Antibodies, antibody-drug conjugates, their preparation and uses

Abstract

Antibodies that specifically target CD142, antibody-drug conjugates, and their preparation and use are provided. The CD142-binding antibody or antigen-binding fragment thereof is covalently linked to a cytotoxic payload via a linker. The CD142-binding antibody or antigen-binding fragment thereof and antibody-drug conjugate exhibit cytotoxic effects against tumor cells.

Description

[Technical Field]

[0001] The present disclosure relates to antibodies that specifically target CD142, antibody-drug conjugates, their preparation and uses. [Background technology]

[0002] The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.

[0003] Antibody-drug conjugates (ADCs) are vectored chemotherapy drugs that selectively deliver cytotoxic drugs into tumor / cancer cells (Antibody-Drug Conjugates: The Last Decade, Nicolas Joubert, et al., Pharmaceuticals (Basel). 2020 Sep 14;13(9):245). The commercially available ADC drugs Enhertz and sacituzumab govitecan have excellent efficacy in treating tumors, especially malignant tumors. Both Enhertz and sacituzumab govitecan use DNA topoisomerase inhibitors (camptothecin derivatives) as cytotoxic drugs, which are more hydrophobic than tubulin inhibitors (e.g., MMAE, MMAF). Sacituzumab govitecan uses MCC-triazole spacer-PEG7-lysine-PABC as a linker to degrade and release camptothecin SN38 in cell lysosomes (US Pat. No. 13 / 948,732). Enhertz, developed by AstraZeneca / Daiichi Sankyo, uses the cathepsin B-activating GGFG (an amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked by a peptide bond) tetrapeptide as a linker, introducing a self-cleaving structure to release the exatecan derivative Dxd (Yusuke Ogitani et al., Clin Cancer Res (2016) 22(20):5097-5108). However, the above-mentioned cytotoxic drugs MMAE, SN38, and Dxd are all substrates of P-glycoprotein (P-gp) (Front Pharmacol 2019;10:749), and some tumors with high P-gp expression may be resistant to the drugs.

[0004] Tissue factor (TF), also known as CD142, is a transmembrane glycoprotein. In complex with its ligand FVIIa, CD142 activates protease-activated receptor 2, thereby activating intracellular signaling pathways exploited by tumors to promote malignant cell survival, tumor growth, angiogenesis, and metastasis. In contrast to limited surface expression on normal tissue cells, CD142 exhibits membranous CD142 expression on various solid tumor cells, including pancreatic, lung, cervical, prostate, bladder, ovarian, breast, and colon cancers. CD142 is significantly expressed on tumor cells and tumor vasculature and has been reported to be associated with poor disease prognosis and increased metastatic potential. These characteristics suggest CD142 as a potential ADC target. CD142-targeting ADCs to date include tisotumab vedotin, ICON-2 (XB002), and MRG004A.

[0005] Tisotumab vedotin, which uses the linker MC-VC-PABA and the payload MMAE (PCT / EP2014 / 075326, PCT / EP2009 / 066755), was approved for manufacture and sale in the United States in September 2021. Its indication is cervical cancer. According to the FDA (US Food and Drug Administration) recommendation, tisotumab vedotin is administered by injection every three weeks at a recommended dose of 2 mg / kg. This drug has serious adverse reactions, including cutaneous and ocular toxicity, and bleeding, and its therapeutic window is limited to 3 mg / kg (BLA Multi-disciplinary Review and Evaluation {Biologics License Application (BLA) 761208} {tisotumab vedotin}).

[0006] ICON-2 is currently undergoing Phase I clinical trials in the United States, and its indications are adenocarcinoma, bladder cancer, fallopian tube cancer, and head and neck tumors. The relevant patent is PCT / US2019 / 012427. The drug linker-payload uses Zymeworks' proprietary ZymeLink Auristatin (ZLA) technology. Similar to tisotumab vedotin, severe or significant dermal toxicity was observed in non-human primate (NHP) toxicity studies of ICON-2 (Thi-Sau Migone, et al., "ICON-2, a Tissue Factor-Targeted Antibody-Drug Conjugate for the Treatment of Solid Tumors." Presented at World ADC Digital, September 15-18, 2020).

[0007] Dose escalation and dose expansion in a Phase I / II clinical trial of MRG004A (related patent PCT / CN2017 / 087779) is currently underway in China and the United States to evaluate safety, tolerability, pharmacokinetic properties, and preliminary efficacy. Preclinical studies have shown that MRG004A also inhibits coagulation (Oncotarget, 2017, Vol. 8, (No. 35), pp: 59086-59102).

[0008] Therefore, there remains a need to develop new CD142-targeting antibodies and antibody-drug conjugates. Summary of the Invention

[0009] The present disclosure provides a novel isolated antibody or antigen-binding fragment thereof that binds to CD142, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, and methods for preparing and using the same, with the aim of reducing or eliminating serious adverse reactions and improving anti-tumor activity.

[0010] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to CD142, comprising at least a heavy chain variable region (VH) and at least a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 1, HCDR2 comprises the amino acid sequence represented as IYPGX1GDX2 (SEQ ID NO: 2), HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 3, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 4, LCDR2 comprises the amino acid sequence represented as LTS, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 5, wherein X1 is D or Q and X2 is S or A.

[0011] In some embodiments, X1 is D and X2 is S. In some embodiments, X1 is Q and X2 is S. In some embodiments, X1 is Q and X2 is A.

[0012] The present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to CD142, comprising at least a heavy chain variable region (VH) and at least a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence represented by SEQ ID NO: 20, HCDR2 comprises the amino acid sequence represented as IRNRAX3X4YTT (SEQ ID NO: 21), HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 22, LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 23, LCDR2 comprises the amino acid sequence represented as YTS, and LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 24, wherein X3 is N or Q, and X4 is G or A.

[0013] In some embodiments, X3 is N and X4 is G. In some embodiments, X3 is N and X4 is A. In some embodiments, X3 is Q and X4 is G.

[0014] In one aspect, the present disclosure provides a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to CD142 as described above.

[0015] In one aspect, the disclosure provides a vector, the vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to CD142.

[0016] In one aspect, the disclosure provides a host cell, the cell comprising the nucleic acid or vector described above.

[0017] In one aspect, the present disclosure provides a compound of formula I: Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, wherein: Ab is an isolated antibody or antigen-binding fragment thereof that binds to CD142; L is a linker covalently binding Ab and D, respectively; D is the payload, n is an integer from 1 to 10.

[0018] In one aspect, the disclosure provides a method for preparing an antibody-drug conjugate of Formula I, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, the method comprising reducing an isolated antibody or antigen-binding fragment thereof that binds to CD142, such that disulfide bonds are at least partially reduced, and reacting with a reactive group of a linker in a linker-payload to obtain an antibody-drug conjugate of Formula I.

[0019] In some embodiments, the method comprises reducing the antibody such that disulfide bonds of the antibody are at least partially reduced and reacted with the carbon atom at position 3 of the maleimide-N-yl of the linker of Formula IV in the linker-payload; TIFF2025535258000002.tif39132 here In the linker-payload, the carbonyl group in the ester group of the linker of formula IV is linked to the amino group of the payload, In Formula IV, R1 and R2 are independently selected from hydrogen, methyl, and isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is hydrogen or benzyl, n 1 represents an integer of 0 to 2, and n 2 represents an integer from 0 to 2, R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 1 to 20.

[0020] In one aspect, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to CD142, or an antibody-drug conjugate of Formula I, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient.

[0021] In one aspect, the present disclosure provides a kit comprising an isolated antibody or antigen-binding fragment thereof that binds to CD142, or an antibody-drug conjugate as described above.

[0022] In one aspect, the present disclosure provides the use of an isolated antibody or antigen-binding fragment thereof that binds to CD142, an antibody-drug conjugate of Formula I, an antibody-drug conjugate of Formula I prepared by a method herein, the pharmaceutical composition described above, or a kit in the manufacture of a therapeutic agent for the diagnosis, prevention, and treatment of tumor diseases.

[0023] In some embodiments, the tumor comprises a solid tumor that expresses CD142.

[0024] In one aspect, the present disclosure provides a method for reducing the number of CD142-expressing cells, comprising administering to a subject a therapeutic dose of a therapeutic agent, the therapeutic agent comprising an isolated antibody or antigen-binding fragment thereof that binds to CD142, an antibody-drug conjugate of Formula I, an antibody-drug conjugate of Formula I prepared by a method herein, a pharmaceutical composition, or a kit as described above.

[0025] The isolated antibody or antigen-binding fragment thereof that binds to CD142 and the antibody-drug conjugate of Formula I provided herein have improved or excellent in vivo efficacy and safety, and HNSTD (no severe toxicity seen at the maximum dose) can reach 30 mg / kg. Furthermore, the isolated antibody or antigen-binding fragment thereof that binds to CD142 and the antibody-drug conjugate of Formula I provided herein have a one-week effect on blood coagulation function, thus avoiding the possibility of bleeding-related adverse reactions.

[0026] The following is a brief description of the drawings, which are presented for the purpose of illustrating exemplary embodiments disclosed herein and are not intended to limit the present specification. [Brief explanation of the drawings]

[0027] [Figure 1] A and B show the size exclusion chromatography and hydrophobic interaction chromatography detection graphs, respectively, of the Hu01-L3H1 naked antibody prepared according to Example 4. The Hu01-L3H1 naked antibody is used as a quality control substance. [Figure 2] A and B show the size exclusion chromatography and hydrophobic interaction chromatography detection graphs, respectively, of the Hu02-L1H2 naked antibody prepared according to Example 5. The Hu02-L1H2 naked antibody is used as a quality control substance. [Figure 3] 1A and 1B show the size exclusion chromatography and hydrophobic interaction chromatography detection graphs, respectively, of the antibody-drug conjugate Hu01-L3H1-LP1-DAR8 prepared according to Example 7. [Figure 4]1A and 1B show the size exclusion chromatography and hydrophobic interaction chromatography detection graphs, respectively, of the antibody-drug conjugate Hu01-L3H1-LP1-DAR4 prepared according to Example 8. [Figure 5] 1A and 1B show the size exclusion chromatography and hydrophobic interaction chromatography detection graphs, respectively, of the antibody-drug conjugate Hu02-L1H2-LP1-DAR8 prepared according to Example 9. [Figure 6] 1A and 1B show the size exclusion chromatography and hydrophobic interaction chromatography detection graphs, respectively, of the antibody-drug conjugate HuIgG-LP1-DAR8 prepared according to Comparative Example 1. [Figure 7] A and B show the size exclusion chromatography and hydrophobic interaction chromatography detection graphs, respectively, of the reference ADC prepared according to Comparative Example 2. [Figure 8] 1 shows flow cytometry graphs of endocytosis of BxPC3 cells toward antibody Mu01 prepared according to Example 4 and antibody Mu02 prepared according to Example 5. MFI is the abbreviation for mean fluorescence intensity. [Figure 9] 1 shows cell viability-concentration change curves of MDA-MB-231 cells for antibody Mu01 prepared in Example 4 and antibody Mu02 prepared in Example 5. [Figure 10] 1 shows the affinity curves of BxPC3 cells obtained by flow cytometry for humanized antibody candidate molecules L5H2, L5H1, L3H1, and L3H2 derived from Mu01 and the chimeric antibody Ch01. [Figure 11] 1 shows flow cytometry affinity curves of BxPC3 cells for humanized antibody candidate molecules L1H1, L1H2, L1H3, L1H4, L1H5, L1H6, L2H1, L2H2, L2H3, L2H5, and L2H6 derived from Mu02, and chimeric antibody Ch02. [Figure 12] 1 shows the peak change curves of human plasma thrombin affected by the antibody Hu01-L3H1 prepared in Example 4 and the antibody Hu02-L1H2 prepared in Example 5. [Figure 13] 1 shows a killing assay of KYSE150 cells by Hu01-L3H1-LP1-DAR8 prepared according to Example 7 and Hu02-L1H2-LP1-DAR8 prepared according to Example 9. [Figure 14] 1 shows a killing assay of 5637 cells by Hu01-L3H1-LP1-DAR8 prepared according to Example 7 and Hu02-L1H2-LP1-DAR8 prepared according to Example 9. [Figure 15] 1 shows a killing assay of SW780 cells by Hu01-L3H1-LP1-DAR8 prepared according to Example 7 and Hu02-L1H2-LP1-DAR8 prepared according to Example 9. [Figure 16] 1 shows a killing assay of Detroit 562 cells by Hu01-L3H1-LP1-DAR8 prepared in Example 7, Hu01-L3H1-LP1-DAR4 prepared in Example 8, and Hu02-L1H2-LP1-DAR8 prepared in Example 9. [Figure 17] 1 shows the in vivo efficacy of the ADC in the NCI-H292 cell-derived xenograft mouse model. [Figure 18] 1 shows the weight-time change curve after treatment in a NCI-H292 cell-derived xenograft mouse model. [Figure 19] 1 shows the in vivo efficacy of the ADC in a cell-derived xenograft mouse model of NCI-H226. [Figure 20] 1 shows the in vivo efficacy of the ADC in a cell-derived xenograft mouse model of IGROV1. [Figure 21] 1 shows the in vivo efficacy of the ADC in an SW780 cell-derived xenograft mouse model. [Figure 22] The in vivo efficacy of the ADC is shown in a patient-derived tumor xenograft mouse model constructed with human lung cancer tissue. [Figure 23] The in vivo efficacy of the ADC is shown in a patient-derived tumor xenograft mouse model constructed with human cervical cancer tissue. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention is described in further detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention can be implemented or all the features that can be added to the invention. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be omitted from that embodiment. Moreover, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure without departing from the invention. Thus, the following description is intended to illustrate some specific embodiments of the invention, but is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in carrying out tests of the present disclosure, the preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terms are used:

[0030] Unless otherwise defined, all numerical values ​​used in the specification and claims to express contents, concentrations, ratios, masses, volumes, times, temperatures, thicknesses, technical effects, and the like, are to be understood as being modified in all instances by the term "about" or "approximately." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties and effects sought to be obtained by the present disclosure, and each numerical parameter should be construed in accordance with the number of significant digits and conventional rounding approaches, or as understood by one of ordinary skill in the art.

[0031] Notwithstanding that the numerical ranges and parameters setting forth the scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range provided throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0032] [antibody] The present disclosure provides examples of isolated antibodies or antigen-binding fragments thereof that bind to CD142, also known as tissue factor (TF). CD142 exhibits membranous CD142 expression on various solid tumor cells, compared with restricted surface expression on normal tissue cells, and is associated with poor tumor prognosis and increased metastatic potential. Therefore, CD142 can be used as a target and / or biomarker for the treatment and diagnosis of targeted tumors.

[0033] The term "antibody" or "antibodies" refers to immunoglobulin molecules capable of specifically binding to targets, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., via at least one antigen recognition site located within the variable region of the immunoglobulin molecule. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL). The variable region is a region at the N-terminus of the antibody molecule that exhibits significant variation in amino acid composition and arrangement. The specific binding site, i.e., the antigen-binding site, is used to determine the specificity of antibody recognition. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity-determining regions" (CDRs), interspersed with more conserved regions known as "framework regions" (FRs). Each VH and VL generally consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely defined using methods known in the art, such as the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al. (1997) J. Mol. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. Antibodies can be intact (i.e., full-length) polyclonal or monoclonal antibodies.Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of a particular class. Immunoglobulins can be divided into different classes depending on the antibody amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0034] As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab'), Fv, single-chain (scFv), variants thereof, antibody portions, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single-chain antibodies, fusion proteins including multispecific antibodies (e.g., bispecific antibodies), and any other modified constructs of immunoglobulin molecules that contain an antigen recognition site with a desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0035] The present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to CD142, comprising at least a heavy chain variable region (VH) and at least a light chain variable region (VL), wherein the VH comprises heavy chain CDRs (HCDRs) 1, 2, and 3, and the VL comprises light chain CDRs (LCDRs) 1, 2, and 3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence set forth as IYPGX1GDX2 (SEQ ID NO: 2), HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, LCDR2 comprises the amino acid sequence set forth as LTS (leucine-threonine-serine), and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 5, wherein X1 is D or Q and X2 is S or A. The CDRs are defined / numbered according to the IMGT system.

