as used in this article, refers to the dissociation equilibrium constant of a specific antibody-antigen interaction." D =k d / k a In some embodiments, based on the K-axis of the interaction between this antibody and its antigen... D Describe the antibody affinity. For clarity, as is known in the art, the smaller K... D A higher K value indicates a higher affinity interaction, while a larger K value indicates a lower affinity interaction. D The value indicates a lower affinity interaction. If the antibody binds to antigen X, the K value... D Less than or equal to 5 × 10 -8 If M, then the antibody is said to have "specifically bound" to antigen X. Ideally, it should be 1 × 10⁻⁶. -8 M or less, 6×10 is more ideal. -9 M or less, 3×10 is more ideal. -9 M or less, 2×10 is more ideal. -9 M or below. The antibody may be chimeric, humanized, or preferably human.

[0173] In this application, the term "K" A (M) refers to the binding equilibrium constant of a specific antibody-antigen interaction, K A =k a / k d .

[0174] In this application, the percentage of identity (degree of homology) between sequences can be determined by comparing the two sequences, for example, using a computer program (e.g., BLASTp or BLASTn with default settings) that is commonly available for this purpose on the World Wide Web (e.g., www.ncbi.nlm.nih.gov).

[0175] In this application, the terms “antibody-drug conjugate”, “antibody-drug conjugate”, and “ADC” are used interchangeably.

[0176] In this application, the terms "linker," "linker," "L," "linker structure," or "moiety" generally refer to a chemical structural fragment with one end for linking to a ligand and the other end for linking to a cytotoxic drug. It may also refer to a segment that is linked to other linkers before being used to link to a cytotoxic drug. The direct or indirect linking to the ligand can refer to the group directly linking the ligand via a covalent bond, or it can refer to the linking of the ligand via a linker structure. Linkers may be readily induced or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and / or disulfide bond cleavage under conditions that maintain the activity of the compound or antibody.

[0177] In this application, the term "peptide unit" refers to a peptide structural unit comprising at least two covalently attached amino acids. The term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, the two or more covalently attached amino acids are linked by peptide bonds and may consist of naturally occurring amino acids and peptide bonds. Alternatively, it may include synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and leucine) or peptide-mimicking structures, i.e., "peptides or protein analogs," such as peptide-like structures. Peptides-like structures are an exemplary class of peptide mimics whose side chains are attached to the nitrogen atom of the peptide backbone, rather than the α-carbon (as in amino acids), and which have different hydrogen bonding and conformational features compared to peptides. Thus, peptide-like structures can resist proteolysis or other physiological or storage conditions and efficiently penetrate cell membranes. Such synthetic amino acids can be incorporated, particularly when antibodies are synthesized in vitro using conventional methods well known in the art. Additionally, any combination of peptide mimics, synthetic, and naturally occurring residues / structures can be used. "Amino acid" also includes imino acid residues, such as proline and hydroxyproline. The amino acid "R group" or "side chain" can be in (L)- or (S)- configuration. In a particular embodiment, the amino acid is in (L)- or (S)- configuration.

[0178] In this application, the term "pharmaceutical unit" or "D" generally refers to any compound having desired biological activity and reactive functional groups that can be used to incorporate a drug into the conjugate of this application.

[0179] In this application, the terms "cytotoxic drug" or "cytotoxic agent" generally refer to a toxic drug, which can be a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations. The "cytotoxic drug" may include, but is not limited to, radioactive isotopes (e.g., At...). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 (and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, donomycin or other inserts); growth inhibitors; enzymes and fragments thereof, such as nucleotide degraders; antibiotics; toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and the various antitumor or anticancer agents disclosed below.

[0180] In this application, the term "pharmaceutically acceptable salt" generally refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a linker drug, or a conjugate). In some aspects, the compound may contain at least one amino group and thus may form an acid addition salt with the amino group. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, tartrates, oleates, tannates, pantothenates, hydrogen tartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, sucrose salts, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pyrates (i.e., 1,1'-methylenebis-(2-hydroxy-3-naphthylcarbamate)). Pharmaceutically acceptable salts may involve the introduction of another molecule, such as an acetate ion, a succinate ion, or other counterions. Counterions can be any organic or inorganic component that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, multiple counterions may be present. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0181] In this application, the term "derivative" generally refers to a chemical compound or molecule made from a parent compound through one or more chemical reactions.

[0182] In this application, the term "tumor" generally refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "carcinoma," "cell proliferation disorder," "proliferative disorder," and "tumor" are not mutually exclusive when used herein. In this application, a tumor can be a solid tumor or a non-solid tumor.

[0183] In this application, the term "prevention and / or treatment" includes not only the prevention and / or treatment of disease, but also generally includes preventing the onset of disease, slowing or reversing the progression of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated with it and / or preventing a further increase in the severity of the disease and / or any symptoms associated with it, preventing, reducing or reversing any physiological damage caused by the disease, and any pharmacological effects that are generally beneficial to the patient being treated. The compositions of this application do not need to achieve a complete cure or eradication of any symptoms or manifestations of the disease to form a viable therapeutic agent. As recognized in the relevant art, a medicine used as a therapeutic agent may reduce the severity of a given disease state, but does not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically constitutes a viable preventive agent without completely and effectively preventing the onset of the condition; it is sufficient simply to reduce the effects of the disease in the subject (e.g., by reducing the amount or severity of their symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) or reduce the likelihood of the disease occurring or worsening.

[0184] Example

[0185] The present application will be further described below with reference to the embodiments. However, those skilled in the art will understand that the scope of protection of the present application is not limited to these embodiments.

[0186] Unless otherwise specified, the reagents, materials or instruments used in the following examples are commercially available.

[0187] Example 1: Preparation of GPC1 antigen and control antibody Miltuximab

[0188] The amino acid sequence from positions 24 to 530 of human GPC-1 (Uniport ID: P35052) was selected, with a 6×His tag added to the C-terminus, named huGPC1-his; and an Fc (human IgG1) sequence added to the C-terminus, named huGPC1-hFc. The control antibody Miltuximab was derived from Minomic International Ltd. patent US11198738B2 (heavy chain variable region sequence shown in SEQ ID NO:45, light chain variable region sequence shown in SEQ ID NO:46). The constant region of the antibody adopted the heavy chain constant region of human IgG1 and the light chain constant region of human Kappa chain. The target genes of the above-mentioned human GPC-1 antigens (huGPC1-his and huGPC1-hFc) and the codon-optimized control antibody coding sequences were synthesized, and the coding sequences of each target gene and control antibody were constructed into the pcDNA3.4 vector. The antigens were transiently expressed using Expi 293 cells, and the proteins were purified after 5 days of expression. The control antibody was transiently expressed using Expi CHO cells, and the proteins were purified after 7 days of expression. The purified proteins were then subjected to physicochemical and biochemical tests.

[0189] Example 2: Preparation of GPC1-HEK293 overexpressing cells

[0190] The full-length protein sequences of human GPC-1 (Uniport ID: P35052) and monkey GPC-1 (Uniport ID: A0A2K5UZ91) were obtained from the Uniport database. The corresponding GPC-1 gene sequences were synthesized and constructed into overexpression cell vectors. HEK293 cells in good logarithmic growth phase were selected and seeded at 8E6 cells per culture dish. Cells were cultured at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 70%–80% as observed under a microscope, transfection was performed to prepare the viral solution. The viral solution was collected 48–72 hours after transfection, filtered through a 0.45 μm syringe filter to remove residual cells, and HEK293 cells were infected with the virus at an MOI of 10. The medium was changed 24 hours after viral infection to obtain a stable cell pool with high GPC1 expression. Two overexpressing cell lines, HEK293-huGPC1 and HEK293-CynoGPC1, were constructed. FACS analysis confirmed that both cell lines highly expressed GPC1 antigen on their surface.

[0191] Example 3: Establishment of a mouse immune and phage library

[0192] GPC1 Immunization. Balb / C mice were immunized using protein and cells, employing three different immunization strategies. For group 1, five Balb / C mice were immunized with 100 μg huGPC1-hFc antigen via intraperitoneal and subcutaneous injection at multiple sites. Subsequently, on day 14, five Balb / C mice received booster immunizations with 50 μg huGPC1-hFc antigen via intraperitoneal and subcutaneous injection at multiple sites. Further booster immunizations with 50 μg huGPC1-hFc antigen were administered on days 28 and 42, and a final booster immunization with 100 μg huGPC1-hFc antigen was given on day 56. For group 2, five Balb / C mice were immunized with 100 μg huGPC1-hFc antigen via intraperitoneal and subcutaneous injection at multiple sites. Subsequently, booster immunizations were administered with 50 μg huGPC1-his antigen on days 14 and 21, with 50 μg huGPC1-hFc antigen on day 28, with 50 μg huGPC1-his antigen on days 42 and 49, and a final booster immunization with 100 μg huGPC1-hFc antigen on day 56. For group 3, 100 μg huGPC1-hFc antigen was administered to 5 Balb / C mice via intraperitoneal and subcutaneous multiple-point injections. Subsequently, on day 14, five Balb / C mice were boosted with 5E6 HEK293-huGPC1 overexpressing cells via intraperitoneal injection. A follow-up booster was given on day 28 with 50 μg huGPC1-hFc antigen, and again on day 42 with 5E6 HEK293-huGPC1 overexpressing cells. Finally, a final booster was given on day 56 with 100 μg huGPC1-hFc antigen. Serum titers from the third, fourth, and final immunizations were measured. Results showed that the serum titer after the final immunization was >64000, meeting the requirements for subsequent experiments.

[0193] Construction of the phage library. RNA was extracted from spleen cells of immunized mice using the chloroform method. The RNA was reverse transcribed into cDNA using a reverse transcription kit. The VH and VL gene fragments of the antibody were amplified from the cDNA using specific primers. The full-length Fab fragment was amplified using fusion PCR and then recovered via gel electrophoresis. The full-length Fab fragment and the phage display vector were double-digested with Nco I and Not I. The digestion products were identified and recovered by agarose gel electrophoresis. The Fab digestion products were ligated into the digested vector. After desalting and purification, the ligation products were mixed thoroughly with competent *E. coli* and transformed. After electroporation, the bacterial culture was plated and incubated overnight at 37°C. The next day, the bacterial growth on the plates was scraped off, and glycerol was added to 10% and stored at -80°C. The library size, antibody variable region insertion accuracy, and CDR3 sequence diversity were analyzed. The results showed that the antibody libraries all had a larger capacity than the designed library and a correct insertion rate of over 80%. CDR3 sequence analysis indicated that the constructed antibody libraries had high sequence diversity, meeting the expected requirements.

[0194] Preparation of phage suspension. The antibody library bacteria were inoculated into 2YT(C) glucose-containing medium at a specific inoculation rate. + -K + In a culture medium, shake and incubate until OD600≈0.5. Add helper phage for 1-1.5 hours of infection, then centrifuge, discard the supernatant, and pour the precipitate into 2YT (C + -K + Incubate overnight in culture medium. On the second day, centrifuge and collect the supernatant, add PEG6000, mix well, and place on ice to precipitate. After 1 hour, centrifuge and discard the supernatant, add pre-cooled PBS, and dissolve by pipetting to prepare a phage suspension for subsequent phage library screening and enrichment.

[0195] Example 4: Enrichment, screening, and construction and expression of chimeric antibodies from phage libraries

[0196] The constructed mouse antibody library was panned using a combination of solid-phase, liquid-phase, and cell-based screening methods. The process primarily involved incubating, washing, and eluting the phage library with antigens. After three rounds of panning, monoclonal antibodies specifically binding to the antigens were enriched in large quantities. Specific Fab antibodies against huGPC1-his and HEK293-huGPC1 were enriched using trypsin and glycine hydrochloride elution. Following three rounds of screening, initial screening of monoclonal antibodies against the GPC1 antigen was performed.

[0197] Liquid phase panning. The specific steps of liquid phase panning are as follows: The prepared phage suspension is incubated with magnetic beads blocked with 2.5% BSA. First, the streptavidin-conjugated magnetic beads are incubated with biotin-labeled huGPC1 antigen (huGPC1-his and HEK293-huGPC1) to allow the biotin-labeled huGPC1 antigen to bind to the magnetic beads. The magnetic beads bound to huGPC1 antigen are then incubated with the phage suspension at room temperature for 2 hours. After washing 10 times with PBST to remove non-specifically adsorbed phages, the number of washes depends on the screening round. Trypsin is added to elute the specifically bound antibodies and display the phages. Subsequently, the eluted phages were used to infect logarithmic-phase *E. coli* SS320 cells and incubated for 30 min, then cultured at 220 rpm for 1 h. Helper phages were added and incubated for another 30 min, followed by another 1 h of culture at 220 rpm. The culture was then centrifuged and transferred to 2YT (C+-K+) medium. The resulting phages were used for the next round of panning. A total of three rounds of screening were performed, with antigen concentrations of 300 nM, 100 nM, and 30 nM used in each round, respectively. In addition, the washing intensity of PBST was gradually increased, with 10, 15, and 20 washes used successively, to screen for antibody clones with higher affinity.

[0198] Solid-phase panning. The specific steps of solid-phase panning are as follows: Add 1 mL of 100 μg / mL huGPC1 antigen to an immunotube and coat overnight at 4°C. The next day, discard the coating solution, add 5% milk PBS for blocking for 2 hours, wash twice with PBS, add phage suspension, and incubate for 2 hours. Wash 8 times with PBS, then wash twice with PBST to remove non-specifically bound phages. The number of washes depends on the screening round. Then, add trypsin to the immunotube to elute specifically bound antibodies and display phages. Subsequently, infect logarithmic-phase SS320 cells with the eluted phages and incubate for 30 minutes, then culture at 220 rpm for 1 hour. Add helper phages and incubate for 30 minutes, then continue to culture at 220 rpm for 1 hour. Centrifuge and transfer to 2YT (C+-K+) medium. The obtained phages are then used for the next round of panning. A total of three rounds of screening were conducted, with the antigen dosage used in each round being 100 μg / mL, 30 μg / mL, and 10 μg / mL, respectively. In addition, the washing intensity of PBS was gradually increased, with the number of PBS washes being 8, 13, and 18, respectively, in order to screen for antibody clones with higher affinity.

[0199] Cell panning. The specific steps for cell panning are as follows: Add 5 × 10⁵ cells to the cell culture flask. 6HEK293 cells were incubated with phage suspension blocked with 5% FBS at room temperature in a shaker at 80 rpm for 1 h. The phage suspension was then used for negative screening of HEK293 cells. 5 × 10⁶ cells were added to the culture flask. 6 HEK293-huGPC1 cells were incubated with a phage suspension obtained after negative screening at 80 rpm for 2 hours at room temperature on a shaker. The supernatant in the culture flask was discarded, and the cells were washed with 5% FBS-PBS to remove non-specifically bound phages. The number of washes depended on the screening round. 100 mM glycine-HCl (pH 2.2) was added to elute the phages, and 1 M Tris-base was added to neutralize to pH 7–7.2 (the volume added needs to be adjusted when preparing the solution). Subsequently, the eluted phages were used to infect SS320 cells in the logarithmic growth phase and incubated for 30 min. Then, the cells were cultured at 220 rpm for 1 hour. Helper phages were added and the cells were incubated for another 30 min. The cells were then cultured at 220 rpm for 1 hour. After centrifugation, the cells were transferred to 2 YT (C+-K+) medium, and the resulting phages were used for the next round of screening.

[0200] Enrichment of different output sets was detected using pool ELISA. This panning process yielded several well-enriched output sets at the ELISA level. Single clones from the first, second, and third rounds of panning were selected for initial screening using Phage ELISA. A total of 502 clones were picked from six plates, resulting in 268 positive clones binding to huGPC1-his and 138 positive clones binding to CynoGPC1-his. 135 positive clones exhibiting human-monkey cross-activity were sent for sequencing analysis, yielding 46 molecules with unique sequences. Based on sequence diversity analysis, 44 were selected for the construction of full-length chimeric antibody plasmids. The constructed antibody plasmids were transfected into Expi CHO cells for transient expression over 7 days at a volume of 10 mL. After expression, protein purification, aliquoting, and analysis were performed.

[0201] The binding activity of chimeric antibodies to their family proteins huGPC2-his, huGPC3-his, and huGPC5-his was detected (see Figures 1-1 to 1-3, antibody specificity recognition test diagrams for family proteins). The species cross-binding activity with overexpressing cells HEK293-huGPC1 and HEK293-CynoGPC1 was also assessed (see Table 3 and Figures 2-1 to 2-8, antibody cross-binding test diagrams for human and monkey target antigens), antigen affinity, and endocytic activity were also evaluated to screen for preferred chimeric antibodies. These preferred chimeric antibodies were then conjugated with vcMMAE, followed by cell-killing activity and efficacy testing. Ultimately, two candidate antibodies, mAb02 and mAb07, were selected.

[0202] Table 1 shows the CDR sequences of the candidate antibodies, and Table 2 shows the heavy chain variable region, heavy chain constant region, light chain variable region, light chain constant region, heavy chain and light chain amino acid sequences of the candidate antibodies.

[0203] Table 1. CDR sequences of chimeric antibodies

[0204] Table 2. Heavy and light chain amino acid sequences of chimeric antibodies.

[0205] Table 3

[0206] Example 5: Antibody Humanization and Characterization

[0207] Example 5.1 Antibody Humanization

[0208] Humanization of mAb02 and mAb07 antibodies was performed using CDR transplantation and reverse mutation methods. The CDRs of the murine antibody sequences were divided using the Kabat numbering system. The VH and VL sequences of the murine antibodies were compared with existing human Ig gene sequences to obtain the best-matching human germline Ig gene sequence as the humanization backbone. The murine CDR sequence was then spliced ​​with the optimal human germline Ig gene framework sequence to obtain the preliminary humanized sequence. Simultaneously, homology modeling was performed on the murine antibody Fv to identify key amino acid sites, such as classical residues and loop interaction residues, that would maintain the original conformation of the antibody sequence. Reverse mutations were then performed on these key amino acids to obtain the final humanized sequence. Multiple sequences were designed for each light and heavy chain sequence to create multiple humanized antibodies. Antibody production was performed using the Expi CHO expression system. The affinity and endocytosis of the humanized antibodies were analyzed to screen for the best humanized antibodies. After conjugating the selected humanized antibodies with vcMMAE, cell killing activity and efficacy were tested, and two humanized antibodies were finally screened out, namely mAb02H8L4 and mAb07H4L1.

[0209] Humanization of mAb02. The light and heavy chains of the mAb02 antibody were classified using the Kabat numbering system. The amino acid sequences of the heavy and light chains, as well as the CDR1, CDR2, and CDR3 sequences, are shown in Table 1. For the light chain of mAb02, IGKV3-11*01 was the optimal germline gene, and was selected as the humanization backbone. For the heavy chain of mAb02, IGHV3-7*01 was the optimal germline gene, and was selected as the humanization backbone. Then, CDRL1, CDRL2, and CDRL3 were transplanted into the frame sequence of IGKV3-11*01, and CDRH1, CDRH2, and CDRH3 were transplanted into the frame sequence of IGHV3-7*01. Homology modeling was then used to generate a 3D model to identify the amino acids crucial for antibody binding and conformation in the FR region sequence of the chimeric antibody. For the light chain of the mAb02 antibody, four humanized sequences were designed: mAb02-L1, mAb02-L2, mAb02-L3, and mAb02-L4. For the heavy chain of the mAb02 antibody, eight humanized sequences were designed: mAb02-H1, mAb02-H2, mAb02-H3, mAb02-H4, mAb02-H5, mAb02-H6, mAb02-H7, and mAb02-H8. The eight humanized heavy chains and four humanized light chains were then synthesized and cloned into the pcDNA3.4 vector. After plasmid extraction, transient transfection expression of the antibodies was performed using Expi CHO cells. Pairing of human VH and human VL resulted in 18 humanized antibodies (see Table 4).

[0210] Humanization of mAb07. The light and heavy chains of the mAb07 antibody were divided into CDRs using the Kabat numbering system. The amino acid sequences of the heavy and light chains, as well as the CDR1, CDR2, and CDR3 sequences, are shown in Table 1. For the mAb07 light chain, IGKV2-29*02 was the optimal germline gene, and it was selected as the humanization backbone. For the mAb07 heavy chain, IGHV1-46*01 was the optimal germline gene, and it was selected as the humanization backbone. Then, CDRL1, CDRL2, and CDRL3 were transplanted into the frame sequence of IGKV2-29*02, and CDRH1, CDRH2, and CDRH3 were transplanted into the frame sequence of IGHV1-46*01. Homology modeling was then used to generate a 3D model to determine the amino acids in the FR region sequence of the chimeric antibody that are crucial for antibody binding and conformation. Two humanized sequences, mAb07-L1 and mAb07-L2, were designed for the light chain of the mAb07 antibody; four humanized sequences, mAb07-H1, mAb07-H2, mAb07-H3, and mAb07-H4, were designed for the heavy chain of the mAb07 antibody. These four humanized heavy chains and two humanized light chains were then synthesized and cloned into the pcDNA3.4 vector. After plasmid extraction, the antibodies were transiently expressed using Expi CHO cells. Pairing of human VH and human VL cells yielded eight humanized antibodies (see Table 4).

[0211] Table 4. Heavy and light chain amino acid sequences of humanized antibodies.

[0212] Example 5.2

[0213] The affinity of the humanized antibody for the huGPC1 antigen was measured using a Biacore 8K. The binding of the humanized antibody to HEK293-huGPC1 overexpressing cells was detected by FACS analysis. After conjugating the humanized antibody to vcMMAE, cell killing activity and CDX efficacy were tested. Finally, two humanized antibodies were screened out, namely mAb02-H8L4 and mAb07-H4L1 (as shown in Table 4).

[0214] 5.2.1 Detection of antibody affinity for cells using FACS method

[0215] The three antibodies described in this application were incubated with NCI-H1703 tumor cells (Nanjing Kober) at low temperature, and then a secondary antibody (Alexa Fluo 647-conjugated AffiniPure Goat Anti-Human IgG, Fc Fragment Specific, Cat#109-605-098, Jackson) was added. The fluorescence value of the cell surface was detected on a CytoFLEX flow cytometer (manufacturer: Beckman Coulter Biotechnology (Suzhou) Co., Ltd., model: A00-1-1102) to assess the affinity of the antibodies for tumor cells.

[0216] Table 5. Results of FACS binding affinity assay for humanized antibodies.

[0217] As shown in Table 5, the activity (i.e., affinity for tumor cells) of the humanized antibody of this application is superior to that of the positive control antibody Miltuximab.