[0036] In some embodiments, X1 is D and X2 is S. In some embodiments, X1 is Q and X2 is S. In some embodiments, X1 is Q and X2 is A. Amino acids are referred to herein by single-letter codes well known to those of skill in the art, e.g., "D," "Q," "S," and "A" represent aspartic acid, glutamine, serine, and alanine, respectively.

[0037] Antibodies with the same light / heavy chain CDR1, CDR2, and CDR3 regions as the exemplary antibodies targeting CD142 are within the scope of this disclosure.

[0038] The antibody or antibody framework region may have mutations that do not affect the binding of the antibody variable region to the antigen, and these mutations may increase the binding affinity of the antibody to the antigen or may remain substantially unchanged. In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds to CD142 further comprises conservatively modified variants, which include individual substitutions, deletions, or additions to the polypeptide sequence, resulting in the replacement of amino acids with chemically similar amino acids. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles. The following eight groups contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence.

[0039] In some embodiments of the isolated antibody or antigen-binding fragment thereof that binds to CD142, the VH comprises the amino acid sequence represented by SEQ ID NO: 8, 9, 10, 11, or 12, and the VL comprises the amino acid sequence represented by SEQ ID NO: 13, 14, 15, 16, or 17.

[0040] In some embodiments, the variable region of the isolated antibody or antigen-binding fragment thereof that binds to CD142 is selected from the following (a-1) to (e-1): (a-1) a VH having at least 70%, 86%, 87%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 6, and at least 70%, 79%, 80%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 7. (b-1) a VL having 87%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 8; (b-2) a VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 15; (c-3) a VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 8; (d-1) a VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 9 and a VL having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 17; (d-2) a VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 9 and a VL having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 15; and (e-1) a VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 9, and a VL having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 17.

[0041] In this disclosure, the term "identity" in the context of two or more nucleic acid or polypeptide sequences refers to the degree to which two or more sequences or subsequences are identical. Two sequences are "identical" if they have the same sequence of amino acids or nucleotides over the region being compared. Two sequences are "substantially identical" if they have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length. Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms (described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1997; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively).

[0042] In addition to the above-mentioned sequence identity percentage, another indicator that two polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibody produced against the polypeptide encoded by the second nucleic acid, as described below.In this case, the polypeptide is typically substantially identical to the second polypeptide, for example, the two peptides differ only by conservative substitutions.Another indicator that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize with each other under stringent conditions.Another indicator that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0043] In some embodiments, the VH of the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises the amino acid sequence set forth in SEQ ID NO:6, and the VL comprises the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:8, and the VL comprises the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:8, and the VL comprises the amino acid sequence set forth in SEQ ID NO:17. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:9, and the VL comprises the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:9, and the VL comprises the amino acid sequence set forth in SEQ ID NO:17.

[0044] In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises at least a heavy chain variable region (VH) and at least a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20, HCDR2 comprises the amino acid sequence set forth as IRNRAX3X4YTT (SEQ ID NO: 21), HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, LCDR2 comprises the amino acid sequence set forth as YTS (tyrosine-threonine-serine), and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24, wherein X3 is N or Q and X4 is G or A. CDRs are defined according to the IMGT system.

[0045] In some embodiments, X3 is N and X4 is G. In some embodiments, X3 is N and X4 is A. In some embodiments, X3 is Q and X4 is G.

[0046] In some embodiments, the VH of the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises the amino acid sequence represented by SEQ ID NO: 27, 28, 29, 30, 31 or 32, and the VL comprises the amino acid sequence represented by SEQ ID NO: 33 or 34.

[0047] In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises a VH and a VL selected from the following (a-2) to (l-2): (a-2) a VH having at least 70%, 85%, 87%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, and a VL having at least 70%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 26; (b-2) a VH having at least 70%, 85%, 87%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 26; (c-2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 27, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 33; (c-3) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 28, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 33; (d-4) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 33; (e-2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by sequence number 29, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 33; (f- 2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 31, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 33; (g-2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 32, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 33;(h-2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 27, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 34; (i-2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 28, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 34; (j-2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 29, and and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 34; (k-2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 31, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 34; and (l-2) a VH having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 32, and a VL having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 34.;

[0048] In some embodiments, the VH of the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises the amino acid sequence set forth in SEQ ID NO:25, and the VL comprises the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:27, and the VL comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:28, and the VL comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:29, and the VL comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:30, and the VL comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:31, and the VL comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:32, and the VL comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:27, and the VL comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:28, and the VL comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:29, and the VL comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:31, and the VL comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:32, and the VL comprises the amino acid sequence set forth in SEQ ID NO:34.

[0049] The isolated antibody or antigen-binding fragment thereof that binds to CD142 provided by the present disclosure can bind to mammalian (e.g., human or mouse) CD142 protein. In some embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to human CD142. In some embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to mouse CD142.

[0050] The isolated antibodies or antigen-binding fragments thereof provided herein that bind to CD142 comprise heavy and light chain constant regions, wherein the constant chains are derived from immunoglobulin classes IgM, IgG, IgA, IgD, or IgE, or subclasses thereof. In some embodiments, the isolated antibodies or antigen-binding fragments thereof that bind to CD142 are of the IgG class, optionally of the IgG1, IgG2, IgG3, or IgG4 subclass. In some embodiments, the isolated antibodies or antigen-binding fragments thereof that bind to CD142 are of the human IgG1 subclass.

[0051] In some embodiments, the heavy chain of any isolated antibody or antigen-binding fragment thereof that binds to CD142 described herein may further comprise a heavy chain constant region (CH) or a portion thereof, and the light chain may further comprise a light chain constant region (CH) or a portion thereof. The constant region may be of any suitable origin, e.g., human, mouse, rat, or rabbit. Antibody heavy and light chain constant regions are well known in the art, such as those provided in the IMGT database (imgt.org) or vbased2.org, both of which are incorporated herein by reference.

[0052] In some embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:35, and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:36.

[0053] Optionally, an isolated antibody or antigen-binding fragment thereof that binds to CD142 described herein can comprise a modified constant region. For example, it can comprise a modified constant region that is immunologically inert, e.g., does not induce complement-mediated lysis or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity can be assessed using the methods disclosed in U.S. Patent No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624, PCT Application No. PCT / GB99 / 01441, and / or UK Patent Application No. 9809951.8.

[0054] The isolated antibody or antigen-binding fragment thereof that binds to CD142 described herein can be a human antibody, a humanized antibody, or a chimeric antibody.

[0055] In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds CD142 described herein is a human antibody.

[0056] The term "human antibody" is one that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies strictly exclude humanized antibodies.

[0057] In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds CD142 described herein is a humanized antibody.

[0058] The term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues of the recipient's complementarity-determining regions (CDRs) are derived from mouse, rat, or rabbit CDRs (donor antibody), substituting residues from a non-human species having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are present neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further improve and optimize antibody performance. Generally, humanized antibodies comprise substantially the entire variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin consensus sequence. A humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region or domain (Fc), typically a human immunoglobulin constant region or domain. Antibodies may have altered Fc regions as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and / or six) that are altered relative to the original antibody, also referred to as "derived from" one or more CDRs of the original antibody.

[0059] In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds CD142 described herein is a chimeric antibody, which may contain heavy and light chain constant regions from a human antibody. A chimeric antibody refers to an antibody having a variable region or portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from one mammal (e.g., a non-human mammal such as mouse, rabbit, or rat), while the constant regions are homologous to sequences in antibodies from another mammal, such as a human. In some embodiments, amino acid modifications may be made in the variable and / or constant regions.

[0060] The isolated antibodies or antigen-binding fragments thereof that bind to CD142 described herein can be prepared by any method known in the art, e.g., Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0061] Isolated antibodies or antigen-binding fragments thereof that bind to CD142 can be obtained by immunizing animals with CD142 or any polypeptide selected from the amino acid sequence of CD142 and collecting and purifying the antibodies produced in vivo, according to methods commonly practiced in the art. In this case, antibodies applicable to human diseases can be selected by examining the cross-reactivity of the obtained antibodies that bind to heterologous CD142 with human CD142. Alternatively, they can be obtained by the following known method (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennet, R. eds., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)), in which antibody-producing cells that produce antibodies against CD142 are fused with myeloma cells to establish hybridomas, and monoclonal antibodies are obtained from the hybridomas. CD142 used as an antigen can be obtained by expressing the CD142 gene in host cells using genetic engineering.

[0062] Hybridomas can be engineered to obtain chimeric antibodies, such as those in which antibody variable regions of mouse or rat origin are linked to constant regions of human origin (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0063] Humanized antibodies can be exemplified by antibodies obtained by incorporating only the complementarity-determining regions (CDRs) into an antibody of human origin (see Nature (1986) 321, pp. 522-525), and by antibodies obtained by CDR grafting, for example, antibodies obtained by grafting a portion of framework amino acid residues in addition to the CDR sequences (WO90 / 07861). Human antibodies can be obtained by a method using a human antibody-producing mouse carrying a human chromosomal fragment containing heavy and light chain genes of a human antibody (see, for example, Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. eds., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727).

[0064] The isolated antibodies or antigen-binding fragments thereof provided herein specifically bind to CD142 and exhibit good affinity for CD142-expressing cells. The isolated antibodies or antigen-binding fragments thereof have a clear killing effect on tumor cells. In some embodiments, the isolated antibodies or antigen-binding fragments thereof provided herein that bind to CD142 specifically bind to diseased cells, e.g., CD142. + Tumor cells may be reduced / eliminated, thereby treating and / or diagnosing CD142-targeted tumors.

[0065] The isolated antibodies or antigen-binding fragments thereof provided herein have reduced effects on blood coagulation function and reduced coagulation toxicity. In some embodiments, the isolated antibodies or antigen-binding fragments thereof provided herein have reduced effects on clotting time compared to prior art antibodies targeting CD142, such as tisotumab vedotin, and are effective in avoiding bleeding side effects.

[0066] The present disclosure provides nucleic acids encoding the above-described isolated antibody or antigen-binding fragment thereof that binds to CD142, wherein the nucleic acid encodes a VH and / or a VL. In some embodiments, the nucleic acid encoding the VH comprises the nucleotide sequence set forth in SEQ ID NO: 18 and the nucleic acid encoding the VL comprises the nucleotide sequence set forth in SEQ ID NO: 19, or the nucleic acid encoding the VH comprises the nucleotide sequence set forth in SEQ ID NO: 37 and the nucleic acid encoding the VL comprises the nucleotide sequence set forth in SEQ ID NO: 38.

[0067] The present disclosure also provides a vector comprising the above-described nucleic acid, while the present disclosure provides a host cell comprising the above-described nucleic acid or vector.

[0068] [Antibody-drug conjugates] The present disclosure provides an antibody-drug conjugate having the structure of Formula I, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, Ab-(LD)n (I) wherein Ab is an isolated antibody or antigen-binding fragment thereof that binds to CD142; L is a linker covalently binding Ab and D, respectively; D is the payload, n is an integer from 1 to 10.

[0069] The term "antibody drug conjugate," also known as "ADC," refers to an antibody or antigen-binding conjugate thereof described herein that binds to CD142 and is covalently linked to a payload. Typically, an antibody drug conjugate may include an antibody, a payload, and optionally a linker between the antibody and the payload. An ADC is a conjugate of an antibody or antigen-binding conjugate that binds to CD142 and is covalently linked to a payload. + cells, especially CD142 + Therapeutic effects can be provided by delivering a payload to tumor cells. Antibody-drug conjugates can be prepared by a variety of methods known to those skilled in the art.

[0070] The term "linker" refers to the linking structure connecting the antibody and the payload. The molecular design and properties of the linker are crucial determinants of the efficacy of an ADC in terms of pharmacokinetics (PK) / pharmacodynamics (PD) and therapeutic window. For optimal efficacy, an ideal linker should possess the following properties: (1) The linker should be sufficiently stable in plasma so that the ADC can circulate in the bloodstream and localize to the tumor site without premature cleavage. Linker instability can lead to premature release of the toxic payload and unwanted damage to non-target healthy cells, resulting in systemic toxicity and adverse effects. (2) The linker should be capable of rapid cleavage to release the liberated toxic payload once the ADC is internalized into the target tumor cells. (3) Another property to consider in linker design is hydrophobicity. A hydrophobic linker coupled to a hydrophobic payload often promotes ADC aggregation. Such molecules are undesirable in the pursuit of therapeutically useful ADCs and may cause liver toxicity or elicit unwanted immune responses (Kyoji Tsuchikama et al., Antibody-drug conjugates: recent advances in conjugation and linker chemistry, Protein Cell. 2018 Jan;9(1):33-46).

[0071] The term "isomer" refers to compounds with the same molecular formula but different structures, also known as structural isomers, and generally includes structural isomers and stereoisomers. Structural isomers refer to isomers caused by differences in the connection order of atoms in a molecule or by different bonding properties, and preferably include tautomers. Tautomers refer to functional group isomers resulting from the rapid movement of atoms at two positions in a molecule. Stereoisomers refer to isomers caused by atoms or atomic groups in a molecule that are connected to each other in the same order and bond but have different spatial arrangements, and preferably include optical isomers. Optical isomers refer to stereoisomers that have different optical properties due to the lack of reverse axial symmetry in the molecule, such as enantiomers, diastereomers, racemates, and meso isomers.

[0072] The term "prodrug" refers to a compound obtained by modifying the chemical structure of a drug, which is inactive or less active in vitro and exerts a pharmacological effect by releasing the active drug through enzymatic or non-enzymatic conversion in vivo. In the present disclosure, the prodrug may be an ADC molecule or a payload.

[0073] In some embodiments, the linker is a cleavable linker or a non-cleavable linker.

[0074] In some embodiments, the linker comprises a cleavable peptide.

[0075] In some embodiments, the cleavable peptide is enzymatically cleavable.

[0076] In some embodiments, the enzyme comprises cathepsin B.

[0077] In some embodiments, the cleavable peptide or L comprises an amino acid unit.

[0078] In some embodiments, the amino acid unit comprises a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

[0079] In some embodiments, the amino acid unit is selected from the group consisting of Val-Cit, Val-Ala, Glu-Val-Cit, Ala-Ala-Asn, Gly-Val-Cit, Gly-Gly-Gly, and Gly-Gly-Phe-Gly, or combinations thereof. Amino acids represented by three letter codes are well known to those of skill in the art and include, but are not limited to, Val for valine, Cit for citrulline, Ala for alanine, Glu for glutamic acid, Asn for asparagine, Gly for glycine, and Phe for phenylalanine.

[0080] In some embodiments, L comprises at least one spacer that can provide distance between the payload and the antibody.

[0081] In some embodiments, the spacer comprises a self-immolative spacer.

[0082] In some embodiments, the self-immolative spacer comprises p-aminobenzoxycarbonyl (PABC) or p-aminobenzyl (PAB).

[0083] A self-immolative spacer can be defined as a bifunctional chemical moiety that can covalently link two spaced chemical moieties into a generally stable tripartite molecule, e.g., that can be cleaved enzymatically to release one of the spaced chemical moieties from the tripartite molecule, and that, following cleavage (e.g., enzymatic cleavage), can spontaneously cleave from the remainder of the molecule to release the other spaced chemical moiety.

[0084] In some embodiments, the cleavable peptide is spliced ​​directly to the spacer.

[0085] In some embodiments, the spacer is -NH-(CH2)n 4 -La-Lb-Lc-, wherein La represents -O- or a single bond, and Lb represents -CR 2 (-CR3 )-, or a single bond, where R 2 and R 3 are each independently C1-C6 alkyl, -(CH2)n a -NH2, -(CH2)n b -COOH, or -(CH2)n c represents -OH, n 4 represents an integer from 0 to 6, and n a , n b and n c each independently represents an integer of 1 to 4, and R 2 and R 3 is n a is 0, Lc represents -C(=O)-.

[0086] In some embodiments, the spacer comprises -NH-(CH2)3-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-(CH2)2-O-CH2-C(=O)-.

[0087] In some embodiments, the linker comprises a structure represented by -L1-L2-L3-, where L1 is -(succinimidyl-3-yl-N)-(CH2)m 1 -C(=O)-, -CH2-C(=O)-NH-(CH2)m 2 -C(=O)- or -C(=O)-(CH2)m 3 represents -C(=O)-, where m 1 represents an integer from 2 to 8, and m 2 represents an integer from 1 to 8, and m 3 represents an integer of 1 to 8, L2 represents an amino acid unit, and L3 represents a self-immolative spacer.

[0088] In some embodiments, m 1 represents 2, 3, 4, 5, 6, 7, or 8. In some embodiments, m 2 represents 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, m 3 represents 1, 2, 3, 4, 5, 6, 7, or 8.

[0089] In some embodiments, L is selected from the group consisting of: -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-GGFG-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-PABC-; -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-PABC-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-VA-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2o-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-PABC-; -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-PABC-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-PABC-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2-O-CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-O-CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(Succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; and -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-.

[0090] In some embodiments, the p-aminobenzoxycarbonyl (PABC) or p-aminobenzyl (PAB) comprises a polysarcosine (poly-N-methylglycine) residue or a methylamino group.

[0091] In some embodiments, the linker comprises Formula II: TIFF2025535258000003.tif31134 In Formula II, R1 and R2 are independently selected from hydrogen, methyl, and isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond; R5 is selected from hydrogen or benzyl; n 1 represents an integer of 0 to 2, and n 2 represents an integer of 0 to 2; R4 represents a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 1 to 20.

[0092] In some embodiments, in the linker of Formula II, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 1 to 20. 3may be selected from any integer of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0093] In some embodiments, in the linker of Formula II, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer of 8 to 15. In some embodiments, in the linker of Formula II, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 10 to 12.

[0094] In some embodiments, in the linker of formula II, R4 represents a methylamino group.

[0095] In the present disclosure, the introduction of R4 containing a hydrophilic amino group (such as a polysarcosine group or a methylamino group) is beneficial for increasing the hydrophilicity of the antibody-drug conjugate, especially when a hydrophobic payload is conjugated to the antibody-drug conjugate. Increasing the hydrophilicity of the ADC helps reduce aggregation of the ADC during the preparation process, thereby improving the stability, homogeneity, and purity of the ADC.

[0096] In some embodiments, in the linker of formula II, R3 represents a single bond.

[0097] In some embodiments, in the linker of Formula II, R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 -, R5 is benzyl, and n 1 represents an integer of 1 or 2, and n 2 represents the integer 1 or 2.

[0098] In some embodiments, in the linker of formula II, R3 represents -CR5HCONH-, -CH2CONH-, -CR5HCONH-CH2CONH-; -(CR5HCONH)2-CH2CONH-; -CR5HCONH-(CH2CONH)2-; or -(CR5HCONH)2-(CH2CONH)2-; and R5 is benzyl.

[0099] In some embodiments, in the linker of Formula II, R is hydrogen. In some embodiments, in the linker of Formula II, R is isopropyl.

[0100] In some embodiments, in the linker of Formula II, R2 is hydrogen. In some embodiments, in the linker of Formula II, R2 is methyl.

[0101] In some embodiments, the linker of the antibody drug conjugate is selected from the group consisting of: TIFF2025535258000004.tif115142

[0102] In the present disclosure, in the antibody-drug conjugate, the succinimidyl group of the linker of Formula II is covalently linked to the antibody. In some embodiments, the terminal succinimidyl group of the linker of Formula II forms a thioether bond with the sulfhydryl group obtained by reduction of the interchain disulfide chain of the antibody. The succinimidyl group TIFF2025535258000005.tif4044, which forms a thioether bond with the sulfhydryl moiety obtained by reduction of the interchain disulfide chain of the antibody by the carbon atom at position 3. A bond by represents a chemical bond that is connected to another group. TIFF2025535258000006.tif2525

[0103] In the present disclosure, disulfide bonds of an antibody include interchain disulfide bonds and intrachain disulfide bonds, preferably interchain disulfide chains that are processed, e.g., activated to sulfhydryls, and then attached to a linker. The amino acids in the antibody that are chemically attached to succinimidyl groups include one or a combination of lysine, histidine, tyrosine, and cysteine, preferably cysteine.

[0104] The term "payload" includes compounds that are cytotoxic or capable of killing cells when released from an antibody-drug conjugate, compounds, radionuclides, or polypeptides that have a radiolabel, a fluorophore, a chromophore, an imaging agent, and / or a metal ion as a detection label or have a cell-killing effect, and compounds, nucleic acids, polypeptides or proteins, enzymes, hormones, or nucleic acids that are capable of modulating immune activity in the body (including activating or inhibiting effects).

[0105] Under ideal conditions, the conjugated payload in an antibody-drug conjugate has little or no cytotoxicity, or its cytotoxicity is so low that administration of a therapeutically effective dose of the ADC does not cause systemic toxicity in a subject due to the conjugated payload. The payload can be a clinically validated drug for the treatment of a particular disease, or a compound, radionuclide, nucleic acid, protein, or polypeptide with acceptable pharmacological activity under conditions of clinical use.

[0106] In some embodiments, the payload in the antibody-drug conjugate is a label containing a radiolabel, a luminescent material, a chromophore, an imaging agent, and / or a metal ion as a detection label. Labels include, but are not limited to, chemically synthesized organic compounds, radionuclides, metal complexes, or polypeptides. Here, a radiolabel refers to a labeled compound in which one or more atoms in the compound molecule are replaced with a radionuclide so that the compound can be identified and used as a tracer. Radiolabels include amino acids, polypeptides, proteins, carbohydrates, nucleotides, nucleosides, purines, pyrimidines, steroids, lipid compounds, as well as tumor antigens, hormones, receptors, vitamins, and drugs used in medical research. Radionuclides are typically nuclei that can naturally emit radiation, including, but not limited to, tritium, iodine-125, iodine-131, sulfur-35, phosphorus-32, and carbon-14. The luminescent material is typically a group containing a conjugated double bond, and fluoresces when the molecule falls back from an excited state to the ground state. A chromophore refers to an unsaturated group and its associated chemical bond contained in a molecule that can absorb light radiation and has a transition. An imaging agent typically refers to a radiopharmaceutical in nuclear medicine that can image organs, tissues, or molecules when introduced into the body.

[0107] In some embodiments, the payload in the antibody drug conjugate is a nucleic acid, which can be a ribonucleic acid and / or a deoxyribonucleic acid.

[0108] In some embodiments, the payload in the antibody-drug conjugate is a hormone, a growth factor, a clotting factor, or a plasminase (e.g., a prodrug-converting enzyme, a ribonuclease, that can convert a prodrug into an active drug).

[0109] In some embodiments, the payload in an antibody-drug conjugate is an immunomodulatory agent (including cytokines and chemokines that can affect immunity), or a biologically active agonist or antagonist antibody.

[0110] In some embodiments, the payload in the antibody-drug conjugate is a cytotoxic compound. In some embodiments, the payload in the antibody-drug conjugate has antitumor activity or is an antitumor drug. The payload is selected from a DNA topoisomerase inhibitor or a tubulin inhibitor. The DNA topoisomerase inhibitor can be a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0111] The term "topoisomerase inhibitor" generally refers to a compound that inhibits topoisomerase activity. Compounds known as topoisomerase I inhibitors have activity against topoisomerase I, and topoisomerase II inhibitors have activity against topoisomerase II. Some compounds have activity against both topoisomerase I and topoisomerase II and are known as topoisomerase I / II inhibitors.

[0112] The term "tubulin inhibitor" generally refers to compounds that inhibit the microtubule system of eukaryotic cells, disrupting cell division and inhibiting cell proliferation.

[0113] In some embodiments, the payload is camptothecin or a derivative thereof that has a topoisomerase inhibitory effect. The term "derivative" refers to a compound formed by replacing an atom or group of atoms in the molecule of a parent compound with another atom or group of atoms, and is called a derivative of the parent compound. The term "camptothecin and its derivatives" generally includes camptothecin and camptothecin derivatives. Camptothecin exerts its pharmacological effect by irreversibly inhibiting topoisomerase I. Camptothecin derivatives include exatecan, irinotecan, topotecan, lurtotecan, siratecan, etirinotecan pegol, TAS103, 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methylenedioxycamptothecin, 9-amino-10,11-methylenedioxycamptothecin, 9-chloro-10,11-methylenedioxycamptothecin, and 9-chloro-10,11-methylenedioxycamptothecin. Camptothecins include 1-methylenedioxycamptothecin, (7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin, 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecin, and 7-(2-N-isopropylamino)ethyl)-(20S)-camptothecin, as well as stereoisomers, salts, and esters thereof. Methods for synthesizing camptothecin and its camptothecin analogs or derivatives are known and are summarized and described in U.S. Pat. No. 5,244,903, which is incorporated herein by reference in its entirety.

[0114] In some embodiments, the payload is an auristatin or a derivative thereof, or maytansine or a derivative thereof, which has a tubulin inhibitory effect. The term "auristatin or a derivative thereof" generally includes auristatin F and auristatin F derivatives. Auristatin F derivatives include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). The term "maytansine or a derivative thereof" generally includes maytansine and maytansine derivatives. Maytansine derivatives include maytansine DM1, maytansine DM2, and maytansine DM4.

[0115] In some embodiments, the payload is exatecan, a camptothecin derivative, which, as a topoisomerase inhibitor, can act throughout the cell cycle and has strong permeability and good therapeutic effect on slow-growing solid tumors. Furthermore, the number of intracellular targets is much smaller than that of tubulin inhibitors, so a better killing effect can be achieved when the ADC molecule carries the same amount of payload into cells. Meanwhile, the exatecan molecule is not a substrate of P-gp, which is beneficial in reducing or alleviating the problem of drug resistance.

[0116] In some embodiments, the payload is a camptothecin of formula III or a pharmaceutically acceptable salt thereof, which is linked to the linker by the nitrogen atom of the amino group on its cyclohexane ring. TIFF2025535258000007.tif7193

[0117] Due to its rigid structure and low hydrophilicity, exatecan molecules easily undergo polymerization when linked to the GGFG tetrapeptide linker commonly used in the prior art to prepare ADCs, which does not meet the requirements for ADC drug development (Bioorg. Med. Chem. Lett. 26 (2016) 1542-1545). Therefore, the selection and matching of linkers and payloads affects the safety and stability of ADC drugs.

[0118] Without being bound by any theory, multiple hydrophilic groups in the linker of an antibody-drug conjugate can improve the hydrophilicity of the linker-payload structure and reduce aggregation and precipitation of the ADC caused by a hydrophobic payload.

[0119] After the ADC molecule is endocytosed into a cell, the linker is degraded, releasing the payload compound or the linker (or part of the linker)-payload structure. In some embodiments, the amino group on the cyclohexane ring of exatecan of Formula III is bonded to a carbonyl group in the ester group of the linker of Formula I, forming a carbamate-containing linker-payload structure. Without being bound by any theory, after the ADC molecule is endocytosed into a cell, the linker is cleaved by a cathepsin (e.g., cathepsin B) to form an intermediate or active metabolite as shown in Formula V below: TIFF2025535258000008.tif71109 R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 1 to 20.

[0120] The PABC group in the intermediate or active metabolite of Formula V then undergoes 1,6-elimination to release exatecan. The mechanism of 1,6-elimination of PABC is described in Angew. Chem. Int. Ed. 2015, 54, 7492-7509. Therefore, the linker-payload structure in the ADC provided by the present disclosure has good in vivo stability and biological activity.

[0121] Without being bound by any theory, the cleavage site in the linker-payload structure may be an amide bond in the linker, for example, an amide bond between the carbon atom on which the substituent represented by R2 is located and the group represented by R3, or an amide bond in the group represented by R3.

[0122] In antibody-drug conjugates, n, i.e., the ratio of the number of molecules of conjugated payload to each molecule of antibody (DAR), is 1 to 10. In some embodiments, n is 1 to 10, 1 to 2, 2 to 4, 4 to 6, 2 to 8, 4 to 8, 4 to 10, 6 to 10, 7 to 10, or 8 to 10, illustratively n is 4.66, 7.67, or 7.83.

[0123] The term "DAR (drug-to-antibody ratio)" refers to the average number of conjugated payload or drug molecules per antibody molecule, i.e., the average number of conjugated drug molecules. In an antibody-drug conjugate, the number of conjugated payload molecules per antibody molecule is an important factor affecting its efficacy and safety. The production of an antibody-drug conjugate is carried out by specifying reaction conditions, such as the amounts of starting materials and reagents used in the reaction, to achieve a certain number of conjugated payload molecules. When an antibody-drug conjugate is prepared, a mixture containing different numbers of conjugated payload molecules is usually obtained. Unless otherwise specified, in the present disclosure, the number of conjugated payload or drug molecules per antibody molecule is defined as the average value, i.e., the average number of conjugated payload or drug molecules.

[0124] In some embodiments, ADCs with the above-described linkers are chemically coupled to antibodies targeting CD142, resulting in higher DAR values ​​(e.g., DAR8).

[0125] In some embodiments, the antibody-drug conjugate comprises any one of the following structures: TIFF2025535258000009.tif127129TIFF2025535258000010.tif127162Ab represents an isolated antibody or antigen-binding fragment thereof that binds to CD142, and n is equal to the ratio of the number of molecules of payload to each molecule of antibody, i.e., DAR.

[0126] The antibody in the antibody-drug conjugate specifically targets CD142, and the antibody forms a reactive sulfhydryl group through a disulfide bond and is then linked to a linker. In some embodiments, a disulfide bond in the hinge region of the antibody forms a reactive sulfhydryl group and is then linked to a linker.

[0127] In some embodiments, the antibody in the antibody-drug conjugate is an antibody or antigen-binding fragment thereof provided above. In some embodiments, the antibody or antigen-binding fragment thereof in the antibody-drug conjugate comprises a VH sequence set forth in SEQ ID NO: 8 and a VL sequence set forth in SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment thereof in the antibody-drug conjugate comprises a VH sequence set forth in SEQ ID NO: 28 and a VL sequence set forth in SEQ ID NO: 33.

[0128] Improving the hydrophilicity of an antibody-drug conjugate may also increase its homogeneity, and therefore, antibody-drug conjugates with improved hydrophilicity and / or homogeneity may have improved cytotoxic effects on target cells, and improve or maintain biological activity, safety, and other pharmaceutical properties.