[0218] 5.2.2 SPR method for detecting antibody binding kinetics

[0219] On a Biacore T200 (Cytiva) chip, ligands (antibodies) were captured onto the chip using a Protein A chip, with a serially diluted analyte (GPC1 antigen) as the mobile phase. The antibody binding kinetics were calculated based on the system response values.

[0220] Table 6. Results of Humanized Antibody Affinity Kinetics Detection

[0221] As shown in Table 6, the binding ability of the humanized antibody in this application is comparable to that of the positive control antibody Miltuximab.

[0222] 5.2.3 FACS method for detecting antibody endocytosis efficiency

[0223] The antibody was conjugated to fluorescein Cy5 with disulfide bonds, and then cultured at a cell density of 2.0 × 10⁶ cells per tube. 6100 μL or 500 nM fluorescein Cy5-labeled antibody was added to NCI-H1703 cells per mL and incubated at 4°C for 1 h. After binding, a portion of the cells was removed and treated with 100 μL of 100 mM DTT. DTT reduction can separate the antibody from the Cy5 bound to the cell surface, thereby eliminating the Cy5 signal on the cell surface. This group was used as the background for detection. The remaining cells were immediately incubated at 37°C for 1 h for internalization. After internalization, the cells were washed three times with pre-cooled PBS to remove the supernatant containing the fluorescein-labeled antibody. The internalized cells were divided into two groups: one group was not treated with DTT, and the other group was treated with 100 μL of 100 mM DTT to eliminate the un-endocytosed Cy5 signal on the cell surface, while retaining the intracellular Cy5 signal. DTT was applied at 4℃ for 30 min to ensure the removal of fluorescent dyes from the cell surface. Fluorescence intensity was measured in DTT-treated and untreated cells using a CytoFLEX flow cytometer (manufacturer: Beckman Coulter Biotechnology (Suzhou) Co., Ltd., model: A00-1-1102). The level of antibody internalization on the cell surface was calculated based on the fluorescence values. Endocytosis efficiency = 100 * [FI(1h+DTT) - FI(+DTT)] / [FI(1h) - FI(+DTT)].

[0224] The results of the endocytosis efficiency test are shown in Figure 3 (test results of endocytosis efficiency after antibody binding to antigen) and Table 7.

[0225] Table 7 Results of humanized antibody endocytosis efficiency detection

[0226] As shown in Table 7, the endocytosis efficiencies of the two antibodies mAb02-H8L4 and mAb07-H4L1 in this application and the positive control antibody Miltuximab in NCI-H1703 cells were 79.66%, 52.10%, and 65.08%, respectively. The results show that both antibodies in this application have good endocytosis efficiency.

[0227] Example 6: Synthesis of linker-payload intermediates

[0228] Example 6.1 4-((6S,9S,12S,15S,18S)-1-amino-9-(2-amino-2-oxoethyl)-6-((S)-1-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)glycyl)pyrrolidine-2-carboxamido)-1-imino-12-isobutyl-15-isopropyl-7,10,13, Synthesis of 16-tetraoxo-18-(3-ureapropyl)-2,8,11,14,17-pentazanonadecan-19-amido)benzyl(2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)ureo)phenethoxy)ethyl)(methyl)carbamate (L015-CC885)

[0229] 6.1.1 Synthesis of 2-(2-chloro-4-nitrophenyl)ethane-1-ol (1-2)

[0230] Compound 1-1 (4.00 g, 18.5 mmol, 1.00 eq) was dissolved in THF (40.0 mL), cooled to 0 °C, and BH3-Me2S (10 M, 4.64 mL, 2.50 eq) was added dropwise under N2 protection. The mixture was stirred for 30 minutes, then heated to 70 °C and stirred for 2 hours under N2 protection. TLC (petroleum ether / ethyl acetate = 1:1, product Rf = 0.4) showed that compound 1-1 was completely consumed. The mixture was cooled to room temperature. The reaction mixture was quenched with MeOH (100 mL) at 0 °C and extracted with EtOAc (100 mL * 3). The residue was purified by column chromatography (SiO2, petroleum ether / EtOAc = 10 / 1 to 5 / 1) after concentration to give a yellow oily compound 1-2 (2.80 g, 13.8 mmol, yield: 74.8%). 1 HNMR (400MHz, CDCl3) δ: 8.21-8.17 (m, 1H), 8.02 (dd, J = 2.3, 8.4Hz, 1H), 7.47 (d, J = 8.5Hz, 1H), 3.90 (t, J = 6.5Hz, 2H), 3.07 (t, J = 6.5Hz, 2H), 2.11 (s, 1H).

[0231] 6.1.2 Synthesis of 1-(2-chloro-4-nitrophenylethoxy)-4,4-dimethylpentan-2-one (1-3)

[0232] To a solution of compounds 1-2 (3.00 g, 14.8 mmol, 1.0 eq) and tert-butyl bromoacetate (23.2 g, 119 mmol, 17.6 mL, 8.00 eq) in toluene (90.0 mL), Bu₄NHSO₄ (4.04 g, 11.9 mmol, 0.80 eq) and NaOH (5 M, 297 mL, 100 eq) were added. The mixture was stirred at 25 °C for 2 hours. TLC monitoring (petroleum ether / ethyl acetate = 2:1, product Rf = 0.6) indicated that compounds 1-2 were completely consumed. The reaction mixture was diluted with H₂O (50.0 mL) and extracted with EtOAc (100 mL * 2). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a yellow oily compound 1-3 (9.50 g, crude).

[0233] 1 H NMR(400MHz, CDCl3)δ:8.22-8.17(m,1H),8.03(dd,J=2.3,8.4Hz,1H),7.57(d,J=8.4 Hz,1H),3.94-3.91(m,2H),3.79(s,2H),3.12(t,J=6.4Hz,2H),1.44(d,J=1.0Hz,9H)

[0234] 6.1.3 Synthesis of 2-(2-chloro-4-nitrophenylethoxy)acetic acid (1-4)

[0235] TFA (28.1 g, 246 mmol, 18.3 mL, 8.20 eq) was added to a DCM (40.0 mL) solution of compounds 1-3 (9.50 g, 30.0 mmol, 1.00 eq). The mixture was stirred at 25 °C for 1 hour. TLC monitoring (petroleum ether / ethyl acetate = 1:1, product Rf = 0.3) indicated that compounds 1-3 were completely consumed. The reaction mixture was diluted with H₂O (50.0 mL) and extracted with DCM (100 mL * 2). The combined organic layers were washed with brine (50.0 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give yellow oil compounds 1-4 (5.50 g, crude product).

[0236] 6.1.4 Synthesis of 2-(2-chloro-4-nitrophenylethoxy)-N-methylacetamide (1-5)

[0237] To a DMF (50.0 mL) solution of compounds 1-4 (5.50 g, 21.1 mmol, 1.00 eq) and HATU (12.1 g, 31.7 mmol, 1.50 eq), methylamine hydrochloride (1.72 g, 25.4 mmol, 1.20 eq) and DIEA (10.9 g, 84.7 mmol, 14.8 mL, 4.00 eq) were added. The mixture was stirred at 25 °C for 1 hour. TLC monitoring of the reaction (DCM / MeOH = 10:1, product Rf = 0.4) indicated that compounds 1-4 were completely consumed. The reaction mixture was diluted with H₂O (50.0 mL) and extracted with DCM (200 mL * 2). The combined organic layers were washed with brine (30.0 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM / EtOAc = 30 / 1 to 1 / 1) to give yellow oily compounds 1-5 (5.00 g, 18.3 mmol, yield: 86.6%).

[0238] 1 H NMR (400MHz, CDCl3) δ: 8.23 ​​(d, J = 2.3Hz, 1H), 8.07 (dd, J = 2.3, 8.4Hz, 1H), 7.48 (d, J = 8.4Hz, 1H), 6.44 (br s,1H),3.93(s,2H),3.79(t,J=6.4Hz,2H),2.86(s,2H),2.80(s,3H)

[0239] 6.1.5 Synthesis of 2-(2-chloro-4-nitrophenethoxy)-N-methylethane-1-amine (1-6)

[0240] Compounds 1-5 (5.00 g, 18.35 mmol, 1.00 eq) were dissolved in THF (50.0 mL), cooled to 0 °C, and BH3·THF (1 M, 36.7 mL, 2.00 eq) was added dropwise under N2 protection and stirred for 30 min. The mixture was then stirred at 70 °C under N2 protection for 2 h. TLC monitoring of the reaction (DCM / MeOH = 5:1) indicated that the reactants were completely consumed. The mixture was cooled to room temperature. The reaction mixture was quenched by adding MeOH (50.0 mL) at 0 °C, and then refluxed at 80 °C for 30 min. The residue was acidified to pH 6 with 1 M HCl, and the resulting mixture was extracted with EtOAc (100.0 mL). The aqueous phase was alkalized to pH 8 with a saturated NaHCO3 aqueous solution. The resulting mixture was extracted with EtOAc (100.0 mL * 3), washed with brine (50.0 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give yellow oily compounds 1-6 (3.60 g, crude product). ESI-MS (m / z): 258.9 [M + H] + . 1 H NMR (400MHz, CDCl3)δ:8.24(d,J=2.1Hz,1H),8.07(dd,J=2.2,8.4Hz,1H),7.51(d,J=8.4Hz,1H),3.74(t,J=6.5Hz,2H),3.64-3.64(m,1H),3.62(br t,J=5.0Hz,1H),3.13(br t,J=6.5Hz,2H),2.84-2.79(m,2H),2.49(s,3H).

[0241] 6.1.6 Synthesis of tert-butyl (2-(2-chloro-4-nitrophenoxy)ethyl)(methyl)carbamate (1-7)

[0242] To a solution of compounds 1-6 (3.30 g, 12.7 mmol, 1.0 eq.) in THF (10.0 mL), Boc₂O (3.34 g, 15.3 mmol, 3.52 mL, 1.2 eq.) and TEA (1.55 g, 15.3 mmol, 2.13 mL, 1.2 eq.) were added. The mixture was stirred at 25 °C for 2.0 h. LCMS showed that compounds 1-6 reacted completely. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (SiO₂, petroleum ether / DCM = 100 / 1 to 1 / 10, DCM / MeOH = 200 / 1 to 50 / 1) to give yellow oily compounds 1-7 (1.56 g, 4.35 mmol, yield: 34.0%). ESI-MS (m / z): 259.0 [M-Boc₂] + .1 H NMR, (400MHz, CDCl3) δ: 8.22 (d, J = 2.13Hz, 1H), 8.04 (dd, J = 8.44, 2.06Hz, 1H), 7.45 (d, J = 8.38Hz, 1H), 3.70 (br t, J = 6.38Hz, 2H), 3.53 (br s,2H),3.34(br s,2H),3.08(t,J=6.38Hz,2H),2.83(br s,3H),1.43(s,9H).