[0129] The antibody-drug conjugate of the present disclosure has an improved HNSTD (maximum non-severe toxicity dose). Furthermore, it may exhibit significant tumor suppression effects without causing obvious gastrointestinal toxicity. It should be noted that the antibody-drug conjugate of the present disclosure may absorb and retain water, or may become a hydrate upon exposure to air or recrystallization. Such hydrated compounds and their salts are also included in the present disclosure. Furthermore, isotopically variant compounds labeled with various radioactive or non-radioactive isotopes are also included in the present disclosure. Two or more atoms constituting the antibody-drug conjugate of the present disclosure may contain atomic isotopes in unnatural proportions. Examples of atomic isotopes include deuterium (2H), tritium (3H), iodine-125 (125I), and carbon-14 (14C). For example, compounds of the present disclosure can be radiolabeled with radioactive isotopes, such as, for example, tritium (H), iodine-125 (I), or carbon-14 (C). Radiolabeled compounds can be used as therapeutic or prophylactic agents, research reagents, such as test reagents, and diagnostic agents, e.g., in vivo diagnostic imaging agents. All isotopic variants of the antibody-drug conjugates of the present disclosure, whether radioactive or not, are included within the scope of the present disclosure.

[0130] [Pharmaceutical composition] The present disclosure provides a pharmaceutical composition comprising the above-described isolated antibody or antigen-binding fragment thereof that binds to CD142, and a pharmaceutically acceptable excipient.

[0131] The present disclosure provides a pharmaceutical composition comprising the above-described antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient.

[0132] Pharmaceutical compositions can be administered in an appropriate manner depending on the specific application form, physicochemical properties, etc. of the pharmaceutically acceptable excipient. In some embodiments, pharmaceutical compositions can be formulated in the form of a lyophilized preparation or a liquid preparation, which can contain appropriate formulation additives known in the art. For example, the pharmaceutical compositions typically contain a variety of pharmaceutical carriers, such as sterile liquids, such as water and oils (including oils of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). For intravenous administration of the pharmaceutical compositions, water is a more typical carrier. In addition, physiological saline, aqueous glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The pharmaceutical compositions can also contain minor amounts of wetting agents, emulsifiers, or pH buffering agents, as needed. The mode of administration of pharmaceutical compositions is typically parenteral, and can be, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous injection. For example, the pharmaceutical compositions can be administered by infusion or bolus injection. See, e.g., Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0133] The pharmaceutical composition may comprise an active agent, i.e., an isolated antibody or antigen-binding fragment thereof that binds to CD142 or the antibody-drug conjugate described above, and a second therapeutic agent (e.g., a cancer therapeutic agent). In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds to CD142 of the present disclosure may be administered together with other cancer therapeutic agents to enhance the anti-cancer effect. In some embodiments, the antibody-drug conjugate of the present disclosure may be administered together with other cancer therapeutic agents to enhance the anti-cancer effect. The other anti-cancer agent used for this purpose may be administered to an individual simultaneously, separately, or sequentially with the antibody-drug conjugate of the present disclosure, or may be administered at different intervals. Exemplary other cancer therapeutic agents may be, for example, paclitaxel, cisplatin, vinblastine, etc., but are not limited thereto, as long as they have anti-tumor activity.

[0134] In accordance with the present disclosure, the active agent or a pharmaceutical composition comprising the same can be administered to a subject via any suitable route of administration. For example, the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following description of the administration route is provided solely to illustrate various embodiments and should not be construed as limiting the scope in any way.

[0135] The present disclosure also provides kits comprising an isolated antibody or antigen-binding fragment thereof that binds to CD142, or the antibody-drug conjugates described above, which are useful for detecting CD142 or CD142-expressing cells.

[0136] [Preparation method] The present disclosure provides a method for preparing an antibody-drug conjugate, the method comprising the steps of: reducing the antibody or antigen-binding fragment thereof so that its disulfide bonds are at least partially reduced, and reacting with a reactive group of the linker in the linker-payload to form an antibody-drug conjugate having the structure of Formula I: Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof; wherein Ab is the isolated antibody or antigen-binding fragment thereof that binds to CD142 as described above, L is a linker that covalently binds Ab and D, respectively, D is a payload, and n is an integer from 1 to 10.

[0137] The reactive group is not particularly limited, so long as it contains a moiety that can react with a sulfhydryl group of the resulting antibody.

[0138] In some embodiments, the carbon atom at position 3 of the maleimide-N-yl in LD reacts with and covalently attaches to a reduced antibody to prepare an ADC.

[0139] In some embodiments, the method comprises the steps of: reducing an antibody such that the interchain disulfide bonds of the antibody are at least partially reduced; and reacting the carbon atom at position 3 of the maleimido-N-yl of a linker represented by formula IV; TIFF2025535258000011.tif39136 The carbonyl group in the ester group of the linker of Formula IV is linked to the amino group of the payload in the antibody-drug conjugate; In Formula IV, R1 and R2 are each independently selected from hydrogen, methyl, and isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is selected from hydrogen or benzyl, n 1 represents an integer of 0 to 2, and n 2 represents an integer from 0 to 2, R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 1 to 20.

[0140] In antibody-drug conjugates, in most practical cases, a linker having the structure of Formula IV above, which bears a payload, is linked to the same antibody molecule bearing a reactive thiol group. In some embodiments, the antibody is reacted with a reducing agent, such as dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP), to break down disulfide bonds in the antibody chains and form reactive sulfhydryl groups. The amount of reducing agent can be 0.3 to 10 times the molar equivalent of the antibody, for example, 1 to 10, 3 to 10, 5 to 10, or 7 to 10 times the molar equivalent of the antibody.

[0141] In some embodiments, the method further comprises reacting the antibody with a reducing agent in a buffer solution containing a chelator, followed by adding a linker-payload solution to carry out the reaction. The linker-payload is a compound formed by bonding a linker of Formula IV with a payload, in which an amino group (primary amino group) in the payload is linked to a carbonyl group in the ester group of the linker of Formula IV. The term "chelator" refers to a complex that can form a complex with a cyclic structure through a coordinate bond with a metal atom or ion.

[0142] In some embodiments, the reducing agent reacts with the antibody in a buffer solution containing a chelating agent to produce an antibody with partially or completely reduced interchain disulfide bonds. Chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). The chelating agent is used at a concentration of 1 mM to 20 mM, e.g., 2 mM to 20 mM, 5 mM to 20 mM, 8 mM to 20 mM, 1 mM to 15 mM, or 1 mM to 10 mM. Components of the buffer solution can be buffer salts commonly used in the art, such as sodium phosphate, sodium borate, sodium acetate, or similar buffer salts.

[0143] The reaction of the antibody with the reducing agent is carried out at a controlled pH. In some embodiments, when the antibody reacts with the reducing agent, the pH of the solution is 5 to 9, optionally 6 to 8, 6 to 7, 6.5 to 7.5, or 7 to 8. For example, the reaction is carried out when the pH of the solution is about 7. The pH of the solution can be adjusted using either acidic or basic chemicals, examples of which include acetic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sodium bicarbonate, sodium carbonate, sodium hydroxide, and triethylamine.

[0144] The reaction of the antibody with the reducing agent is carried out under a controlled temperature, and exemplary reaction temperatures are -10 to 40°C, -5 to 40°C, 0 to 40°C, 5 to 40°C, 25 to 40°C, 30 to 40°C, 35 to 38°C, for example, about 37°C.

[0145] The linker-payload may be dissolved in an organic solvent selected from any one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP), or a combination thereof.

[0146] In some embodiments, the linker-payload solution is added to a buffered solution of a reduced antibody or an antibody having a reactive thiol group in an amount of 1% to 20% by volume based on the volume of the buffered antibody solution, hi some embodiments, the volume ratio of the added linker-payload solution is 1% to 20%, 2% to 20%, 5% to 20%, 10% to 20%, 15% to 20%, 1% to 18%, 1% to 15%, 1% to 13%, 1% to 10%, or 5% to 15% based on the volume of the buffered antibody solution.

[0147] In some embodiments, the DAR is 4 to 20, optionally 8 to 20. In some embodiments, the DAR is 10 to 20, 14 to 20, 16 to 20, or 18 to 20.

[0148] In some embodiments, the temperature at which the antibody reacts with the linker-payload is -10 to 40°C, -5 to 40°C, 0 to 40°C, 5 to 40°C, 10 to 40°C, 15 to 40°C, 20 to 40°C, or 0 to 37°C. In some embodiments, the reaction temperature is 5 to 37°C, 10 to 37°C, 10 to 25°C, or 15 to 30°C.

[0149] In some embodiments, the antibody is reacted with the linker-payload for 0.5 to 2 hours, 0.5 to 1.75 hours, 0.5 to 1.5 hours, 0.5 to 1.25 hours, 0.75 to 2 hours, or 1 to 2 hours.

[0150] The reaction can be terminated by inactivating any unreacted linker-payload using a thiol-containing reagent, such as, but not limited to, cysteine ​​or N-acetyl-(L)-cysteine ​​(NAC). More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of the thiol-containing reagent to the reaction solution relative to the linker-payload and incubating at room temperature (10 to 25°C) for 10 to 30 minutes.

[0151] When the antibody has a sulfhydryl group, the antibody-drug conjugate can also be obtained by reacting the compound using known methods (e.g., methods described in Patent Publication US2016 / 297890 (e.g., methods described in paragraphs

[0336] to

[0374] ). Antibodies having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)).

[0152] The antibody-drug conjugates provided by the present disclosure can be obtained by the above-mentioned preparation methods. In some embodiments, the prepared antibody-drug conjugates are subjected to a purification process, including, but not limited to, gel filtration, for example, purification using a gel column.

[0153] [How to use] The present disclosure also provides use of the above-described antibody, the above-described antibody-drug conjugate, the above-described pharmaceutical composition, or the antibody-drug conjugate prepared by the above-described method, or the above-described kit in the manufacture of a therapeutic agent for the diagnosis, prevention, and treatment of tumor diseases, including benign tumors and malignant tumors (e.g., cancer).

[0154] The present disclosure provides methods for diagnosing, preventing, and treating tumor diseases, comprising administering a therapeutically effective amount of an antibody, an antibody-drug conjugate, an antibody-drug conjugate prepared by the above-described method, a pharmaceutical composition, or the above-described kit to a subject in need thereof.

[0155] The tumor disease is not limited to the above diseases, as long as the cells at the lesion site express a protein that can be recognized by the CD142-targeting antibody. In some embodiments, the tumor disease includes ovarian cancer, gastric cancer, esophageal cancer, cervical cancer, prostate cancer, pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer, bladder cancer, melanoma, and kidney cancer.

[0156] In some embodiments, the neoplastic disease is a CD142-associated disease, such as a CD142-expressing solid tumor. In some embodiments, CD142-associated diseases include pancreatic cancer, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, and cervical cancer.

[0157] The present disclosure provides use of the above-described antibody, the above-described antibody-drug conjugate, the above-described pharmaceutical composition, or the antibody-drug conjugate prepared by the above-described method in the manufacture of a therapeutic agent targeting CD142.

[0158] The present disclosure provides a method for reducing the number of CD142-expressing cells, comprising administering to a subject in need thereof a therapeutically effective amount of the above-described antibody, antibody-drug conjugate, pharmaceutical composition, or antibody-drug conjugate prepared by the above-described method. Elimination of CD142-expressing cells in the subject is effective in treating a CD142-associated disease.

[0159] As used herein, a subject can be a non-human mammal or a human. Non-human mammals include, but are not limited to, livestock, game animals, pets, primates, horses, dogs, cats, mice, and rats. In some embodiments, the subject is a human. A human subject in need of treatment can be a human subject who has, is at risk of, or is suspected of having a tumor or a target disease / disorder associated with a CD142-associated tumor.

[0160] Subjects with target diseases or disorders can be identified by routine medical examinations (for example, clinical tests, organ function tests, CT scans, or ultrasound).Subjects suspected of having any such target disease / disorder can show one or more symptoms of the disease / disorder.Subjects at risk of having disease / disorder can be subjects with one or more risk factors for the disease / disorder.

[0161] The term "therapeutically effective amount" refers to the amount of each active agent, alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject. In some embodiments, the therapeutic effect is a reduction in CD142 activity or a reduction in CD142 activity. +It refers to a decrease in cellular activity. It will be clear to those skilled in the art that the amount of antibody or ADC containing an antibody determines whether a therapeutic effect is achieved. As those skilled in the art will recognize, the effective amount will depend on factors such as the specific condition being treated, the severity of the condition, individual patient parameters (including age, health, size, sex, and weight), the duration of treatment, the nature of concomitant therapy (if any), the specific route of administration, and the knowledge and expertise of the medical professional. These factors are well known to those skilled in the art and can be accounted for by no more than routine experimentation. In general, it is preferable to use the maximum amount of each component or combination thereof, i.e., the maximum safe amount according to sound medical judgment.

[0162] In some embodiments, the dosage of an antibody or antigen-binding fragment thereof, or antibody-drug conjugate can be empirically determined in an individual who has received one or more administrations of the antibody. In some embodiments, acceptable therapeutic doses of an antibody-drug conjugate are 0.1-30 mg / kg, 0.5-30 mg / kg, 1-30 mg / kg, 1-25 mg / kg, 0.1-25 mg / kg, 0.1-20 mg / kg, 1-20 mg / kg, or 0.5-20 mg / kg. In some embodiments, the dosing frequency is once every 12 hours, once daily, once weekly, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks; or once monthly, once every two months, or once every three months or less. The therapeutic dose and administration frequency can vary depending on the treatment regimen.

[0163] The various embodiments and preferred embodiments of the present disclosure may be combined with one another unless they are essentially inconsistent with one another, and the various embodiments formed by the combination are considered to be part of the disclosure of this application.

[0164] The technical solutions of the present disclosure are explained more clearly and specifically below with reference to embodiments as examples. It should be understood that these embodiments are for illustrative purposes only and are in no way intended to limit the protection scope of the present disclosure. The protection scope of the present disclosure is limited only by the claims. [Example]

[0165] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0166] Example 1: Preparation of compound LP-1 TIFF2025535258000012.tif144143Step 1: Synthesis of intermediate 11-1 DCM (dichloromethane):MeOH (methanol) (v:v = 2:1, 90 mL) and EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; 1.86 g, 7.55 mmol) were added to a mixed solution of compound 11-1A (Mc-Val-Ala-OH, purchased from MedChemExpress Shanghai; 2.4 g, 6.29 mmol) and compound 11-1B (3.18 g, 6.29 mmol) at room temperature (20 °C-30 °C). The reaction solution was stirred at room temperature for 24 h, and the solvent therein was removed in vacuo. The crude residue was then further purified by flash chromatography to give compound 11-1 (3.9 g, 71%). LC-MS (ESI, m / z): 868.49 (M+H).

[0167] Step 2: Synthesis of intermediate 11-2 Compound 11-1 (2 g, 2.3 mmol) was dissolved in anhydrous THF (tetrahydrofuran; 50 mL), and hydrogen fluoride-pyridine (4.6 g, 46 mmol) was added thereto under an argon atmosphere at 0 °C. The reaction mixture was then stirred at 0 °C for 2 hours. The reaction was quenched by adding water. The resulting mixture was extracted with DCM, and the organic phase therein was dried and concentrated. The residue was purified by silica gel column chromatography to give compound 11-2 (1.1 g, 76%). LC-MS (ESI, m / z): 630.31 (M+H).

[0168] Step 3: Synthesis of intermediate 11-3 Compound 11-2 (700 mg, 1.11 mmol) was dissolved in anhydrous DMF (N,N-dimethylformamide; 4 mL), and DIPEA (N,N-diisopropylethylamine; 0.39 ml, 2.23 mmol) and 4,4'-dinitrodiphenyl carbonate (406 mg, 1.33 mmol) were added thereto at room temperature under an argon atmosphere. The reaction mixture was then stirred overnight at ambient temperature. The solvent in the reaction mixture was removed by concentration, and the resulting product was precipitated using MTBE (methyl tert-butyl ether). The yellow solid was collected by filtration, washed with diethyl ether, and dried to give compound 11-3. LC-MS (ESI, m / z): 795.41 (M+H).