[0243] 6.1.7 Synthesis of tert-butyl (2-(4-amino-2-chlorophenethoxy)ethyl)(methyl)carbamate (1-8)

[0244] To a mixed solution of compounds 1-7 (1.55 g, 4.32 mmol, 1.0 eq.) in ethanol (12.5 mL) and water (2.50 mL), NH4Cl (693 mg, 12.9 mmol, 3.0 eq.) was added, followed by Fe powder (1.21 g, 21.6 mmol, 5.0 eq.) with stirring. The mixture was stirred at 80 °C for 2.0 h. TLC (DCM:MeOH = 50:1) showed that compounds 1-7 (Rf = 0.40) were completely consumed. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (SiO2, DCM / MeOH = 200 / 1 to 10 / 1) to give a yellow solid compound 1-8 (1.10 g, 3.35 mmol, yield: 77.4%). ESI-MS (m / z): 351.0 [M+Na] + . 1 H NMR (400MHz, CDCl3) δ: 7.00 (d, J = 8.13Hz, 1H), 6.68 (d, J = 2.38Hz, 1H), 6.51 (dd, J = 8.19, 2.31Hz, 1H), 3.56-3.60 (m, 2H), 3.54 (br s, 2H), 3.35 (br s,2H),2.87-2.91(m,2H),2.83-2.87(m,3H),1.45(s,9H).

[0245] 6.1.8 Synthesis of tert-butyl (2-(2-chloro-4-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate (1-9)

[0246] To a THF (13.0 mL) solution of compounds 1-8 (1.20 g, 3.65 mmol, 1.0 eq.), trichloromethyl chloroformate (1.44 g, 7.30 mmol, 880 μL, 2.0 eq.) was added. The mixture was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure and redissolved in DMF (13.0 mL). At 25 °C, the above solution was added dropwise to a stirred mixture of compounds 3-[5-(aminomethyl)-1-oxo-2-isoindolinyl]piperidine-2,6-dione hydrochloride (CAS: 1158264-69-7, 1.36 g, 4.38 mmol, 1.2 eq.) and TEA (3.69 g, 36.4 mmol, 5.08 mL, 10 eq.) in DMF (13.0 mL). The mixture was stirred at 25 °C for 1.0 h. LCMS showed that compounds 1-8 reacted completely. The reaction mixture was diluted with ice water (100 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (100 mL x 5), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give yellow oily compounds 1-9 (2.90 g, crude). ESI-MS (m / z): 528.3 [M-Boc] + .

[0247] 6.1.9 Synthesis of 1-(3-chloro-4-(2-(2-(methylamino)ethoxy)ethyl)phenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)urea (1-10)

[0248] TFA (10.3 g, 90.8 mmol, 6.75 mL, 21.1 eq) was added to a DCM (27.0 mL) solution of compounds 1-9 (2.70 g, 4.30 mmol, 1.0 eq). The mixture was stirred at 25 °C for 1.0 h. LCMS showed that compounds 1-9 reacted completely. The reaction mixture was diluted with H₂O (10.0 mL) and extracted with DCM (10.0 mL x 3). The combined organic layers were washed with brine (10.0 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by preparative HPLC to give compound 1-10 (984 mg, 1.85 mmol, yield: 43.0%) as a white solid. ESI-MS (m / z): 528.2 [M+H] + .

[0249] 6.1.1 Synthesis of 9H-fluorene-9-yl)methyl((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (1-12)

[0250] EEDQ (19.9 g, 80.5 mmol, 2.0 eq.) and PAB (9.92 g, 80.5 mmol, 2.0 eq.) were added to a solution of compound Fmoc-Val-Cit-OH (1-11, 20.0 g, 40.3 mmol, 1.0 eq.) in DCM (200 mL) and MeOH (50.0 mL). The mixture was stirred at 25 °C for 16 hours. The reaction was detected by LC-MS. The reaction product was filtered, and the filtrate was added to 2000 mL of EtOAc / MTBE (8 / 1) and stirred twice for 10 min. The mixture was filtered, and the filtrate was washed with EtOAc to give a white solid compound 1-12 (20 g, crude product). ESI-MS (m / z): 602.3 [M+H] + .

[0251] 6.1.11 Synthesis of (9H-fluorene-9-yl)methyl((S)-3-methyl-1-((S)-1-((4-((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate (1-13)

[0252] DIEA (8.59 g, 66.4 mmol, 11.5 mL, 2.0 eq.) was added to a solution of compounds 1-12 (20.0 g, 33.2 mmol, 1.0 eq.) and PNP (20.2 g, 66.4 mmol, 2.0 eq.) in DMF (200 mL). The mixture was stirred at 25 °C for 3 hours. The reaction was confirmed by LC-MS. The reaction mixture was added dropwise to MTBE on ice and filtered. The filtered solid was dissolved in DCM and then poured into MTBE / EtOAc (1:1) and stirred for 30 min. The mixture was then filtered and concentrated under reduced pressure to give a white solid compound 1-13 (21 g, crude). ESI-MS (m / z): 767.4 [M+H] + .

[0253] 6.1.12 Synthesis of 4-((S)-2-((S)-2-(((9H-fluorene-9-yl)methoxy)carbonyl)-3-methylbutylamino)-5-ureidopentamid)benzyl(2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)ureido)phenethoxy)(methyl)carbamate (1-14)

[0254] HOBt (79.8 mg, 590 μmol, 1.2 eq.) and 2,6-dimethylpyridine (211 mg, 1.97 mmol, 229 μL, 4.0 eq.) were added to a solution of compounds 1-10 (260 mg, 488.8 μmol, 1.0 eq.) in DMF (3.00 mL). The mixture was stirred at 50 °C for 6.0 h. LCMS showed that the reaction was complete. The reaction mixture was purified by preparative HPLC to give compound 1-14 (187 mg, 145 μmol, yield: 29.5%) as a yellow solid. ESI-MS (m / z): 1155.6 [M+H] + .

[0255] 6.1.13 Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutylamino)-5-ureidopentamine)benzyl(2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate (1-15)

[0256] TEA (72.7 mg, 718 μmol, 0.10 mL, 17.3 eq.) was added to a solution of compound 1-14 (48.0 mg, 41.5 μmol, 1.0 eq.) in DMF (0.40 mL). The mixture was stirred at 25 °C for 5.0 h. LC-MS showed that the reaction was complete. The reaction mixture was added dropwise to isopropyl ether (5.00 mL) and centrifuged to give a brown oily compound 1-15 (101 mg, crude product). ESI-MS (m / z): 933.5 [M+H] + .

[0257] 6.1.14 Synthesis of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)glycyl-L-prolyl-L-arginyl-L-asparagine-L-leucine (1-16)

[0258] Solid-phase peptide synthesis:

[0259] The polypeptide was synthesized using standard Fmoc chemical methods.

[0260] 1) Resin Preparation: DIEA (4.00 eq) was added dropwise to a container containing 2-CTC resin (3.00 mmol, 3.00 g, Sub = 1.00 mmol / g) and Fmoc-Leu-OH (1.06 g, 3.00 mmol, 1.00 eq) in 80 mL of DCM and stirred for 2 hours. Then, MeOH (3.0 mL) was added to the resin under bubbling N2 and stirred for 30 minutes. The resin was washed with DMF (80 mL x 5), and then a 20% piperidine DMF solution (80 mL) was added, followed by stirring with N2 at 25°C for 12 minutes for Fmoc deprotection. The mixture was filtered, and the resin was washed with DMF (80 mL x 5) before proceeding to the next step.

[0261] 2) Coupling: A solution of Fmoc-Asn(Trt)-OH (3.00 eq), HATU (2.85 eq), and DIEA (6.00 eq) in DMF (80 mL) was added to the resin, and the mixture was stirred under nitrogen at 25 °C for 30 minutes. The coupling reaction was monitored by a ninhydrin test; if the result was colorless, the coupling was complete. The resin was then washed with DMF (80 mL * 5).

[0262] 3) Deprotection: Add 80 mL of DMF solution containing 20% ​​piperidine to the resin and stir the mixture with N2 at 25 °C for 12 minutes. Then wash the resin with DMF (80 mL * 5). Monitor the deprotection reaction by the ninhydrin test; if it shows a blue or brownish-red color, the reaction is complete.

[0263] 4) Repeat steps 2) to 3) to perform the next amino acid coupling.

[0264] 5) Coupling: A solution of DIEA (12.00 eq) and N-hydroxysuccinimide 6-maleimide hexanoate (6.00 eq) in DMF (80 mL) was added to the resin, and the mixture was stirred with N2 at 25 °C for 16 hours. The coupling reaction was monitored using tetrachlorobenzoquinone reagent; if it turned colorless, the coupling was complete. The resin was then washed with DMF (80 mL * 5).

[0265] Table 8

[0266] Peptide cleavage and purification:

[0267] 1) The lysis mixture (TFA / Tis / H2O, 95 / 2.5 / 2.5, v / v / v, 100 mL) was added to a flask containing the side-chain protected peptide, and the mixture was stirred at 25 °C for 2 hours. LCMS showed the desired MS.

[0268] 2) Filter the mixture. Precipitate the filtrate with cold isopropyl ether (500 mL) and centrifuge (3000 rpm for 2 minutes), then wash twice with isopropyl ether (500 mL * 2). Vacuum dry the residue for 2 hours.

[0269] 3) The residue was purified by preparative HPLC (A: 0.10% TFA in H2O, B: ACN) to give a white solid compound 1-16 (1.3 g, yield: 57.9%, purity: 98.6%). ESI-MS (m / z): 749.6 [M+H] + .

[0270] 6.1.15 4-((6S,9S,12S,15S,18S)-1-amino-9-(2-amino-2-oxoethyl)-6-((S)-1-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)glycyl)pyrrolidine-2-carboxamido)-1-imino-12-isobutyl-15-isopropyl-7,10,13, Synthesis of 16-tetraoxo-18-(3-ureapropyl)-2,8,11,14,17-pentazanonadecan-19-amido)benzyl(2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)ureo)phenethoxy)ethyl)(methyl)carbamate (L015-CC885)

[0271] To a solution of compounds 1-15 (101 mg, 40.6 μmol, 1.0 eq) and 1-16 (30.4 mg, 40.6 μmol, 1.0 eq) in DMF (1.00 mL), HATU (18.5 mg, 48.8 μmol, 1.2 eq) and DIEA (7.89 mg, 61.0 μmol, 10.6 μL, 1.5 eq) were added. The mixture was stirred at 25 °C for 0.5 h. LC-MS showed that compound 1-15 reacted completely. The reaction mixture was purified by prep-HPLC to give a white solid compound L015-CC885 (32.0 mg, 18.3 μmol, yield: 44.9%, purity: 95.2%). ESI-MS (m / z): 1664.4 [M+2H] + .

[0272] Example 6.2 Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexylamino)-3-methylbutyramido)-5-ureidopentamido)benzyl(2-(2-chloro-4-(3-((2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate (MC-VC-PAB-N3-CC885)

[0273] 6.2.1 Synthesis of (S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-5-ureidovalerate (2-1)

[0274] Solid-phase peptide synthesis:

[0275] The polypeptide was synthesized using standard Fmoc chemical methods.

[0276] 1) Resin preparation: DIEA (6.00 eq) was added to a solution of 2-CTC resin (20.0 mmol, 1.00 eq, Sub 0.80 mmol / g) and Fmoc Cit-OH (1.00 eq) in DCM (50.0 mL), and the mixture was stirred with N2 at 25 °C for 4.0 h. MeOH (25.0 mL) was added to the resin, and the mixture was stirred with N2 at 25 °C for 0.5 h. The mixture was then filtered to obtain the resin. The resin was washed with DMF (300 mL * 5).

[0277] 2) Deprotection: Add DMF solution (300 mL) containing 20% ​​piperidine, stir with N2 at 25°C for 30 min, wash the resin with DMF (300 mL * 5), and filter to obtain the resin.