[0169] Step 4: Synthesis of intermediate 11-4 Compound 11-3 (300 mg, 0.44 mmol) was dissolved in anhydrous DMF (4 mL), and dry pyridine (1 mL) was added, followed by the addition of exatecan mesylate (purchased from MedChemExpress Shanghai; 234 mg, 0.44 mmol) and HOBt (1-hydroxybenztriazole; 60 mg, 0.44 mmol). The reaction mixture was stirred overnight at room temperature under argon. The product thus obtained was purified by preparative HPLC (preparative high-performance liquid chromatography) to give intermediate 11-4 (230 mg, 48%). LC-MS (ESI, m / z): 1091.53 (M+H).

[0170] Step 5: Synthesis of intermediate 11-5 Compound 11-4 (200 mg, 0.183 mmol) was dissolved in 1 mL of anhydrous DCM, and 300 μL of TFA was added thereto at 0 °C. The reaction mixture was stirred at room temperature for 30 minutes, and the solvent therein was removed by concentration to obtain the TFA salt of intermediate 11-5, which could be used in the next step without further purification. LC-MS (ESI, m / z): 991.47 (M+H).

[0171] Step 6: Synthesis of compound LP-1 Compound 11-5 (120 mg, 0.109 mmol) was dissolved in 1 mL of anhydrous DMF, and Ac-Sar10-COOH (N-acetyldecasarcosine; 84 mg, 0.109 mmol) was added thereto, followed by HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; 50 mg, 0.130 mmol) and DIPEA (38 μL, 0.22 mmol). The reaction mixture was stirred overnight at room temperature, and the solvent therein was removed by concentration. The crude product was purified by preparative HPLC to give compound LP-1 (74 mg, 38%). LC-MS (ESI, m / z): 1743.85 (M+H).

[0172] Example 2: Preparation of compound LP-2 TIFF2025535258000013.tif43144 The synthesis of compound LP-2 was carried out according to the synthesis steps of compound LP-1. Starting material 11-1A was replaced with Mc-GGFG-OH (purchased from MedChemExpress Shanghai) to obtain compound LP-2 as a beige amorphous solid. LC-MS (ESI, m / z): 1891.90 (M+H).

[0173] Example 3: Preparation of compound LP-3 TIFF2025535258000014.tif61148 Compound LP-3 was an intermediate of LP-1. By eliminating step 6, intermediate 11-5 becomes LP-3.

[0174] Example 4: Preparation of monoclonal antibodies Mu01 and Hu01 Balb / c mice (8-12 weeks old) were immunized with a fragment of the extracellular domain of human CD142 protein antigen (purchased from ACROBiosystems, product number TF3-H52H5), and their serum titers were monitored to determine the immunization response. After the initial immunization, mice received three or four booster immunizations, and serum was collected for titer detection. Mice with a suitable titer received one booster immunization, and whole spleens and half lymph nodes were harvested and fused with the myeloma SP2 / 0 cell line for PEG fusion. The fused cells were cultured in plates. The supernatants were collected and screened for antigen by ELISA. Positive cells were transferred to 96-well plates for further culture. After 7 days, the supernatants were collected and assayed for antigen reactivity using ELISA. Positive cells were further tested for antigen-binding affinity at different dilutions. The 20 parent clones with the highest immunogenic affinity were subcloned. Monoclonal hybridoma cells were obtained by limiting dilution and ELISA screening. To prepare ascites, the hybridoma cells were injected into the abdomen of mice. After collection and purification, a monoclonal antibody designated Mu01 was obtained. The variable regions and CDRs of Mu01 were sequenced, as shown in Table 1. The amino acid sequences of the CDRs are underlined. [Table 1]

[0175] The DNA sequence encoding VH is shown as SEQ ID NO:18, and the DNA sequence encoding VL is shown as SEQ ID NO:19.

[0176] The VH and VL of the hybridoma sequences were modified to obtain a humanized 01 variable region (referred to as "Hu01") as shown in Table 2. H1, H2, H3, H4, and H5 represent the sequence codes for VH, respectively. L1, L2, L3, L4, and L5 represent the sequence codes for VL, respectively. L3H1 (or Hu01-L3H1") represented a humanized antibody comprising a light chain variable region encoded by L3 and a heavy chain variable region encoded by H1. L3H2 represented a humanized antibody comprising a light chain variable region encoded by L3 and a heavy chain variable region encoded by H2. L5H1 represented a humanized antibody comprising a light chain variable region encoded by L5 and a heavy chain variable region encoded by H1. L5H2 represented a humanized antibody comprising a light chain variable region encoded by L5 and a heavy chain variable region encoded by H2. [Table 2]

[0177] The CDRs of Mu01 and Hu01 were defined and numbered according to the IMGT system.

[0178] For the purpose of preparation and / or detection in the Examples, the constant region of human IgG1 was selected as the constant region of the above-mentioned Hu01 antibody, and the heavy chain constant region and light chain constant region are shown as SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

[0179] Example 5: Preparation of monoclonal antibodies Mu02 and Hu02 Balb / c mice (8-12 weeks old) were immunized with a fragment of the extracellular domain of human CD142 protein antigen (purchased from ACROBiosystems, product number TF3-H52H5), and their serum titers were monitored to determine the immunization response. After the initial immunization, mice received three or four booster immunizations, and serum was collected for titer detection. Mice with a suitable titer received one booster immunization, and whole spleens and half lymph nodes were harvested and fused with the myeloma SP2 / 0 cell line for PEG fusion. The fused cells were cultured in plates. The supernatants were collected and screened for antigen by ELISA. Positive cells were transferred to 96-well plates for further culture. After 7 days, the supernatants were collected and assayed for antigen reactivity using ELISA. Positive cells were further tested for antigen-binding affinity at different dilutions. The 20 parent clones with the highest immunogenic affinity were subcloned. Monoclonal hybridoma cells were obtained by limiting dilution and ELISA screening. To prepare ascites, hybridoma cells were injected into the abdominal cavity of mice. After collection and purification, a monoclonal antibody designated Mu02 was obtained. The amino acid sequence, variable regions, and CDRs of Mu02 were identified as shown in Table 3. The CDRs are underlined. [Table 3]

[0180] The DNA sequence encoding VH is shown as SEQ ID NO:37, and the DNA sequence encoding VL is shown as SEQ ID NO:38.

[0181] The VH and VL of the hybridoma sequence were modified to obtain the humanized 02 variable region (referred to as "Hu02") as shown in Table 4. H1, H2, H3, H4, H5, and H6 represent the sequence codes for VH, respectively. L1 and L2 represent the sequence codes for VL, respectively. L1H1 represents a humanized antibody comprising a light chain variable region encoding L1 and a heavy chain variable region encoding H1. L1H2 (or Hu02-L1H2) represents a humanized antibody comprising a light chain variable region encoding L1 and a heavy chain variable region encoding H2. L1H3 represents a humanized antibody comprising a light chain variable region encoding L1 and a heavy chain variable region encoding H3. L1H4 represents a humanized antibody comprising a light chain variable region encoding L1 and a heavy chain variable region encoding H4. L1H5 represents a humanized antibody comprising a light chain variable region encoding L1 and a heavy chain variable region encoding H5. L1H6 represented a humanized antibody comprising a light chain variable region encoding L1 and a heavy chain variable region encoding H6. L2H1 represented a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H1. L2H2 represented a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H2. L2H3 represented a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H3. L2H4 represented a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H4. L2H5 represented a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H5. L2H6 represented a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H6. [Table 4] HCDR2 is underlined and amino acid substitutions are in bold italics.

[0182] The CDRs of Mu02 and Hu02 were defined and numbered according to the IMGT system.

[0183] For the purposes of preparation and / or detection in the examples, the heavy chain constant region sequences and light chain constant region sequences of the Hu02 antibody are the same as those of the humanized antibody in Example 4.

[0184] Example 6: Method for detecting antibody-drug conjugates The antibody-drug conjugates were identified by concentrating, media changing, and purifying them, measuring the antibody concentration, and calculating the average number of drug molecules carried by each antibody according to the following methods.

[0185] Procedure A: Concentration of antibody or antibody-drug conjugate An ultrafiltration tube (Amicon Ultra, 50,000 MWCO, Millipore Corporation) was taken out and the antibody or antibody-drug conjugate solution to be concentrated was added thereto. The ultrafiltration tube was centrifuged until the antibody or antibody-drug conjugate solution therein reached the required volume, and then taken out.

[0186] Procedure B: Measurement of antibody concentration The absorbance of the antibody was measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific) according to the manufacturer's instructions. The antibody concentration is the ratio of the absorbance value to the absorption coefficient of the antibody at the detection wavelength.

[0187] Procedure C: Antibody medium exchange Zeba spin desalting columns (5 mL, 40K MWCO) were pre-equilibrated with phosphate-buffered saline containing 50 mM sodium chloride and 2 mM EDTA (referred to as "PBS7.0 / EDTA," 50 mM, pH 7.0) according to the manufacturer's instructions (Thermo Fisher Scientific). A 2 mL sample was loaded onto each Zeba spin desalting column and centrifuged (1000 g, 4 min). The flow-through fraction was then collected and concentrated as per procedure A, and the antibody concentration was determined as per procedure B, after which the antibody concentration was adjusted with PBS7.0 / EDTA.

[0188] Procedure D: Purification of antibody-drug conjugates A Zeba spin desalting column (5 mL, 40K MWCO) was pre-equilibrated with storage buffer according to the manufacturer's instructions (Thermo Fisher Scientific). Histidine-acetate buffer (20 mM histidine, pH 5.5) containing 150 mM NaCl or phosphate buffer (50 mM, pH 7.0) containing 50 mM NaCl was used as the storage buffer. The reaction solution containing the antibody-drug conjugate (approximately 2 mL) was added to the Zeba spin desalting column (5 mL) and centrifuged (1000 g, 4 min). The flow-through fraction (approximately 2 mL) was then collected, and the elution process was repeated twice to remove low molecular weight compounds, including unbound linker-payload and reducing agent.

[0189] Procedure E: Determination of antibody concentration in antibody-drug conjugates and average number of drug molecules linked to each antibody (DAR value) - (1) The concentration of the drug conjugated to the antibody-drug conjugate can be obtained by measuring the UV absorption values ​​of an aqueous solution of the antibody-drug conjugate at 280 nm and 370 nm and calculating using the following formula:

[0190] At any given wavelength, the total absorbance of the system is equal to the sum of the absorbances of all light-absorbing chemicals present in the system (additivity of absorbance). Therefore, assuming that the molar absorption coefficients of the antibody and drug remain unchanged before and after conjugation of the antibody and drug, the concentrations of the antibody and drug in the antibody-drug complex can be expressed by the following equations: A 280 = A D,280 + A A,280 = ε D,280 C D + ε A,280 C A Formula (1) A 370 = A D,370 + A A,370 =ε D,370 C D + ε A,370 C A Formula (2)

[0191] A 280represents the total absorbance value of the antibody-drug conjugate aqueous solution at 280 nm, and A 370 represents the total absorbance value of the antibody-drug conjugate aqueous solution at 370 nm. A,280 represents the absorbance value of the antibody at 280 nm, and A A,370 represents the absorbance value of the antibody at 370 nm, and A D,280 represents the absorption value of the drug molecule at 280 nm, and A D,370 represents the absorption value of the drug molecule at 370 nm, and ε A,280 represents the molar extinction coefficient of the antibody at 280 nm, and ε A,370 represents the molar extinction coefficient of the antibody at 370 nm, and ε D,280 represents the molar extinction coefficient of the drug molecule at 280 nm, and ε D,370 represents the molar extinction coefficient of the drug molecule at 370 nm, and C A represents the concentration of antibody in the antibody-drug conjugate, and C D represents the concentration of drug molecules in the antibody-drug conjugate.

[0192] In this case, ε A,280 , ε A,370 , ε D,280 , and ε D,370 are all known values ​​(calculated from the antibody sequence or measured by UV absorption of the compound). For example, ε A,280 can be calculated from the amino acid sequence of the antibody using known methods (Protein Science, 1995, Vol. 4, pp. 2411-2423). Since antibodies usually do not have absorbance at 370 nm, ε A,370 is usually 0. D,280 and ε D,370 The value of C can be calculated by measuring the change in absorbance of the drug molecule at 280 nm and 370 nm with concentration and using the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × light path). A and C D is the absorbance value A of the antibody-drug conjugate at 280 nm and 370 nm 280 and A 370and then solving the simultaneous equations (1) and (2). Furthermore, the average number of drug molecules linked to each antibody (DAR value) can be obtained by calculating C D C A can be obtained by dividing by

[0193] Procedure F: Average number of drug molecules linked to each antibody (DAR value) - (2) In addition to the above "Procedure E," the average number of drug molecules conjugated to each antibody molecule in an antibody-drug conjugate may be determined using a hydrophobic interaction chromatography (HIC) analytical method described below.

[0194] The elution of antibody-drug conjugates from a hydrophobic interaction chromatography column was based on the difference in salt ion concentration in the eluent. As the salt ion concentration decreased, the number of small molecule drugs in the eluted antibody-drug conjugates increased, i.e., antibody-drug conjugates with low DAR values ​​were preferentially eluted. The peak order of each component was D0 (antibody not conjugated to any linker-payload), D2 (antibody conjugated to an average of about two linker-payloads), D4 (antibody conjugated to an average of about four linker-payloads), D6 (antibody conjugated to an average of about six linker-payloads), and D8 (antibody conjugated to an average of about eight linker-payloads). The peak area ratio of each peak was measured to obtain the content percentage of each component. The HIC-DAR of the corresponding sample was then calculated as follows: Average number of conjugated drug molecules = D0 peak area ratio × 0 + D2 peak area ratio × 2 + D4 peak area ratio × 4 + D6 peak area ratio × 6 + D8 peak area ratio × 8

[0195] Procedure G: Measurement of aggregates in antibody-drug conjugates Aggregates in the antibody-drug conjugate were detected using size exclusion chromatography in high performance liquid chromatography as follows. High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system Detector: UV absorption spectrometer (detection wavelength: 280 nm) Column type: TOSOH TSKgel G3000SWXL (7.8 x 300 mm, 5 μm) Mobile phase: 200 mmol / L KHPO4, 150 mmol / L NaCl, 15% (v / v) isopropyl alcohol, pH 7.0 Flow rate: 0.75mL / min Analysis time: 18 minutes Column temperature: room temperature Injection volume: 50 μg Data Analysis: Substances

[0196] The size-exclusion chromatogram of a quality control material (QC, Hu01-L3H1 naked antibody, i.e., a humanized antibody encoding L3H1 derived from Mu01 not conjugated to a linker-payload) is shown in Figure 1A. The size-exclusion chromatogram of a quality control material (QC, Hu02-L1H2 naked antibody, i.e., an antibody encoding L1H2 derived from antibody Mu02 not conjugated to a linker-payload) is shown in Figure 2A. The retention time of the main peak (single peak) of the 150 kDa quality control material was 9.5 to 10.5 min. The retention time of the aggregates should be shorter than that of the monomers mentioned above.

[0197] Procedure H: Comparison of the hydrophobicity of antibody-drug conjugates The hydrophobicity of the antibody-drug conjugates was analyzed using high performance liquid chromatography hydrophobic interaction chromatography (HIC), as follows: High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system Detector: UV absorption spectrometer (detection wavelength: 280 nm) Column type: TOSOH TSKgel Butyl-NPR (4.6 mm ID x 3.5 cm, 2.5 μm) Mobile phase A: mol / L(NH4)2SO4, 50mmol / L KHPO4, pH7.0 Mobile phase B: 50 mmol / L KHPO4, 25% (v / v) isopropanol, pH 7.0 Analysis time: 25 minutes Column temperature: room temperature Elution procedure (B%): 0% to 25% (0 to 1 min), 25% (1 to 3 min), 25% to 80% (3 to 13 min), 80% (13 to 17 min), 80% to 0% (17 to 17.10 min), 0% (17.10 to 25 min) Injection volume: 10 μL Data analysis: The hydrophobic interaction chromatogram of the quality control material (QC, Hu01-L3H1 naked antibody) is shown in Figure 1B. The hydrophobicity chromatogram of the quality control material (QC, Hu02-L1H2 naked antibody) is shown in Figure 2B. Samples with shorter retention times were less hydrophobic. The antibody-drug conjugate was more hydrophobic than the unconjugated naked antibody, and therefore its retention time was longer.

[0198] Example 7: Preparation of antibody-drug conjugate Hu01-L3H1-LP1-DAR8 Antibody reduction: The Hu01-L3H1 antibody medium was diluted with the same solution as in procedure B of Example 6 (the absorbance coefficient of the antibody at 280 nm was 1.658 mL mg -1 cm -1 The medium was then replaced with PBS7.0 / EDTA according to procedure C, resulting in an antibody concentration of 7.353 mg / mL. 19.51 μL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added to 284.24 μL of Hu01-L3H1 antibody aqueous solution, followed by simultaneous addition of 76 μL of 50 mM phosphate buffer (PBS7.0) and 0.26 μL of ultrapure water. The mixed solution was placed in a 37°C environment and allowed to react for 2 hours.

[0199] Conjugation of antibody with linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload LP-1 prepared in Example 1 was dissolved in N,N-dimethylacetamide (DMA), and then 20.90 μL (corresponding to 15 times the equivalent of the antibody content) of the solution was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0200] Purification of antibody-drug conjugate: The above reaction solution was purified according to the method of operation D in Example 6 to obtain antibody-drug conjugate Hu01-L3H1-LP1-DAR8.

[0201] Characterization of antibody-drug conjugates: The obtained antibody-drug conjugates were analyzed by the procedure E(ε) of Example 6. D,280 = 6384 and ε D,370 =16180), and characterized using Procedure F, Procedure G, and Procedure H.

[0202] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 4.93 mg / mL, and the average number of payloads conjugated per antibody measured and calculated by procedure E was 7.67. Figure 3A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate Hu01-L3H1-LP1-DAR8 measured by procedure G was 1.33%. Figure 3B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate Hu01-L3H1-LP1-DAR8, and the retention time of the antibody-drug conjugate measured by procedure H was 7.199 minutes.

[0203] Example 8: Preparation of antibody-drug conjugate Hu01-L3H1-LP1-DAR4 Antibody reduction: The Hu01-L3H1 antibody medium was diluted with the same solution as in procedure B of Example 6 (the absorbance coefficient of the antibody at 280 nm was 1.658 mL mg -1 cm -1The medium was then replaced with PBS7.0 / EDTA according to procedure C, resulting in an antibody concentration of 22.51 mg / mL. 40 μL of 5 mM TCEP solution (equivalent to 2.5 times the antibody content) was added to 533.10 μL of Hu01-L3H1 antibody aqueous solution, followed by simultaneous addition of 320 μL of 50 mM phosphate buffer (PBS7.0) and 706.90 μL of ultrapure water. The mixed solution was placed in a 37°C environment and allowed to react for 2 hours.

[0204] Conjugation of antibody with linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload LP-1 prepared in Example 1 was dissolved in DMA, and 64 μL of the solution (equivalent to 8 times the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0205] Purification of antibody-drug conjugate: The above reaction solution was purified according to the method of operation D in Example 6 to obtain antibody-drug conjugate Hu01-L3H1-LP1-DAR4.

[0206] Characterization of antibody-drug conjugates: The obtained antibody-drug conjugates were analyzed by the procedure E(ε) of Example 6. D,280 = 6384 and ε D,370 =16180), and characterized using Procedure F, Procedure G, and Procedure H.

[0207] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 7.19 mg / mL, and the average number of payloads conjugated per antibody measured and calculated by procedure E was 4.66. Figure 4A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate Hu01-L3H1-LP1-DAR4 measured by procedure G was 1.16%. Figure 4B shows a hydrophobic interaction chromatography detection graph of the retention time distribution of the antibody-drug conjugate measured by procedure H.

[0208] Example 9: Preparation of antibody-drug conjugate Hu02-L1H2-LP1-DAR8 Antibody reduction: The Hu02-L1H2 antibody medium was diluted with the same solution as in procedure B of Example 6 (the absorbance coefficient of the antibody at 280 nm was 1.382 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C, resulting in an antibody concentration of 11.98 mg / mL. 106.67 μL of 5 mM TCEP solution (equivalent to 10 times the antibody content) was added to 667.78 μL of Hu02-L1H2 antibody aqueous solution, followed by simultaneous addition of 320 μL of 50 mM phosphate buffer (PBS 7.0) and 505.50 μL of ultrapure water. The mixed solution was placed in a 37°C environment and allowed to react for 2 hours.

[0209] Conjugation of antibody with linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload LP-1 prepared in Example 1 was dissolved in DMA, and 80 μL of the solution (corresponding to 15 times the equivalent amount of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0210] Purification of antibody-drug conjugate: The above reaction solution was purified according to the method of operation D in Example 6 to obtain antibody-drug conjugate Hu02-L1H2-LP1-DAR8.

[0211] Characterization of antibody-drug conjugates: The obtained antibody-drug conjugates were analyzed by the procedure E(ε) of Example 6. D,280 = 6384 and ε D,370 =16180), and characterized using Procedure F, Procedure G, and Procedure H.

[0212] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 5.03 mg / mL, and the average number of payloads conjugated per antibody measured and calculated by procedure E was 7.83. Figure 5A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate Hu02-L1H2-LP1-DAR8 measured by procedure G was 1.31%. Figure 5B shows a hydrophobic interaction chromatography detection graph of the measured antibody-drug conjugate, and the retention time of the antibody-drug conjugate measured by procedure H was 6.768 minutes.

[0213] Comparative Example 1: Preparation of antibody-drug conjugate HuIgG-LP1-DAR8 Antibody reduction: The human IgG antibody medium was diluted with 1.35 mL of 100% IgG antibody as described in Procedure B of Example 6 (the absorbance coefficient of the antibody at 280 nm was 1.35 mL / min). -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C, resulting in an antibody concentration of 10 mg / mL. 0.267 mL of 5 mM TCEP solution (equivalent to 10 times the antibody content) was added to 1.33 mL of human IgG protein aqueous solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number SP001), followed by simultaneous addition of 0.4 mL of 50 mM PBS 7.0. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0214] Conjugation of antibody with linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload LP-1 was dissolved in DMA, and then 0.2 mL of the solution (corresponding to 15 times the equivalent of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0215] Purification of antibody-drug conjugate: The above reaction solution was purified according to the method of operation D in Example 6 to obtain antibody-drug conjugate HuIgG-LP1-DAR8.

[0216] Characterization of antibody-drug conjugates: The obtained antibody-drug conjugates were analyzed by the procedure E(ε) of Example 6. D,280 = 6384 and ε D,370 =16180), procedure G, and procedure H.

[0217] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 11.08 mg / mL, and the average number of payloads conjugated per antibody measured and calculated by procedure E was 9.15. Figure 6A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate HuIgG-LP1-DAR8 measured by procedure G was 4.38%. Figure 6B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate HuIgG-LP1-DAR8, and the retention time of the antibody-drug conjugate human IgG-LP1 measured by procedure H was 6.213 minutes.

[0218] Comparative Example 2: Preparation of antibody drug conjugate tisotumab-MMAE (referred to as reference ADC) The sequence of the antibody tisotumab (referred to as the reference antibody) is recorded in PCT / EP2009 / 066755 (VH: SEQ ID NO: 9, VL: SEQ ID NO: 65), where the antibody was designated TF-11.

[0219] Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 6 (the absorbance coefficient of the antibody at 280 nm is 1.508 mL mg -1 cm -1 The medium was then replaced with PBS7.0 / EDTA according to procedure C, resulting in an antibody concentration of 7.318 mg / mL. 23.21 μL of 5 mM TCEP solution (equivalent to 2.04 times the antibody content) was added to 1127.36 μL of the reference antibody aqueous solution, followed by simultaneous addition of 300 μL of 50 mM PBS7.0 and 49.43 μL of ultrapure water. The mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0220] Conjugation of antibody with linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload vc-MMAE (DC Chemicals, DC50025) was dissolved in DMA, and then 79.66 μL (corresponding to 7 times the equivalent of the antibody content) of the solution was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0221] Purification of antibody-drug conjugate: The above reaction solution was purified according to the method of operation D in Example 6 to obtain the antibody-drug conjugate, reference ADC.

[0222] Characterization of antibody drug conjugates: The resulting antibody drug conjugates were characterized using Procedure B, Procedure F, Procedure G, and Procedure H of Example 6.

[0223] The antibody-drug conjugate concentration calculated by measurement in procedure B was 5.01 mg / mL. The average number of conjugated payloads per antibody in the reference ADC measured and calculated by procedures F and G was 3.73. Figure 7A shows an aggregate detection graph, and the aggregate content in the reference ADC measured by procedure G was 0.92%. Figure 7B shows a hydrophobic interaction chromatography detection graph of the reference ADC measured by procedure H. The HNSTD (highest non-severe toxicity dose) of the reference ADC in cynomolgus monkeys was 3 mg / kg.

[0224] Experimental Example 1: Affinity assay of Mu01 and Mu02 for BXPC3 cells BxPC3 cells (human pancreatic cancer cells, obtained from the National Collection of Authenticated Cell Cultures) were digested in a culture dish with trypsin (purchased from Gibco, product number 25200072). The digested cells were centrifuged at 1000 rpm for 5 minutes and blocked with 10% goat serum (purchased from SenBeiJia Biological Technology Co., Ltd., product number SRJ-SE-GO012) for 30 minutes. Next, Mu01 and Mu02 antibodies prepared in Examples 4 and 5, respectively, were added at a final concentration of 20 μg / mL and incubated for 1 hour. After washing twice with phosphate buffer (PBS, pH 7.2-7.4), FITC-labeled goat anti-mouse antibody (purchased from Jackson ImmunoResearch, product number 115-545-003) was added at a dilution of 1:500 and incubated for 1 hour. After washing twice with PBS, the resulting products were analyzed by flow cytometry.

[0225] As shown in Figure 8, both Mu01 and Mu02 showed good affinity for BXPC3 cells that were CD142 positive.

[0226] Experimental Example 2: In vitro killing assay of Mu01 and Mu02 against MDA-MB-231 cells MDA-MB-231 (human breast cancer, derived from Zhejiang Meisen Cell Technology Co., Ltd.) cells were cultured to a cell density of 80%. The cells were harvested and added to a 96-well plate, and the cell density was adjusted to 2–5 × 10. 4 The concentration was adjusted to 1 / mL. 100 μL was added to each well. Mu01 prepared in Example 4, Mu02 prepared in Example 5, or negative control IgG (mouse anti-IgG, purchased from Abmart Medical Technology (Shanghai) Co., Ltd., product number M070890) was serially diluted 3-fold from an initial concentration of 30 nM and then added to the cell culture medium. 2 μg / mL of goat anti-mouse antibody conjugated with MMAE (goat anti-mouse antibody purchased from Jackson, catalog number 115-545-003) was added at the same time. The cells were cultured for 3 days. Cell survival was monitored periodically during this period. After 3 days, 15 μL of CCK-8 kit stock solution (purchased from Abbkine, catalog number ATUOC1303) was added directly to the 96-well plate. After incubation in a 37°C incubator for 0.5 to 2 hours, the absorbance at 450 nm was detected using a microplate reader, and a cell survival curve was created according to the absorbance value and the antibody dilution gradient.

[0227] The method for MMAE-conjugated goat anti-mouse antibody was similar to that of Comparative Example 2.

[0228] As shown in Figure 9, in the human breast cancer cell line MDA-MB-231, the cell viability decreased continuously with increasing concentrations of Mu01 and Mu02, indicating that the antibodies Mu01 and Mu02 had obvious killing effects on tumor cells.

[0229] Experimental Example 3: Affinity assay of Hu01 candidate molecules to BXPC3 cells BxPC3 cells (human pancreatic cancer cells, obtained from the National Collection of Authenticated Cell Cultures) were digested in a culture dish with trypsin (purchased from Gibco, product number 25200072). The digested cells were centrifuged at 1000 rpm for 5 minutes and blocked with 10% goat serum (purchased from SenBeiJia Biological Technology Co., Ltd., product number SRJ-SE-GO012) for 30 minutes. Next, the Hu01 antibodies L5H2, L5H1, L3H1, and L3H2 prepared in Example 4 were diluted three-fold from an initial concentration of 500 nM. The Hu01 antibodies were then added to the cells and incubated for 1 hour. After washing twice with PBS, FITC-labeled goat anti-human antibody (purchased from Sigma, product number FS9512-2ML) was added at a dilution of 1:500 and incubated for 1 hour. The products were analyzed by flow cytometry after washing twice with PBS.

[0230] The Ch01 antibody was a chimeric antibody, the variable region of which was the same as that of Mu01 prepared in Example 4, and the constant region of which was the same as that of human IgG1 described in Example 4.

[0231] As shown in FIG. 10, all four candidate molecules of the Hu01 antibody showed good affinity to BXPC3 cells, with L3H1 showing the highest affinity.

[0232] Experimental Example 4: Affinity assay of Hu02 candidate molecules to BXPC3 cells BxPC3 cells (human pancreatic cancer cells, obtained from the National Collection of Authenticated Cell Cultures) were digested in a culture dish with trypsin (purchased from Gibco, product number 25200072). The digested cells were centrifuged at 1000 rpm for 5 minutes and blocked with 10% goat serum (purchased from SenBeiJia Biological Technology Co., Ltd., product number SRJ-SE-GO012) for 30 minutes. Next, Hu02 antibodies L1H1, L1H2, L1H3, L1H4, L1H5, L1H6, L2H1, L2H2, L2H3, L2H5, and L2H6 prepared in Example 5 were serially diluted 3-fold from an initial concentration of 500 nM. The Hu02 antibodies were then added to the cells and incubated for 1 hour. After washing twice with PBS, FITC-labeled goat anti-human antibody (purchased from Sigma, product number FS9512-2ML) was added at 1:500 and incubated for 1 hour. After washing twice with PBS, the products were analyzed by flow cytometry (BD biosciences, product number BD Accuri C6 Plus).

[0233] The Ch02 antibody was a chimeric antibody, the variable region of which was the same as that of Mu02 prepared in Example 5, and the constant region of which was the same as that of human IgG1 described in Example 4.

[0234] As shown in FIG. 11 and Table 5, all 11 candidate molecules of the Hu02 antibody showed good affinity for BXPC3 cells, among which L2H6 showed the highest affinity and L1H2 showed good affinity. [Table 5]

[0235] Experimental Example 5: Effects of Hu01-L3H1, Hu02-L1H2 and Tisotumab on Blood Coagulation The effects of Hu01-L3H1, Hu02-L1H2, and the reference antibody (tisotumab) on blood coagulation were detected by thrombin generation assay (TGA).