[0278] 3) Coupling: Add a solution of HBTU (2.85 eq) and Fmoc Val OH (3.00 eq) in DMF (50.0 mL) to the resin, then add DIEA (6.00 eq), and stir with N2 at 25 °C for 30 min. Wash the resin with DMF (300 mL * 5).

[0279] 4) Deprotection: Add 20% piperidine to DMF (300mL), stir with N2 at 25℃ for 30min, wash the resin with DMF (300mL*5), and filter to obtain the resin.

[0280] 5) Coupling: Add a solution of DIC (2.0 eq) and 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid (2.00 eq) in DMF (50.0 mL) to the resin, then add HOAT (2.00 eq), stir with N2 at 25 °C for 20 hours, and wash the resin with DMF (300 mL * 5).

[0281] Table 9

[0282] Peptide cleavage and purification:

[0283] 1) The resin was washed three times with methanol and then dried under vacuum.

[0284] 2) Add lysis buffer (20% HFIP / DCM) to the peptide resin and stir for 30 min × 3 times.

[0285] 3) Reduced pressure concentration of DCM and TFA.

[0286] 4) The polypeptide was dried in high vacuum for 2 hours to obtain compound 2-1 (5.0 g, purity: 90.1%, crude product) in the form of a white solid.

[0287] ESI-MS (m / z): 468.4 [M+H] + .

[0288] 6.2.2 Synthesis of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureido-5-ureido-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanoamide (2-2)

[0289] HOBt (578 mg, 4.28 mmol, 1.00 eq), (4-aminophenyl)methanol (2.11 g, 17.1 mmol, 4.00 eq), and DIC (2.16 g, 17.1 mmol, 4.00 eq) were added to a solution of compound 2-1 (2.00 g, 4.28 mmol, 1.00 eq) in DMF (20.0 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed that compound 2-1 reacted completely. The reaction mixture was filtered and recrystallized from MTBE (200 mL) at 25 °C to give compound 2-2 (2.89 g, 3.12 mmol, yield 73.0%) as a pale yellow solid. ESI-MS (m / z): 573.4 [M+H] + .

[0290] 6.2.3 Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxy-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3-methylbutamido)-5-ureopentamido)benzyl(4-nitrophenyl)carbonate (2-3)

[0291] To a solution of compound 2-2 (2.89 g, 3.12 mmol, 1.00 eq) in DMF (28.0 mL), DIEA (807 mg, 6.25 mmol, 1.09 mL, 2.00 eq) and bis(4-nitrophenyl) carbonate (1.90 g, 6.25 mmol, 2.00 eq) were added. The mixture was stirred at 25 °C for 2 hours. LC-MS showed that compound 2-2 reacted completely. The reaction mixture was filtered and purified by recrystallization with isopropyl ether (500 mL) to give the residue. The residue was purified by preparative HPLC to give compound 2-3 (670 mg, 853 μmol, yield: 27.3%) as a white solid. ESI-MS (m / z): 738.2 [M+H] + .

[0292] 6.2.4 Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)-3-methylbutamido)-5-ureidopentamido)benzyl(2-(2-chloro-4-(3-((2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate (MC-VC-PAB-N3-CC885)

[0293] To a solution of compounds 1-10 (66 mg, 125 μmol, 1.00 eq.) in DMF (0.60 mL), 2,6-dimethylpyridine (26.7 mg, 250 μmol, 28.9 μL, 2.00 eq.) and HOBt (8.45 mg, 62.5 μmol, 0.50 eq.), along with compounds 2-3 (92.2 mg, 125 μmol, 1.00 eq.), were added. The mixture was stirred at 25 °C for 1 hour. LC-MS showed that compounds 1-10 reacted completely. The reaction mixture was diluted with H₂O (5.00 mL) and extracted with DCM (5.00 mL x 2). The combined organic layers were washed with brine (50.0 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC to obtain a yellow oily substance MC-VC-PAB-N3-CC885 (12 mg, 27.8 μmol, yield: 8.50%, purity: 98.1%). ESI-MS (m / z): 1126.5 [M+H] + .

[0294] Example 7: Preparation and Identification of Antibody-Drug Conjugates

[0295] DAR identification methods:

[0296] Detection of DAR in ADC by HIC-HPLC method

[0297] The samples were analyzed using a TSKgel Butyl-NPR 2.5μm 4.6mm×3.5cm column with 1.5M (NH2)2SO4 and 50mM KH2PO4 as mobile phase A, and 50mM KH2PO4 and 25% IPA as mobile phase B, at a flow rate of 0.8mL / min.

[0298] Injection volume: 50 μg, detection wavelength: 280 nm. Elution gradient is shown in Table 10.

[0299] Table 10

[0300] DAR calculation method:

[0301] Wherein, DAR0 represents an antibody without drug conjugation, DAR2 represents a conjugate with two drugs, DAR4 represents a conjugate with four drugs, DAR6 represents a conjugate with six drugs, and DAR8 represents a conjugate with eight drugs. A represents the peak area of ​​the conjugate on the chromatogram. A(Total) represents the total peak area of ​​the ADC, that is, the total area of ​​the conjugates DAR0, DAR2, DAR4, DAR6, and DAR8.

[0302] LC-MS method for detecting DAR of ADC

[0303] Sample preparation: Take 50 μg of ADC sample, add 2 μL of 0.5M TCEP, add ultrapure water to 100 μL to dilute to a concentration of about 0.5 mg / mL, mix well, reduce at 37℃ for 30 min, and then use LC-MS to detect the sample.

[0304] DAR calculation method:

[0305] In this context, LC represents the antibody light chain; HC represents the antibody heavy chain; L0 represents a conjugate with the light chain uncoupled with a toxin molecule; L1 represents a conjugate with the light chain coupled to one toxin molecule; H0 represents a conjugate with the heavy chain uncoupled with a toxin molecule; H1 represents a conjugate with the heavy chain coupled to one toxin molecule; H2 represents a conjugate with the heavy chain coupled to two toxin molecules; and H3 represents a conjugate with the heavy chain coupled to three toxin molecules. A represents the peak area of ​​each conjugate in the mass spectrometer. A(LC-Total) represents the total peak area of ​​all light chains, whether coupled with or uncoupled with the drug, i.e., the total peak area of ​​L0 and L1. A(HC-Total) represents the total peak area of ​​all heavy chains, whether coupled with or uncoupled with the drug, i.e., the total peak area of ​​H0, H1, H2, and H3.

[0306] Example 7.1: Preparation of Miltuximab-MC-VC-PAB-N3-CC885 sample

[0307] Miltuximab was placed in PBS (pH 6.0) buffer, and EDTA solution was added to a final concentration of 2 mM, resulting in an antibody concentration of 2.68 mg / mL. Reduction was then performed at 37°C for 2 h using 9.50 equivalents of TCEP. After reduction, unreacted TCEP was removed using a 40 kDa MWCO Zeba™ centrifugal desalting column, and the reaction solution was replaced with 20 mM histidine-acetic acid (pH 5.5). Following antibody reduction, N,N-dimethylacetamide (DMA) and MC-VC-PAB-N3-CC885 dissolved in DMA were added at a volume ratio of 10% (organic solvent) and a feed ratio of 10 equivalents of MC-VC-PAB-N3-CC885. The reaction solution was placed at 22℃ for 1 h. After coupling, it was purified in one step using a 40 kDa MWCO Zeba™ centrifugal desalting column. The coupling product was then stored in a 20 mM histidine-acetic acid (pH 5.5) solution to obtain the coupling compound Miltuximab-MC-VC-PAB-N3-CC885. The final DAR was determined to be 4.13 by HIC-HPLC.

[0308] Example 7.2: Preparation of Miltuximab-GGFG-Dxd-DAR4 Sample

[0309] Miltuximab was placed in PBS (pH 6.0) buffer, and EDTA solution was added to a final concentration of 2 mM, resulting in an antibody concentration of 2.68 mg / mL. Reduction was then performed at 37°C for 2 h using 9.50 equivalents of TCEP. After reduction, unreacted TCEP was removed using a 40 kDa MWCO Zeba™ centrifugal desalting column, and the reaction solution was replaced with 20 mM histidine-acetic acid (pH 5.5). Following antibody reduction, N,N-dimethylacetamide (DMA) and GGFG-Dxd dissolved in DMA were added at a volume ratio of 10% (organic solvent) and a feed ratio of 10 equivalents of GGFG-Dxd. The reaction solution was placed at 22℃ for 1 h. After coupling, it was purified in one step using a 40 kDa MWCO Zeba™ centrifugal desalting column. The coupling product was then stored in a 20 mM histidine-acetic acid (pH 5.5) solution to obtain the coupling compound Miltuximab-GGFG-Dxd-DAR4. The final DAR value was determined to be 4.15 by HIC-HPLC.

[0310] Example 7.3: Preparation of Miltuximab-GGFG-Dxd-DAR8 Sample

[0311] Miltuximab was placed in PBS (pH 7.4) buffer, and EDTA solution was added to a final concentration of 2 mM, resulting in an antibody concentration of 2.67 mg / mL. Reduction was performed at 37°C for 2 h using 9.50 equivalents of TCEP. After reduction, unreacted TCEP was removed using a 40 kDa MWCO Zeba™ centrifugal desalting column, and the reaction solution was replaced with PBS (pH 7.4) buffer. Following antibody reduction, N,N-dimethylacetamide (DMA) and GGFG-Dxd dissolved in DMA were added at a volume ratio of 10% (organic solvent) and a feed ratio of 14 equivalents of GGFG-Dxd. The reaction solution was placed at 22°C for 1 h. After coupling, it was purified in one step using a 40 kDa MWCO Zeba™ centrifugal desalting column. The coupling product was then stored in a 20 mM histidine-acetic acid (pH 5.5) solution to obtain the coupling compound Miltuximab-GGFG-Dxd-DAR8. The final DAR value was 7.87 as determined by LC-MS.

[0312] Example 7.4: Preparation of Miltuximab-L015-CC885-DAR8 sample

[0313] Miltuximab was placed in PBS (pH 7.4) buffer, and EDTA solution was added to a final concentration of 2 mM, resulting in an antibody concentration of 1.5 mg / mL. Reduction was performed at 37°C for 2 h using 10 equivalents of TCEP. After reduction, unreacted TCEP was removed using a 40 kDa MWCO Zeba™ centrifugal desalting column, and the reaction solution was replaced with PBS (pH 7.4) buffer. Following antibody reduction, N,N-dimethylacetamide (DMA) and L015-CC885 dissolved in DMA were added at a volume ratio of 10% (organic solvent) and a feed ratio of 14 equivalents of L015-CC885. The reaction solution was placed at 22℃ for 1 h. After coupling, it was purified in one step using a 40 kDa MWCO Zeba™ centrifugal desalting column. The coupling product was then stored in a 20 mM histidine-acetic acid (pH 5.5) solution to obtain the coupling compound Miltuximab-L015-CC885-DAR8. The final DAR value was 7.88 as determined by LC-MS.

[0314] Example 7.5: Sample Preparation of MC-VC-PAB-MMAE-ADC

[0315] 7.5.1: Miltuximab-MC-VC-PAB-MMAE Sample Preparation

[0316] Miltuximab was placed in PBS (pH 6.0) buffer, and EDTA solution was added to a final concentration of 2 mM, resulting in an antibody concentration of 2.68 mg / mL. Reduction was then performed at 37°C for 2 h using 9.50 equivalents of TCEP. After reduction, unreacted TCEP was removed using a 40 kDa MWCO Zeba™ centrifugal desalting column, and the reaction solution was replaced with 20 mM histidine-acetic acid (pH 5.5). Following antibody reduction, N,N-dimethylacetamide (DMA) and MC-VC-PAB-MMAE dissolved in DMA were added at a volume ratio of 10% (organic solvent) and a feed ratio of 10 equivalents of MC-VC-PAB-MMAE. The reaction solution was placed at 22℃ for 1 h. After coupling, it was purified in one step using a 40 kDa MWCO Zeba™ centrifugal desalting column. The coupling product was then stored in a 20 mM histidine-acetic acid (pH 5.5) solution to obtain the coupling compound Miltuximab-MC-VC-PAB-MMAE (hereinafter referred to as Mil-MMAE). The final DAR was determined to be 4.13 by HIC-HPLC.

[0317] 7.5.2: Preparation of mAb02-MC-VC-PAB-MMAE Samples

[0318] mAb02 was placed in PBS (pH 7.4) buffer, and EDTA solution was added to a final concentration of 2 mM, resulting in an antibody concentration of 2.3 mg / mL. Reduction was performed at 37°C for 2 h using 8.5 equivalents of TCEP. After reduction, N,N-dimethylacetamide (DMA) and MC-VC-PAB-MMAE dissolved in DMA were added at a volume ratio of 10% (organic solvent) and a feed ratio of 18 equivalents of MC-VC-PAB-MMAE. The reaction mixture was incubated at 22°C for 1 h. After coupling, the mixture was purified in one step using Amicon (30 kDa, 15 mL), and the conjugate was stored in 20 mM histidine-acetic acid (pH 5.5) solution to obtain the conjugate mAb02-MC-VC-PAB-MMAE. The final DAR value was 4.70 as determined by HIC-HPLC.

[0319] 7.5.3: Preparation of mAb07-MC-VC-PAB-MMAE Samples

[0320] Replacing mAb02 in Example 4.6 with antibody mAb07 yielded the conjugate mAb07-MC-VC-PAB-MMAE, with a DAR of 4.25 as determined by HIC-HPLC.

[0321] 7.5.4: Preparation of mAb02-H1L2-MC-VC-PAB-MMAE Samples

[0322] mAb02-H1L2 was placed in PBS (pH 7.4) buffer, and EDTA solution was added to a final concentration of 2 mM. The antibody concentration was 2 mg / mL, and reduction was performed at 37°C for 2 h using 4 equivalents of TCEP. After reduction, N,N-dimethylacetamide (DMA) and MC-VC-PAB-MMAE dissolved in DMA were added at a volume ratio of 10% and a feed ratio of 8 equivalents of MC-VC-PAB-MMAE. The reaction solution was incubated at 22°C for 1 h. After coupling, one-step purification was performed using Amicon (30 kDa, 15 mL), and the coupling product was stored in 20 mM histidine-acetic acid (pH 5.5) solution to obtain the conjugate mAb02-H1L2-MC-VC-PAB-MMAE. The final DAR was 4.13 as determined by HIC-HPLC.

[0323] 7.5.5: Sample preparation of mAb02-H1L3-MC-VC-PAB-MMAE

[0324] The conjugate mAb02-H1L3-MC-VC-PAB-MMAE was obtained by replacing mAb02-H1L3 with antibody mAb02-H1L3 in Example 7.5.4. The DAR was measured to be 3.83 by HIC-HPLC.

[0325] 7.5.6: Preparation of mAb02-H2L1-MC-VC-PAB-MMAE Samples

[0326] The conjugate mAb02-H2L1-MC-VC-PAB-MMAE was obtained by replacing mAb02-H2L1 with antibody mAb02-H2L1 in Example 7.5.4. The DAR was measured to be 4.01 by HIC-HPLC.

[0327] 7.5.7: Preparation of mAbO2-H2L2-MC-VC-PAB-MMAE Samples

[0328] The conjugate mAb02-H2L2-MC-VC-PAB-MMAE was obtained by replacing mAb02-H2L2 with antibody mAb02-H2L2 in Example 7.5.4. The DAR was 3.09 as determined by HIC-HPLC.

[0329] 7.5.8: Sample preparation of mAb02-H2L3-MC-VC-PAB-MMAE

[0330] The conjugate mAb02-H2L3-MC-VC-PAB-MMAE was obtained by replacing mAb02-H2L3 with antibody mAb02-H2L3 in Example 7.5.4. The DAR was measured to be 3.33 by HIC-HPLC.

[0331] 7.5.9: Preparation of mAb02-H3L2-MC-VC-PAB-MMAE Samples

[0332] The conjugate mAb02-H3L2-MC-VC-PAB-MMAE was obtained by replacing mAb02-H3L2 with antibody mAb02-H3L2 in Example 7.5.4. The DAR was measured to be 3.68 by HIC-HPLC.

[0333] 7.5.10: Preparation of mAb02-H3L3-MC-VC-PAB-MMAE sample

[0334] The conjugate mAb02-H3L3-MC-VC-PAB-MMAE was obtained by replacing mAb02-H3L3 with antibody mAb02-H3L3 in Example 7.5.HPLC, and the DAR was measured to be 4.52.

[0335] 7.5.11: Preparation of mAb02-H5L2-MC-VC-PAB-MMAE sample

[0336] The conjugate mAb02-H5L2-MC-VC-PAB-MMAE was obtained by replacing mAb02-H5L2 with antibody mAb02-H5L2 in Example 7.5.4. The DAR was 4.14 as determined by HIC-HPLC.

[0337] 7.5.12: Preparation of mAb02-H7L4-MC-VC-PAB-MMAE sample

[0338] The conjugate mAb02-H7L4-MC-VC-PAB-MMAE was obtained by replacing mAb02-H7L4 with antibody mAb02-H7L4 in Example 7.5.4. The DAR was 4.14 as determined by HIC-HPLC.

[0339] 7.5.13: Preparation of mAb02-H8L4-MC-VC-PAB-MMAE sample

[0340] The conjugate mAb02-H8L4-MC-VC-PAB-MMAE (hereinafter referred to as 02-H8L4-MMAE) was obtained by replacing mAb02-H8L4 with antibody mAb02-H8L4 in Example 7.5.4. The DAR was measured to be 4.29 by HIC-HPLC.

[0341] 7.5.14: Preparation of mAb07-H3L2-MC-VC-PAB-MMAE sample

[0342] The conjugate mAb07-H3L2-MC-VC-PAB-MMAE was obtained by replacing mAb02-H1L2 in Example 7.5.4 with antibody mAb07-H3L2, and the DAR was measured to be 3.31 by HIC-HPLC.

[0343] 7.5.15: Preparation of mAb07-H4L1-MC-VC-PAB-MMAE sample

[0344] The conjugate mAb07-H4L1-MC-VC-PAB-MMAE (hereinafter referred to as 07-H4L1-MMAE) was obtained by replacing mAb02-H1L2 in Example 7.5.4 with antibody mAb07-H4L1. The DAR was measured to be 3.73 by HIC-HPLC.

[0345] 7.5.16: Preparation of mAb07-H4L2-MC-VC-PAB-MMAE sample

[0346] The conjugate mAb07-H4L2-MC-VC-PAB-MMAE was obtained by replacing mAb02-H1L2 in Example 7.5.4 with antibody mAb07-H4L2, and the DAR was measured to be 3.89 by HIC-HPLC.

[0347] Example 8: Cellular efficacy test

[0348] Lung squamous cell carcinoma, esophageal cancer, and other cell types were seeded into 96-well plates, and different concentrations of conjugated drugs were added. After incubation for 3-5 days, cell viability was tested using a CGT kit. Luminescent Cell Viability Assay, Promega, Cat: G7573).

[0349] The formula for calculating the growth inhibition rate of conjugated drugs on tumor cells is: Inhibition rate % = (Fluorescence reading of cell administration wells - Fluorescence reading of culture medium control wells) / (Fluorescence reading of cell wells - Fluorescence reading of culture medium control wells) × 100%.

[0350] Based on the cell viability inhibition results and the concentration of the added drug, a four-parameter fitting method was used to calculate the half-maximal inhibitory concentration (IC50) of the drug on cell growth.

[0351] The origins of tumor cells are shown in Table 11. The FACS results of GPC1 expression in the lung squamous cell carcinoma cell line are shown in Figure 4; the FACS results of GPC1 expression in the esophageal cancer cell line are shown in Figure 5.

[0352] Table 11 Origin of Tumor Cells

[0353] Table 12 Results of cell killing by bioactive molecules on lung squamous cell carcinoma cell lines

[0354] Table 13 Results of cell killing by bioactive molecules on esophageal cancer cell lines

[0355] Conclusion: Based on the results in Tables 12 and 13, the bioactive molecules in this application exhibit significant inhibitory activity against the proliferation of esophageal cancer cell lines and lung squamous cell carcinoma cells. The molecules in this application all possess similar inhibitory activity against tumor proliferation.

[0356] Example 9: Pharmacokinetic Tests in Rats

[0357] An ADC conjugated with MC-VC-PAB-MMAE was injected into rats via the tail vein. Blood samples were then collected from the rats' orbital sinuses at time points, and the whole blood was processed into rat plasma. The concentrations of the whole antibody and the conjugated drug in the rat plasma were detected using ELISA. After treating the plasma with protease to release the MMAE conjugated to the antibody, the concentration of the conjugated drug was detected using LC-MS / MS.

[0358] The reagents and consumables used in the ELISA experiment are shown in Table 14.

[0359] Table 14 Information on Main Reagents and Consumables

[0360] Example 9.1 Detection of total antibody concentration in rat plasma using ELISA method

[0361] The concentration of total antibodies in rat plasma was detected using an ELISA method. The specific procedure was as follows: The corresponding antigen (GPC1-6xHis, sanyou bio) was diluted to 500 ng / mL with coating buffer (PBS buffer), mixed well, and transferred to a 96-well ELISA plate, 100 μL / well, incubated overnight at 4°C; the antigen coating buffer was discarded, and the plate was washed with PBST, 300 μL / well, repeated 5 times; blocking buffer (PBS buffer containing 3% BSA) was added, 100 μL / well, and the plate was blocked at room temperature for 1 hour; ADC standard curve samples were prepared using dilution buffer (PBST solution containing 1.5% BSA) containing 0.05% monkey plasma, with an initial concentration of 1000 ng / mL, 3-fold dilution, for a total of 10 concentration gradients. Control wells and blank control wells were also set up, containing the detection antibody (Goat Anti-Human IgG Fc Antibody, Horseradish Peroxidase (HRP) Conjugate). Rat plasma samples were diluted with dilution buffer and prepared in replicates. After blocking, the plate was washed repeatedly, and 100 μL of the test sample and ADC standard curve sample were added to each well. Incubation was allowed at room temperature for 1 hour. After incubation with the detection antibody (Goat Anti-Human IgG Fc Antibody, Horseradish Peroxidase (HRP) Conjugate), the plate was washed repeatedly, and 100 μL of TMB chromogenic buffer was added to each well. Incubation was allowed for approximately 15 minutes. Finally, 100 μL of TMB stop solution was added to each well to stop the incubation. The OD value at 450 nm was read using a microplate reader for concentration calculation.