[0236] Thirty microliters of PPP reagent (a mixture of 5 pM tissue factor and 4 μM phospholipids, purchased from Stago, product number TS30.00) was added to human plasma containing 50 μg / mL CTI (corn trypsin inhibitor, purchased from Enzyme Research) and diluted with antibody Hu01-L3H1 prepared in Example 4, Hu02-L1H2 prepared in Example 5, or negative control phosphate-buffered saline (PBS), and incubated at 37°C for 10 minutes. 120 μl of the mixture was added to a reaction cup, followed by the addition of 30 μl of FluCa reagent (containing calcium and thrombin substrate, purchased from Stago, product number TS50.00) to initiate the tissue factor-dependent thrombin formation reaction. Parameters were detected and automatically exported using a TC TechnoClone instrument.

[0237] As shown in Figure 12, the inhibition rates of Hu01-L3H1 and Hu02-L1H2 against thrombin peak values ​​were lower than those of the reference antibody. These results indicated that Hu01-L3H1 and Hu02-L1H2 had less effect on blood coagulation function and lower coagulation toxicity in vitro compared to the reference antibody.

[0238] Experimental Example 6: In vitro killing assay of ADCs Hu01-L3H1-LP1-DAR8, Hu01-L3H1-LP1-DAR4 and Hu02-L1H2-LP1-DAR8 in KYSE150, 5637, SW780 and Detroit562 cells KYSE150 (human esophageal cancer cells, obtained from the National Collection of Authenticated Cell Cultures), 5637 (human bladder cancer cells, obtained from the National Collection of Authenticated Cell Cultures), SW780 (human bladder cancer, obtained from Zhejiang Meisen Cell Technology Co., Ltd.), and Detroit562 (human pancreatic cancer cells, obtained from the National Collection of Authenticated Cell Cultures) were cultured to a cell density of 80%. The cells were harvested and plated in 96-well plates at a cell density of 2–5 × 10. 4 The ADC molecules were adjusted to 1 / ml. 100 μL of cells were plated in each well. The ADC molecules were serially diluted 3-fold from an initial concentration of 300 nM and then added to cell culture medium and incubated for 5 days. During this period, cell apoptosis was monitored periodically. After 5 days, 15 μL of CCK-8 kit stock solution (purchased from Abbkine, product number ATUOC1303) was added directly to the 96-well plate and incubated for 0.5 to 2 hours in a 37°C incubator. Absorbance at 450 nm was measured using a microplate reader, and a cell viability curve was generated based on the absorbance values ​​and the antibody dilution gradient.

[0239] Hu01-L3H1-LP1-DAR8 prepared in Example 7, Hu01-L3H1-LP1-DAR4 prepared in Example 8, Hu02-L1H2-LP1-DAR8 prepared in Example 9, and the isotype control HuIgG-LP1-DAR8 prepared in Comparative Example 1 were selected as ADC molecules.

[0240] Figures 13, 14, and 15 show the cell viability curves of Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 in KYSE150, 5637, and SW780 cells, respectively. The results showed that Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 had obvious cytotoxic effects on these cell lines in vitro, with Hu01-L3H1-LP1-DAR8 having a better cytotoxic effect.

[0241] Figure 16 shows the cell viability curves of Detroit 562 cells treated with Hu01-L3H1-LP1-DAR8, Hu01-L3H1-LP1-DAR4, and Hu02-L1H2-LP-DAR8 in vitro. The results showed that the cytotoxicity of the ADCs was evident in vitro, with the cytotoxic effect, from high to low, being Hu01-L3H1-LP1-DAR8 > Hu01-L3H1-LP1-DAR4 > Hu02-L1H2-LP1-DAR8.

[0242] Experimental Example 7: Tumor-inhibitory effects of Hu01-L3H1-LP1-DAR8, Hu02-L1H2-LP1-DAR8 and reference ADC in NCI-H292 CDX mouse model Six-week-old Balb / c nude mice were purchased from Jiangsu GemPharmatech Co. Ltd. Each mouse received 1.0 × 10 7 NCI-H292 (human lung cancer cells, obtained from the National Collection of Authenticated Cell Cultures) cells were subcutaneously inoculated to establish a CDX (cell-derived xenograft) mouse model. The average tumor volume was approximately 100 mm. 3 At this time, 2.5, 5, and 10 mpk (mg / kg) of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 10 mpk of Hu02-L1H2-LP1-DAR8 prepared in Example 9, 3 mpk of the reference ADC prepared in Comparative Example 2, and 10 mpk of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were intravenously injected. After administration, tumor volumes were measured twice a week with vernier calipers, and tumor volumes were calculated according to the following formula: TV = (length × width).2 / 2.

[0243] Figure 17 shows the in vivo tumor-inhibitory effects of Hu01-L3H1-LP1-DAR8, Hu02-L1H2-LP1-DAR8, and the reference ADC in the NCI-H292 CDX mouse model. Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 were shown to have tumor-inhibitory effects similar to those of the reference ADC. Furthermore, Hu01-L3H1-LP1-DAR8 demonstrated a dose-dependent effect on tumor inhibition, as shown by tumor inhibition curves at different doses.

[0244] Figure 18 shows the effects of Hu01-L3H1-LP1-DAR8, Hu02-L1H2-LP1-DAR8, and the reference ADC on mouse body weight. The results show that the ADC molecules have little effect on mouse body weight, indicating that they do not cause obvious gastrointestinal toxicity at this dose.

[0245] Experimental Example 8: Tumor-inhibitory effects of Hu01-L3H1-LP1-DAR8 and reference ADC in the CDX model of NCI-H226 Six-week-old Balb / c nude mice were purchased from Jiangsu GemPharmatech Co. Ltd. Each mouse received 5.0 × 10 6 CDX mouse models were established by subcutaneous inoculation of NCI-H226 (human lung cancer cells, obtained from the National Collection of Authenticated Cell Cultures) cells. The average tumor volume was approximately 130 mm. 3 At this time, 5 and 10 mg / kg of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 3 mg / kg of the reference ADC prepared in Comparative Example 2, and 10 mg / kg of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were intravenously injected. Tumor volumes were measured and calculated according to the method in Experimental Example 7.

[0246] Figure 19 shows the in vivo tumor-inhibitory effects of Hu01-L3H1-LP1-DAR8 and the reference ADC on the NCI-H226 CDX mouse model. The tumor-inhibitory effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg was shown to be better than that of the reference ADC at a dose of 3 mg / kg. The tumor-inhibitory effect of Hu01-L3H1-LP1-DAR8 at a dose of 5 mg / kg was similar to that of the reference ADC at a dose of 3 mg / kg.

[0247] Experimental Example 9: Tumor-inhibitory effects of Hu01-L3H1-LP1-DAR8 and reference ADC on a mouse model established with IGROV1 cells Six-week-old Balb / c nude mice were purchased from Jiangsu GemPharmatech Co. Ltd. Each mouse received 1.0 × 10 7 IGROV1 cells (human ovarian cancer, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) were subcutaneously inoculated to establish a CDX mouse model. The average tumor volume was approximately 100 mm. 3 At this time, 10 mpk of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 3 mpk of the reference ADC prepared in Comparative Example 2, and 10 mpk of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were intravenously injected. Tumor volumes were measured and calculated according to the method in Experimental Example 7.

[0248] Figure 20 shows the in vivo tumor-suppressing effects of Hu01-L3H1-LP1-DAR8 and the reference ADC on a CDX mouse model established with IGROV1 cells. The tumor-suppressing effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg was shown to be better than that of the reference ADC at a dose of 3 mg / kg.

[0249] Experimental Example 10: Tumor-inhibitory effects of Hu01-L3H1-LP1-DAR8 and reference ADC on a mouse model established with SW780 cells Six-week-old Balb / c nude mice were purchased from Jiangsu GemPharmatech Co. Ltd. Each mouse received 4.0 × 10 6 SW780 cells (human bladder cancer, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) were subcutaneously inoculated to establish a CDX mouse model. The average tumor volume was approximately 100 mm. 3 At this time, 10 mg / kg of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 3 mg / kg of the reference ADC prepared in Comparative Example 2, and 10 mg / kg of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were intravenously injected. Tumor volumes were measured and calculated according to the method in Experimental Example 7.

[0250] Figure 21 shows the in vivo tumor-suppressing effects of Hu01-L3H1-LP1-DAR8 and the reference ADC on a CDX mouse model established with SW780 cells. The tumor-suppressing effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg was shown to be better than that of the reference ADC at a dose of 3 mg / kg.

[0251] Experimental Example 11: Tumor-suppressive effect of Hu01-L3H1-LP1-DAR8 on PDX1 mouse model established with human lung cancer tissue Six-week-old NU / NU nude mice (female, weighing 18–21 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Human lung cancer tissue (obtained from Shanghai Lide Biotechnology Co., Ltd.) was subcutaneously inoculated into each mouse to establish patient-derived tumor xenograft models (PDX). The average tumor volume was approximately 160 mm. 3 At this time, 10 mg / kg of Hu01-L3H1-LP1-DAR8 prepared in Example 7 and PBS were intravenously injected, respectively. The tumor volume was measured and calculated according to the method in Experimental Example 7.

[0252] Figure 22 shows the in vivo tumor-suppressing effects of Hu01-L3H1-LP1-DAR8 and PBS in a PDX1 mouse model. The tumor-suppressing effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg was significantly greater than that of PBS. Furthermore, tumors completely disappeared in five mice by day 22, demonstrating that Hu01-L3H1-LP1-DAR8 had a significant tumor-suppressing effect.

[0253] Experimental Example 12: Tumor-suppressive effect of Hu01-L3H1-LP1-DAR4 on PDX2 mouse model established with human cervical cancer tissue Six-week-old NU / NU nude mice (female, weighing 18-21 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Human lung cancer tissue (obtained from Shanghai Lide Biotechnology Co., Ltd.) was subcutaneously inoculated into each mouse to establish PDX models. The average tumor volume was approximately 160 mm. 3 At this time, 10 mg / kg of Hu01-L3H1-LP1-DAR4 prepared in Example 8, 3 mg / kg of the reference ADC prepared in Comparative Example 2, and PBS were intravenously injected. The tumor volume was measured and calculated according to the method in Experimental Example 7.

[0254] Figure 23 shows the in vivo tumor-suppressing effects of Hu01-L3H1-LP1-DAR4 and the reference ADC in a PDX2 mouse model. The tumor-suppressing effect of Hu01-L3H1-LP1-DAR4 at a dose of 10 mg / kg was shown to be significant compared to the reference ADC, indicating that Hu01-L3H1-LP1-DAR4 has a significant tumor-suppressing effect.

[0255] Experimental Example 13: Toxicology study of Hu01-L3H1-LP1-DAR8 in cynomolgus monkeys Toxicology experiments of Hu01-L3H1-LP1-DAR8 were carried out by repeated administration to cynomolgus monkeys.

[0256] Six cynomolgus monkeys (purchased from Guangxi Frontier Biotechnology Co., Ltd.) were randomly divided into three groups, with one male and one female in each group. Vehicle, 10 mg / kg Hu01-L3H1-LP1-DAR8, and 30 mg / kg Hu01-L3H1-LP1-DAR8 were intravenously injected on days 1, 22, and 43, respectively. During the experiment, animals were observed for abnormalities, and blood samples were collected for hematological and blood biochemistry index analysis. On day 50, animals were euthanized and sampled for pathological analysis. As shown in Table 6, no animal deaths occurred at any dose of the test substance in this experiment. The target organs associated with the test substance were bone marrow, intestine, thymus, and spleen. The HNSTD (maximum dose without severe toxicity) of Hu01-L3H1-LP1-DAR8 was 30 mg / kg, indicating that Hu01-L3H1-LP1-DAR8 is safe. [Table 6]

[0257] The ADCs with the new linkers provided herein have superior safety profiles compared to ADCs with payloads containing MMAE analogs, which are microtubule inhibitor toxins. Specifically, ADCs linked to VC-MMAE (e.g., tisotumab vedotin) have an HNSTD of 3 mpk (data from the US Food and Drug Administration), while ADCs linked to ZymeLink™ auristatins (e.g., XB002 / ICON-2) have an HNSTD of 10 mpk to 18 mpk (World ADC Digital, September 15-18, 2020). The HNSTD of the ADCs provided herein is significantly higher than that of the prior art, providing a wider therapeutic window. At the same time, the ADCs provided herein have superior in vivo antitumor efficacy.

[0258] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are not presently contemplated or may occur to applicant or those skilled in the art. Accordingly, the appended claims as filed, and the appended claims as they may be amended, are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that binds to CD142, comprising at least a heavy chain variable region (VH) and at least a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, and the HCDR2 comprises the amino acid sequence set forth in IYPGX. １ GDX ２ (SEQ ID NO: 2), wherein the HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 4, the LCDR2 comprises the amino acid sequence represented by LTS, and the LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 5, １ is D or Q, and X ２ is S or A.

2. (a1) the X １ is D, and said X ２ is S; (b1) the X １ is Q, and said X ２ is S, or (c1) the X １ is Q, and said X ２ An isolated antibody or antigen-binding fragment thereof that binds to CD142 according to claim 1, wherein is A.

3. 3. The isolated antibody or antigen-binding fragment thereof that binds to CD142 of claim 1 or 2, wherein the VH and the VL are selected from the following: (a-1) the VH having at least 70%, 86%, 87%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 6, and the VL having at least 70%, 79%, 80%, 87%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 7; (b-1) the VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 8, and the VL having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 15; (c-1) the VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 8, and the VL having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence represented by SEQ ID NO: 17; (d-1) the VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 9, and the VL having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 15; and (e-1) The VH has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 9, and the VL has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:

17.

4. The VH comprises the amino acid sequence represented by SEQ ID NO: 6, and the VL comprises the amino acid sequence represented by SEQ ID NO: 7; The VH comprises the amino acid sequence represented by SEQ ID NO: 8, and the VL comprises the amino acid sequence represented by SEQ ID NO: 15; The VH comprises the amino acid sequence represented by SEQ ID NO: 8, and the VL comprises the amino acid sequence represented by SEQ ID NO: 17; The VH comprises the amino acid sequence represented by SEQ ID NO: 9, and the VL comprises the amino acid sequence represented by SEQ ID NO: 15; or An isolated antibody or antigen-binding fragment thereof that binds to CD142 described in any one of claims 1 to 3, wherein the VH comprises the amino acid sequence represented by SEQ ID NO: 9 and the VL comprises the amino acid sequence represented by SEQ ID NO:

17.

5. 1. An isolated antibody or antigen-binding fragment thereof that binds to CD142, comprising at least a heavy chain variable region (VH) and at least a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:

11. ３ X ４ YTT (SEQ ID NO: 21), the HCDR3 comprises the amino acid sequence represented by SEQ ID NO: 22, the LCDR1 comprises the amino acid sequence represented by SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence represented by YTS, and the LCDR3 comprises the amino acid sequence represented by SEQ ID NO: 24, ３ is N or Q, and X ４ is G or A.

6. (a2) Said X ３ is N, and the X ４ is G; (b2) the X ３ is N, and the X ４ is A; (c2) the X ３ is Q, and said X ４ An isolated antibody or antigen-binding fragment thereof that binds to CD142 according to claim 5, wherein is G.

7. 7. The isolated antibody or antigen-binding fragment thereof that binds to CD142 of claim 5 or 6, wherein the VH and the VL are selected from the following: (a-2) the VH having at least 70%, 85%, 87%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 25, and the VL having at least 70%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 26; (b-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 27, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 33; (c-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 28, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 33; (d-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 29, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 33; (e-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 30, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 33; (f-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 31, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 33; (g-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 32, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 33; (h-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 27, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 34; (i-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 28, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 34; (j-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 29, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 34; (k-2) the VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 31, and the VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 34; and (l-2) The VH has at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO: 32, and the VL has at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:

34.