[0362] Example 9.2 Detection of conjugated antibody concentration in rat plasma using ELISA method

[0363] The concentration of the conjugated drug antibody in rat plasma was detected using an ELISA method. The specific procedure was as follows: The corresponding antigen (Monoclonal Anti-MMAE Antibody, Acro, Cat#MME-M5252) was diluted to 500 ng / mL with coating buffer, mixed well, and transferred to a 96-well ELISA plate, 100 μL / well, incubated overnight at 4°C; the antigen coating buffer was discarded, and the plate was washed with PBST, 300 μL / well, repeated 5 times; blocking buffer was added, 100 μL / well, and the plate was blocked at room temperature for 1 hour; the ADC standard curve sample was prepared using Reagent Dilution Buffer containing 0.05% monkey plasma, with an initial concentration of 300 ng / mL, 3-fold dilution, for a total of 10 concentration gradients. Control wells for antibody detection and blank control wells were also included. Rat plasma samples were diluted with Reagent Dilution Buffer and prepared in replicates. After blocking, the plate was washed repeatedly, and 100 μL of the test sample and ADC standard curve sample were added to each well. Incubation was allowed at room temperature for 1 hour. The plate was washed again, and 100 μL of the detection antibody (prepared with Reagent Buffer at a 1:5000 dilution) was added to each well. Incubation was allowed at room temperature for 1 hour. After incubation, the plate was washed again, and 100 μL of TMB chromogenic buffer was added to each well. Incubation was allowed for approximately 15 minutes. Finally, 100 μL of TMB stop solution was added to each well to stop the incubation. The OD value at 450 nm was read using a microplate reader for concentration calculation.

[0364] The results of the rat PK property test of ADC are shown in Figure 6 (Figure of rat PK property test results of ADC).

[0365] Conclusion: As can be seen from the ELISA data in Figure 6, the PK properties of 02-H8L4-MMAE and 07-H4L1-MMAE in this application are basically the same in rats, which is consistent with the general rat pharmacokinetic characteristics of MMAE-ADC.

[0366] Example 10: Drug Efficacy Test in a Mouse CDX Model

[0367] Example 10.1 Efficacy test of anti-GPC1 antibody-drug conjugate against NCI-H1703 CDX model of lung squamous cell carcinoma.

[0368] NCI-H1703 cells were cultured in RPMI 1640 medium containing 10% FBS and maintained in a 37°C, humidified incubator with 5% CO2. NCI-H1703 cells in the logarithmic growth phase were collected and resuspended in RPMI 1640 basal medium containing 50% Matrigel, adjusting the cell concentration to 6 × 10⁻⁶ cells / year. 7Cells / mL. Under aseptic conditions, 0.1 mL of cell suspension was inoculated into the right fat pad of mice at a concentration of 6 × 10⁻⁶ cells / mL. 6 0.1 mL / mouse (cells / 0.1 mL). When the average tumor volume reaches 150-200 mm... 3 Around 10:00 AM, animals were randomly divided into groups of 6 animals each, based on tumor volume, ensuring that the difference in tumor volume between groups was less than 10% of the mean. The day of grouping was designated D0, and drug administration began.

[0369] Both the test substance group and the solvent control group received the drug via tail vein once a week for one week (IV, QW×1), at a dose of 0.5 mpk. Animals were sacrificed on day 28, tumors were harvested, weighed, and photographed; the experiment concluded.

[0370] The tumor-suppressive effect of the ADC in this application on the NCI-H1703 CDX model of lung squamous cell carcinoma is shown in Figure 7.

[0371] The experimental results showed that on day 28, both the test substances 02-H8L4-MMAE and 07-H4L1-MMAE effectively inhibited the growth and proliferation of human lung cancer NCI-H1703 xenografts in BALB / c Nude mice. The average body weight of the animals in each group increased by 2.94%-6.61% (0.70-1.53 ​​g) compared to day 0. No other significant drug-related adverse reactions were observed during the experiment.

[0372] The formula for calculating the percentage change in animal body weight (%BWC) is: (BW... t -BW0) / BW0*100%, where BW t BW0 represents the animal's weight at each measurement, while BW0 represents the animal's weight at the time of grouping.

[0373] Example 10.2 Efficacy test of anti-GPC1 antibody-drug conjugate against SK-MES-1CDX lung squamous cell carcinoma model

[0374] SK-MES-1 cells were cultured in EMEM medium containing 10% FBS and maintained in a 37°C, humidified incubator with 5% CO2. SK-MES-1 cells in the logarithmic growth phase were collected and resuspended in EMEM basal medium containing 50% Matrigel, adjusting the cell concentration to 1×10⁻⁶ cells / cells. 8 Cells / mL. Under aseptic conditions, 0.1 mL of cell suspension was inoculated into the right fat pad of mice at a concentration of 6 × 10⁻⁶ cells / mL. 6 0.1 mL / mouse (cells / 0.1 mL). When the average tumor volume reaches 170-200 mm. 3 Around 10:00 AM, animals were randomly divided into groups of 6 animals each, based on tumor volume, ensuring that the difference in tumor volume between groups was less than 10% of the mean. The day of grouping was designated D0, and drug administration began.

[0375] Both the test substance group and the solvent control group received the drug via tail vein once a week for one week (IV, QW×1), at a dose of 3 mpk. Animals were sacrificed on day 28, tumors were harvested, weighed, and photographed; the experiment concluded.

[0376] The tumor-suppressive effect of the ADC of this application on the SK-MES-1CDX model of squamous cell carcinoma of the lung is shown in Figure 8.

[0377] The experimental results showed that on day 28, the test substances Mil-MMAE, 02-H8L4-MMAE, and 07-H4L1-MMAE effectively inhibited the growth and proliferation of human lung cancer SK-MES-1 xenografts in BALB / c Nude mice, achieving near-complete tumor regression. The average body weight of the animals in each group increased by 4.3%-9.0% (0.61-1.69 g) compared to day 0. No other significant drug-related adverse reactions were observed during the experiment.

[0378] Example 10.3 Efficacy test of anti-GPC1 antibody-drug conjugate in KYSE-520 esophageal cancer model

[0379] Human esophageal squamous cell carcinoma KYSE520 cells were cultured in vitro under conditions specified in the supplier's technical instructions. The cells were passaged twice a week using trypsin-EDTA digestion. When cell confluence reached 80%-90% and the desired number was achieved, the cells were harvested, counted, and seeded. 0.1 mL (5 × 10⁻⁶ cells) was used as the seeding agent. 6 KYSE520 cells were mixed with 0.1 mL of Matrigel and subcutaneously injected into the right posterior dorsal region of mice. The average tumor volume reached ~200 mm². 3 The mice were divided into groups at the beginning of the treatment period. Six mice were enrolled in each treatment group and marked as D0. The administration of the drugs was then initiated.

[0380] Both the test substance group and the solvent control group received the drug via tail vein once a week for a total of 2 weeks (IV, QW×3), at a dose of 3 mpk. Animals were sacrificed on day 28, tumors were harvested, weighed, and photographed; the experiment concluded.

[0381] The tumor-suppressive effect of the ADC in this application on the KYSE-520CDX esophageal cancer model is shown in Figure 9.

[0382] The experimental results showed that on day 28, the test substances Mil-MMAE, 02-H8L4-MMAE, and 07-H4L1-MMAE only partially inhibited the growth of the KYSE-520 xenograft model of esophageal cancer. The tumor inhibition effect was best with 02-H8L4-MMAE, followed by 07-H4L1-MMAE, while Mil-MMAE showed almost no inhibition of tumor growth.

[0383] Example 10.4 Efficacy test of anti-GPC1 antibody-drug conjugate in TE8 esophageal cancer model

[0384] Human esophageal squamous cell carcinoma TE8 cells were cultured in vitro under conditions specified in the supplier's technical instructions. The cells were passaged twice a week using trypsin-EDTA digestion. When cell confluence reached 80%-90% and the desired number was achieved, the cells were harvested, counted, and seeded. 0.1 mL (10 × 10⁻⁶) of the solution was used for seeding. 6 After mixing 100 TE8 cells with 0.1 mL of Matrigel, the mixture was subcutaneously injected into the right posterior dorsal region of mice, resulting in an average tumor volume of approximately 200 mm². 3 The mice were divided into groups at the beginning of the treatment period. Six mice were enrolled in each treatment group and marked as D0. The administration of the drugs was then initiated.

[0385] Both the test substance group and the solvent control group received the drug via tail vein once a week for one week (IV, QW×1), at a dose of 3 mpk. Animals were sacrificed on day 28, tumors were harvested, weighed, and photographed; the experiment concluded.

[0386] The tumor-suppressive effect of the ADC in this application on the TE8 CDX model of esophageal cancer is shown in Figure 10.

[0387] The experimental results showed that on day 28, the test substances Mil-MMAE, 02-H8L4-MMAE, and 07-H4L1-MMAE all inhibited the growth of the TE8 xenograft model of esophageal cancer. The tumor inhibition effect was best with 02-H8L4-MMAE, followed by 07-H4L1-MMAE, while Mil-MMAE had the weakest inhibitory effect on tumor growth.

[0388] Example 11: Toxicity Study of Three-Dosage Administration in Crab-Eating Mammals

[0389] In the pre-toxicology studies of cynomolgus monkeys using 02-H8L4-MMAE and 07-H4L1-MMAE, one male and one female animal were enrolled for each ADC. The ADC was administered every three weeks at a dose of 5 mpk to study the toxicity of the cynomolgus monkeys. During the study, the weight and basic physical signs of the cynomolgus monkeys were observed. Blood samples were collected at the end of the first and third cycles of administration to study the effects of the drug on the monkeys' hematology and blood biochemistry. Gross dissection was performed on the animals 7 days after the third administration to study histopathological changes.

[0390] The effects of the ADC of this application on the body weight of cynomolgus monkeys at a dose of 5 mpk are shown in Figure 11; the effects of the ADC on the main hematological parameters of cynomolgus monkeys at a dose of 5 mpk are shown in Figure 12; and the effects of the ADC on the main blood biochemical parameters of cynomolgus monkeys at a dose of 5 mpk are shown in Figure 13.

[0391] The weight of the cynomolgus monkeys was not affected, and no abnormal changes related to the test product were observed in the general condition of the animals, detailed clinical condition, and injection site.

[0392] Regarding hematological parameters, the main toxicity was a decrease in white blood cells, lymphocytes, and platelets, which returned to normal levels before the second dose. A decrease in hemoglobin was also observed, but it tended to recover before the second dose. A dose of 5 mpk caused a transient increase in liver function parameters, which recovered rapidly. The hematological response in cynomolgus monkeys at this dose was load-related toxicity; no target-related toxicity was observed, confirming that the cynomolgus monkeys tolerated this dose.

[0393] Example 12: Immunohistochemical detection of GPC1 protein expression level in lung squamous cell carcinoma tumor tissue sections

[0394] Slides of squamous cell carcinoma of the lung from cancer patients were baked in an oven at 70°C for 2 hours for slide treatment. Then, they were deaceticated using xylene-anhydrous ethanol-water in sequence, and subjected to high pressure repair at pH 9.0 for 2.5 minutes. After standing for 5 minutes, they were soaked in hydrogen peroxide for 10 minutes and then blocked with blocking solution for 20 minutes to make the slides ready for staining with primary antibody.

[0395] Primary antibody (anti-GPC1: Proteintech, 16700-1-AP) was stained with PBS buffer at a dilution of 1:150 at 35°C for 1 hour. Then, secondary antibody (Poly-HRP anti-rabbit universal antibody) detection system was used for detection. The detection sequence of the secondary antibody detection system was as follows: AMP 20 minutes, HRP 30 minutes, DAB 9 minutes, hematoxylin 40 seconds, hydrochloric acid alcohol differentiation solution 1 second, and lithium carbonate 1 minute.