8. The VH comprises the amino acid sequence represented by SEQ ID NO: 25, and the VL comprises the amino acid sequence represented by SEQ ID NO: 26; The VH comprises the amino acid sequence represented by SEQ ID NO: 27, and the VL comprises the amino acid sequence represented by SEQ ID NO: 33; The VH comprises the amino acid sequence represented by SEQ ID NO: 28, and the VL comprises the amino acid sequence represented by SEQ ID NO: 33; The VH comprises the amino acid sequence represented by SEQ ID NO: 29, and the VL comprises the amino acid sequence represented by SEQ ID NO: 33; The VH comprises the amino acid sequence represented by SEQ ID NO: 30, and the VL comprises the amino acid sequence represented by SEQ ID NO: 33; The VH comprises the amino acid sequence represented by SEQ ID NO: 31, and the VL comprises the amino acid sequence represented by SEQ ID NO: 33; The VH comprises the amino acid sequence represented by SEQ ID NO: 32, and the VL comprises the amino acid sequence represented by SEQ ID NO: 33; The VH comprises the amino acid sequence represented by SEQ ID NO: 27, and the VL comprises the amino acid sequence represented by SEQ ID NO: 34; The VH comprises the amino acid sequence represented by SEQ ID NO: 28, and the VL comprises the amino acid sequence represented by SEQ ID NO: 34; The VH comprises the amino acid sequence represented by SEQ ID NO: 29, and the VL comprises the amino acid sequence represented by SEQ ID NO: 34; The VH comprises the amino acid sequence represented by SEQ ID NO: 31, and the VL comprises the amino acid sequence represented by SEQ ID NO: 34; or An isolated antibody or antigen-binding fragment thereof that binds to CD142 described in any one of claims 5 to 7, wherein the VH comprises the amino acid sequence represented by SEQ ID NO: 32 and the VL comprises the amino acid sequence represented by SEQ ID NO:

34.

9. An isolated antibody or antigen-binding fragment thereof that binds to CD142 according to any one of claims 1 to 8, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to human CD142.

10. An isolated antibody or antigen-binding fragment thereof that binds to CD142 described in any one of claims 1 to 9, wherein the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises constant regions of the heavy chain and the light chain, and the constant regions are derived from immunoglobulin IgA, IgD, IgE, IgG, IgM, or a subclass thereof.

11. An isolated antibody or antigen-binding fragment thereof that binds to CD142 described in any one of claims 1 to 10, comprising a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain constant region comprising the amino acid sequence represented by SEQ ID NO:

36.

12. An isolated antibody or antigen-binding fragment thereof that binds to CD142 according to any one of claims 1 to 11, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

13. A nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to CD142 described in any one of claims 1 to 12, wherein the nucleic acid encodes the VH and / or the VL.

14. The nucleic acid of claim 13, wherein the nucleic acid encoding the VH comprises the nucleotide sequence represented by SEQ ID NO: 18 and the nucleic acid encoding the VL comprises the nucleotide sequence represented by SEQ ID NO: 19; or the nucleic acid encoding the VH comprises the nucleotide sequence represented by SEQ ID NO: 37 and the nucleic acid encoding the VL comprises the nucleotide sequence represented by SEQ ID NO:

38.

15. A vector comprising the nucleic acid of claim 13 or 14.

16. A host cell comprising a nucleic acid according to claim 13 or 14, or a vector according to claim 15.

17. an antibody drug conjugate of Formula I; Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, wherein: Ab is an isolated antibody or antigen-binding fragment thereof that binds to CD142 according to any one of claims 1 to 12; L is a linker covalently binding Ab and D, respectively; D is the payload, n is an integer from 1 to 10; The antibody-drug conjugate.

18. 18. The antibody-drug conjugate of claim 17, wherein L is a cleavable linker or a non-cleavable linker.

19. 19. The antibody-drug conjugate of claim 17 or 18, wherein L comprises a cleavable peptide.

20. 20. The antibody-drug conjugate of claim 19, wherein the cleavable peptide is enzymatically cleavable.

21. 21. The antibody-drug conjugate of claim 20, wherein the enzyme comprises cathepsin B.

22. The antibody-drug conjugate of any one of claims 17 to 21, wherein the cleavable peptide or the L comprises an amino acid unit.

23. 23. The antibody-drug conjugate of claim 22, wherein the amino acid unit comprises a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

24. 24. The antibody-drug conjugate of claim 23, wherein the amino acid unit is selected from the group consisting of Val-Cit, Val-Ala, Glu-Val-Cit, Ala-Ala-Asn, Gly-Val-Cit, Gly-Gly-Gly, and Gly-Gly-Phe-Gly.

25. 25. The antibody-drug conjugate of any one of claims 17 to 24, wherein L comprises at least one spacer, optionally at least one self-immolative spacer.

26. 26. The antibody-drug conjugate of claim 25, wherein the self-immolative spacer is p-aminobenzoxycarbonyl (PABC) or p-aminobenzyl (PAB).

27. The antibody-drug conjugate of any one of claims 19 to 26, wherein the cleavable peptide is directly spliced ​​to the spacer.

28. The spacer is —NH—(CH ２ ) n ４ -La-Lb-Lc-, where La represents -O- or a single bond, and Lb represents -CR ２ (-CR ３ )- or a single bond, R ２ and R ３ are each independently C １ ~C ６ Alkyl, -(CH ２ ) n ａ -NH ２ , -(CH ２ ) n ｂ -COOH, or -(CH ２ ) n ｃ represents —OH, n ４ represents an integer of 0 to 6, and n ａ , n ｂ and n ｃ each independently represents an integer of 1 to 4, ２ and R ３ is n ａ The antibody-drug conjugate of any one of claims 25 to 27, wherein Lc is not the same when Lc is 0 and Lc represents -C(=O)-.

29. The spacer is —NH—(CH ２ ) ３ -C(=O)-, -NH-CH ２ -O-CH ２ -C(=O)-, or -NH-(CH ２ ) ２ -O-CH ２ The antibody-drug conjugate of claim 28, comprising -C(=O)-.

30. The L is -L １ -L ２ -L ３ - including L １ is -(succinimidyl-3-yl-N)-(CH ２ ) m １ -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) m ２ -C(=O)- or -C(=O)-(CH ２ ) m ３ represents —C(═O)—, m １ represents an integer of 2 to 8, m ２ represents an integer of 2 to 8, m ３ The antibody-drug conjugate of any one of claims 17 to 29, wherein L represents an integer of 2 to 8, L2 represents an amino acid unit, and L3 represents a self-immolative spacer.

31. The antibody-drug conjugate of any one of claims 17 to 30, wherein L is selected from the group consisting of: -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-PABC-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-GGFG-PABC-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ -O-CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -O-CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH2CH2o-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-PABC-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-VA-PABC-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ -O-CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -O-CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; and -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-。

32. The antibody-drug conjugate of any one of claims 26 to 31, wherein the p-aminobenzoxycarbonyl (PABC) or the p-aminobenzyl (PAB) comprises a polysarcosine (poly-N-methylglycine) residue.

33. wherein L is as shown in Formula II: R １ and R ２ are independently selected from hydrogen, methyl and isopropyl groups; R ３ is -(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, R ５ is hydrogen or benzyl, and n １ represents an integer of 0 to 2, and n ２ represents an integer from 0 to 2, R ４ is a methylamino group or -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ The antibody-drug conjugate according to any one of claims 17 to 32, wherein represents an integer of 1 to 20.

34. The R ４ But -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer of 8 to 15. The antibody-drug conjugate of claim 33 .

35. The R ４ But -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ The antibody-drug conjugate of claim 33 or 34, wherein represents an integer of 10 to 12.

36. The R ３ But, -(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, The R ４ But -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 8 to 15, R ５ is selected from benzyl, n １ represents 1 or 2, n ２ The antibody-drug conjugate according to any one of claims 33 to 35, wherein represents 1 or 2.

37. The R ３ represents a single bond, and the R ４ But -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ The antibody-drug conjugate according to any one of claims 33 to 36, wherein represents an integer of 8 to 15.

38. The R ３ represents a single bond, and the R ４ The antibody-drug conjugate of any one of claims 33 to 37, wherein represents a methylamino group.

39. The antibody-drug conjugate of any one of claims 33 to 38, wherein L is selected from the group consisting of:

40. The antibody-drug conjugate of any one of claims 33 to 39, wherein the succinimidyl-3-yl-N of the linker shown in Formula II is covalently attached to the antibody.

41. The antibody-drug conjugate of any one of claims 33 to 40, wherein the linker is covalently attached to the antibody by a thioether bond.

42. The antibody-drug conjugate of any one of claims 33 to 41, wherein the payload is at least one selected from the group consisting of a cytotoxic agent, a label, a nucleic acid, a radionuclide, a hormone, an immunomodulator, a prodrug-converting enzyme, a ribonuclease, an agonistic antibody, an antagonistic antibody and a fragment thereof, a fusion protein, or a derivative thereof.

43. 43. The antibody-drug conjugate of claim 42, wherein the cytotoxic agent comprises a tubulin inhibitor and / or a topoisomerase inhibitor, the tubulin inhibitor comprising an auristatin or a derivative thereof, a maytansine or a derivative thereof, and the topoisomerase inhibitor comprising camptothecin and a derivative thereof.

44. The antibody-drug conjugate of any one of claims 17 to 43, wherein the payload comprises a compound of formula III, or a pharmaceutically acceptable salt thereof:

45. The antibody-drug conjugate of any one of claims 17 to 44, wherein a carbonyl group in the ester group of the linker is linked to an amino group of the payload.

46. the antibody-drug conjugate and n represents a DAR value of 1 to 10 or 4 to 10.

47. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to CD142 described in any one of claims 1 to 12, or an antibody-drug conjugate described in any one of claims 17 to 46, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient.

48. A method for preparing the antibody-drug conjugate of any one of claims 17 to 46, comprising reducing the isolated antibody or antigen-binding fragment thereof that binds to CD142 so that disulfide bonds of the antibody or antigen-binding fragment thereof are at least partially reduced; Removing the reactive group of the linker in the linker-payload to form an antibody drug conjugate of Formula I: Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, Ab is an isolated antibody or antigen-binding fragment thereof that binds to CD142 according to any one of claims 1 to 12; L is a linker covalently binding Ab and D, respectively; D is the payload, and n is an integer from 1 to 10.

49. 49. The method of claim 48, wherein the linker is a cleavable linker or a non-cleavable linker.

50. 50. The method of claim 48 or 49, wherein the linker comprises a cleavable peptide.

51. 51. The method of claim 50, wherein the cleavable peptide is enzymatically cleavable.

52. 52. The method of claim 51, wherein the enzyme comprises cathepsin B.

53. 53. The method of any one of claims 48 to 52, wherein the cleavable peptide or the L comprises an amino acid unit.

54. 54. The method of claim 53, wherein the amino acid unit comprises a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

55. 55. The method of claim 54, wherein the amino acid unit is selected from the group consisting of Val-Cit, Val-Ala, Glu-Val-Cit, Ala-Ala-Asn, Gly-Val-Cit, Gly-Gly-Gly, and Gly-Gly-Phe-Gly.

56. 56. The method of any one of claims 48 to 55, wherein the linker comprises at least one spacer, optionally at least one self-immolative spacer.

57. 57. The method of claim 56, wherein the self-immolative spacer is p-aminobenzoxycarbonyl (PABC) or p-aminobenzyl (PAB).

58. 58. The method of any one of claims 50 to 57, wherein the cleavable peptide is directly spliced ​​onto the spacer.

59. The spacer is —NH—(CH ２ ) n ４ -La-Lb-Lc-, where La represents -O- or a single bond, and Lb represents -CR ２ (-CR ３ )- or a single bond, R ２ and R ３ are each independently C １ ~C ６ Alkyl, -(CH ２ ) n ａ -NH ２ , -(CH ２ ) n ｂ -COOH, or -(CH ２ ) n ｃ represents —OH, n ４ represents an integer of 0 to 6, and n ａ , n ｂ and n ｃ each independently represents an integer of 1 to 4, ２ and R ３ is n ａ 59. The method of any one of claims 56 to 58, wherein when is 0, they are not the same and Lc represents -C(=O)-.

60. The spacer is —NH—(CH ２ ) ３ -C(=O)-, -NH-CH ２ -O-CH ２ -C(=O)- or -NH-(CH ２ ) ２ -O-CH ２ 60. The method of claim 59, comprising -C(=O)-.

61. The L is -L １ -L ２ -L ３ -, L １ is -(succinimidyl-3-yl-N)-(CH ２ ) m １ -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) m ２ -C(=O)- or -C(=O)-(CH ２ ) m ３ represents —C(═O)—, m １ represents an integer of 2 to 8, m ２ represents an integer of 2 to 8, m ３ 61. The method of any one of claims 48 to 60, wherein L represents an integer from 2 to 8, L2 represents an amino acid unit, and L3 represents a self-immolative spacer.

62. The method of any one of claims 48 to 61, wherein L is selected from the group consisting of: -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-PABC-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-GGFG-PABC-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ -O-CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -O-CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH2CH2o-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-PABC-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-VA-PABC-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ -O-CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -O-CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimidyl-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; and -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-。

63. 63. The method of any one of claims 57 to 62, wherein the p-aminobenzoxycarbonyl (PABC) or p-aminobenzyl (PAB) comprises a polysarcosine (poly-N-methylglycine) residue.

64. reducing the antibody such that disulfide bonds of the antibody are at least partially reduced; reacting with the carbon atom at position 3 of the maleimide-N-yl of the linker of formula IV in the linker-payload, In the linker-payload, the carbonyl group in the ester group of the linker of formula IV is linked to the amino group of the payload; In the formula IV, R １ and R ２ are independently selected from hydrogen, methyl, and isopropyl groups; R ３ is -(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, R ５ is hydrogen or benzyl, and n １ represents an integer of 0 to 2, and n ２ represents an integer from 0 to 2, R ４ is a methylamino group or -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 1 to 20.

65. 65. The method of claim 64, further comprising reacting the antibody with a reducing agent in a buffered solution containing a chelating agent, then adding a solution of the linker-payload shown in L-D, and adjusting the pH of the reaction solution.

66. 66. The method of claim 64 or 65, wherein the payload is as shown in Formula III and is linked to the carbonyl group of the ester group in the linker of Formula IV by the nitrogen atom of the amino group on its cyclohexane ring:

67. A kit comprising an isolated antibody or antigen-binding fragment thereof that binds to CD142 according to any one of claims 1 to 12, or an antibody-drug conjugate according to any one of claims 17 to 46.

68. Use of an isolated antibody or antigen-binding fragment thereof that binds to CD142 described in any one of claims 1 to 12, an antibody-drug conjugate described in any one of claims 17 to 46, a pharmaceutical composition described in claim 47, an antibody-drug conjugate prepared by the method of any one of claims 48 to 66, or a kit described in claim 68, in the manufacture of a therapeutic agent for the diagnosis, prevention, and treatment of tumor diseases.

69. 69. The use of claim 68, including use in the manufacture of a therapeutic agent that targets CD142.

70. 70. The use of claim 68 or 69, wherein the tumor comprises a solid tumor that expresses CD142.

71. The use according to any one of claims 68 to 70, wherein the tumor disease comprises ovarian cancer, gastric cancer, esophageal cancer, cervical cancer, prostate cancer, pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer, bladder cancer, melanoma, and kidney cancer.

72. A method for diagnosing, preventing, and treating a tumor disease, comprising administering to a subject a therapeutic dose of a therapeutic agent, wherein the therapeutic agent comprises an isolated antibody or antigen-binding fragment thereof that binds to CD142 of any one of claims 1 to 12, an antibody-drug conjugate of any one of claims 17 to 46, a pharmaceutical composition of claim 47, the antibody-drug conjugate prepared by the method of any one of claims 48 to 66, or a kit of claim 68.

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