[0396] After staining, the slides are treated with anhydrous ethanol-xylene-anhydrous ethanol, baked in an oven for 3 minutes, and then mounted and examined.

[0397] The main reagents used in immunohistochemistry are as follows:

[0398] Table 15

[0399] The example results of IHC staining of lung squamous cell carcinoma tumor tissue sections are shown in Figure 14, and the scoring comparison tables are shown in Tables 16 and 17.

[0400] Table 16

[0401] Table 17 H-score = Intensity × Percentage

[0402] The staining evaluation results are shown in Table 18.

[0403] Table 18 Staining Evaluation Results

[0404] Example 13: Immunohistochemical detection of GPC1 protein expression levels in PDX sections of lung squamous cell carcinoma and esophageal cancer.

[0405] PDX models were constructed by inoculating mice with lung squamous cell carcinoma tissue and esophageal cancer tissue derived from patients. Tumor tissue was taken and corresponding sections were prepared. The sections of the PDX model were stained with GPC1 using the same immunohistochemical staining method as in Example 12.

[0406] Example results of PDX sections of lung squamous cell carcinoma and esophageal cancer are shown in Figure 15, and the scoring results are shown in Tables 19 and 20.

[0407] Table 19 IHC staining scores of PDX sections of lung squamous cell carcinoma and esophageal cancer.

[0408] Table 20

[0409] The sequence of this application is listed in Table 21 below.

[0410] Table 21 Serial Number of this Application

[0411] For purposes of description and disclosure, all patents, patent applications, and other publications are expressly incorporated herein by reference. These publications are provided solely because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, in any country, any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0412] Those skilled in the art will recognize that the scope of this application is not limited to the various specific implementations and embodiments described above, but rather that various modifications, substitutions, or recombinations can be made without departing from the spirit of this application, all of which fall within the protection scope of this application.

Claims

An anti-GPC1 antibody or its antigen-binding fragment, comprising: (a) Heavy chain variable regions containing the following complementary determinant regions: HCDR1, comprising the amino acid sequence shown in SEQ ID NO:1 or 7, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:1 or 7, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:1 or 7 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:1 or 7. HCDR2, comprising the amino acid sequence shown in SEQ ID NO:2 or 8, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:2 or 8, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:2 or 8 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:2 or 8. HCDR3, comprising the amino acid sequence shown in SEQ ID NO:3 or 9, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:3 or 9, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:3 or 9 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:3 or 9. (b) Light chain variable regions containing the following complementary determining regions: LCDR1, comprising the amino acid sequence shown in SEQ ID NO:4 or 10, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:4 or 10, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:4 or 10 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:4 or 10. LCDR2, comprising the amino acid sequence shown in SEQ ID NO:5 or 11, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:5 or 11, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:5 or 11 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:5 or 11. LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6 or 12, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:6 or 12, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:6 or 12 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:6 or 12. The anti-GPC1 antibody or its antigen-binding fragment according to claim 1 comprises: (1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively; or (2) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9 respectively, and LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12 respectively. The anti-GPC1 antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The anti-GPC1 antibody or its antigen-binding fragment comprises: (1) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

32. (2) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

36. (3) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

14. (4) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

16. (5) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

24. (6) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:25, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

24. (7) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

24. (8) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

24. (9) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

24. (10) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

24. (11) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

30. (12) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:25, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

30. (13) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

30. (14) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

30. (15) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

30. (16) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

30. (17) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

31. (18) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

32. (19) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

31. (20) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

31. (21) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

32. (22) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

36. (23) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

36. (24) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

36. (25) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

40. (26) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

40. (27) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:40; or (28) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

40. Preferably, the amino acid sequences having at least 80% identity with any of the amino acid sequences shown in SEQ ID NOs: 13, 14, 15, 16, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 have different amino acids that are mainly or entirely located in the FR region. The anti-GPC1 antibody or its antigen-binding fragment according to any one of claims 1-3, wherein, The anti-GPC1 antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; The heavy chain constant region is the heavy chain constant region of IgG, IgA, IgM, IgE or IgD or a variant thereof; the heavy chain constant region is preferably the heavy chain constant region of IgG or a variant thereof; the heavy chain constant region is more preferably the heavy chain constant region of human IgG1 or a variant thereof. The light chain constant region is the constant region of the human κ chain or λ chain or a variant thereof; Preferably, the heavy chain constant region comprises a full-length heavy chain constant region or a segment thereof, wherein the segment may be selected from the CH1 region, CH2 region, CH3 region or Fc region; More preferably, the Fc region is the human IgG1 Fc region. More preferably, the human IgG1 Fc region further has one or more mutations selected from the following: 329A, 329G, 329Y, 331S, 236F, 236R, 238A, 238E, 238G, 238H, 238I, 238V, 238W, 238Y, 248A, 254D, 254E, 254G, 254H, 254I, 254N, 254P, 2 54Q, 254T, 254V, 264S, 265H, 265K, 265S, 265Y, 265A, 267G, 267H, 267I, 267K, 434I, 438G, 439E, 439H, 439Q, 440A, 440D, 440E, 440F, 440M, 440T, and 440V, according to Kabat numbering; More preferably, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:17, or an amino acid sequence having at least 80% identity with it; and / or The light chain constant region contains an amino acid sequence as shown in SEQ ID NO:18 or an amino acid sequence having at least 80% identity with it. The anti-GPC1 antibody or its antigen-binding fragment according to any one of claims 1-4, wherein, The anti-GPC1 antibody or its antigen-binding fragment comprises: Heavy chain, the heavy chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 41, 43, 19 and 21 or an amino acid sequence having at least 80% identity with it; and A light chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 42, 44, 20 and 22 or an amino acid sequence having at least 80% identity with it; Preferably, the anti-GPC1 antibody or its antigen-binding fragment comprises: (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:41 or an amino acid sequence having at least 80% identity with it, and a light chain comprising the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having at least 80% identity with it. (2) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:43 or an amino acid sequence having at least 80% identity with it, and Light chain comprising the amino acid sequence shown in SEQ ID NO:44 or an amino acid sequence having at least 80% identity with it; (3) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 80% identity with it, and Light chain, comprising the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 80% identity with it; or (4) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 80% identity with it, and The light chain comprises the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 80% identity with it. The anti-GPC1 antibody or its antigen-binding fragment according to any one of claims 1-5, wherein, The antibody is a murine antibody, a monkey antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; preferably, the anti-GPC1 antibody is a humanized antibody; and / or The antigen-binding fragment is selected from at least one of the following: Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, and scFv. A nucleic acid molecule that encodes an anti-GPC1 antibody or an antigen-binding fragment thereof as described in any one of claims 1-6. A recombinant vector comprising the nucleic acid molecule of claim 7. A host cell comprising the nucleic acid molecule of claim 7 or the recombinant vector of claim 8. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, comprising the anti-GPC1 antibody or its antigen-binding fragment as described in any one of claims 1 to 6 and a drug; wherein... The antibody-drug conjugate has the structure shown in general formula (I): in: Bp is the anti-GPC1 antibody or its antigen-binding fragment as described in any one of claims 1 to 6; L stands for connector; D represents a drug unit, wherein the drug is selected from cytotoxic agents, radiolabelers, fluorophores, chromophores, imaging agents, immunomodulators, protein degraders, angiogenesis inhibitors, cell proliferation inhibitors, apoptosis promoters, cell lysing enzymes, and any combination thereof. p is any integer between 1 and 20. The antibody-drug conjugate according to claim 10, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, wherein... The connector is a breakable connector; Preferably, the linker is an enzyme-responsive, breakable linker; Preferably, the linker comprises peptide units that can be cleaved by proteases; Preferably, the linker comprises peptide units that can be cleaved by lysosomal enzymes; Preferably, the peptide unit is selected from -valine-citrulline-, -glycine-glycine-phenylalanine-glycine-, -glycine-proline-arginine-asparagine-leucine-valine-citrulline- (SEQ ID NO:47); More preferably, the linker is maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxy (MC-VC-pAB). The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 10 or 11, or in the form of its tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, wherein... The drug unit is selected from immunomodulators, protein degraders, and cytotoxic agents; Preferably, the drug unit comprises: amanitins, anthracyclines, auristatins, baccatins, calicheamicins, camptothecins, cemadotins, colchicine, colcimids, comprbetastatins, cryptophycins, discormolides, duocarmycins, docetaxel, doxorubicin, duocarmycins, echinomycins, and arugula. Elutherobins, epothilones, estramustines, lexitropsins, maytansines, maytansinoids, methotrexate, netropsins, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), puromycins, rhizoxins, SN-38, taxanes, tubulolysins, vincaalkaloids, tetrahydroisoquinoline alkaloids or their derivatives, protein degrading agents or their derivatives; And / or, the pharmaceutical unit includes maytansine alkaloids or derivatives thereof, camptothecin or derivatives thereof, auristatin or derivatives thereof, tetrahydroisoquinoline alkaloids or derivatives thereof, BTK protein degraders or derivatives thereof, GSPT1 protein degraders or derivatives thereof, and CRBN protein degraders or derivatives thereof. More preferably, the drug unit includes CC885, DX8951, MMAE, DM1, DM4, Dxd, SN-38, Trabectedin (ET743), Lurbinectedin, or CC-90009. The antibody-drug conjugate according to any one of claims 10-12, or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso compound, racemic mixture, enantiomer, diastereomer, or mixture thereof, wherein, The antibody-drug conjugate has the following structure: in, Anti-GPC1 is the anti-GPC1 antibody or its antigen-binding fragment; L1 is the connection segment between P and Anti-GPC1; P is a peptide unit that can be cleaved by proteases, selected from -valine-citrulline-, -glycine-glycine-phenylalanine-glycine-, -glycine-proline-arginine-asparagine-leucine-valine-citrulline- (SEQ ID NO:47); L2 is the connection segment between P and D; D is selected from MMAE, DX-8951, or CC885; n is any integer between 3 and 8; Preferably, L1 is MC-; And / or, L2 is -PAB-; Preferably, the DAR value of the antibody-drug conjugate is an integer or decimal of 2-8, 2.5-8, 3-8, 3-7, 3-6.5, 3-6, 3-5.5, 3-5, or 3-4.8; More preferably, the antibody-drug conjugate is selected from the following structures: Application of antibody-drug conjugates targeting GPC1 in the preparation of drugs for the diagnosis, prevention and / or treatment of squamous cell carcinoma of the lung. The use of any anti-GPC1 antibody or antigen-binding fragment thereof as claimed in any one of claims 1-6, the nucleic acid molecule as claimed in claim 7, the recombinant vector as claimed in claim 8, the host cell as claimed in claim 9, or the antibody-drug conjugate or solvate as claimed in any one of claims 10-13, or their tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof in the preparation of a medicament for the diagnosis, prevention, and / or treatment of GPC1-related diseases or conditions; Preferably, the disease or symptom is cancer; More preferably, the cancer is a solid tumor; More preferably, the cancer is squamous cell carcinoma of the lung, esophageal cancer, pancreatic cancer, cervical cancer, bladder cancer, colorectal cancer, liver cancer, glioma, lung cancer, head and neck cancer, thyroid cancer, endometrial cancer, breast cancer, or ovarian cancer.

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