Antibodies, antibody drug conjugates, preparation and uses thereof

Abstract

The present disclosure provides antibodies specifically targeting CD142, antibody drug conjugates, preparation and uses thereof. An antibody, or antigen-binding fragment thereof, that binds to CD142 is covalently linked to a cytotoxic payload via a linker. Antibodies, or antigen-binding fragments thereof, and antibody drug conjugates that bind to CD142 exhibit a cytotoxic effect on tumor cells.

Description

Technical Field

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

[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 carrier-based chemotherapy drugs that selectively deliver cytotoxic drugs to tumors / cancer cells ("Antibody-Drug Conjugates: The Last Decade", Nicolas Joubert et al., Pharmaceuticals (Basel)., September 14, 2020, Vol. 13, No. 9: Page 245). The commercially available ADC drugs trastuzumab and gosatuzumab have excellent effects in treating tumors, especially malignant tumors. Both trastuzumab and gosatuzumab use DNA topoisomerase inhibitors (camptothecin derivatives) that are more hydrophobic than tubulin inhibitors (such as MMAE, MMAF) as cytotoxic drugs. Gosatuzumab uses MCC-triazole spacer-PEG7-lysine-PABC as a linker to decompose and release camptothecin SN38 in cell lysosomes (US13 / 948,732). Detrastuzumab, developed by AstraZeneca / Daiichi Sankyo Co., Ltd., uses a cathepsin B-activated GGFG (an amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked by peptide bonds) tetrapeptide as a linker and introduces a self-cleaving structure to release the exotecan derivative Dxd (Yusuke Ogitani et al., Clin Cancer Res (2016), Vol. 22, No. 20: pp. 5097–5108). However, the above-mentioned cytotoxic drugs MMAE, SN38, and Dxd are all substrates of P-glycoprotein (P-gp) (Front Pharmacol, 2019, Vol. 10: p. 749), and may be resistant to some tumors with high P-gp expression.

[0004] Tissue factor (TF), also known as CD142, is a transmembrane glycoprotein. In a complex with its ligand FVIIa, CD142 can activate protease-activated receptor 2, thereby activating intracellular signaling pathways that tumors can use to promote malignant cell survival, tumor growth, angiogenesis, and metastasis. In contrast to the restricted surface expression in normal tissue cells, CD142 exhibits membranous CD142 expression on a variety of solid tumor cells, including pancreatic cancer, lung cancer, cervical cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, and colon cancer. It has been reported that CD142 is significantly expressed on tumor cells and tumor vasculature and is associated with poor disease prognosis and increased metastasis. The above characteristics indicate that CD142 is a potential ADC target. So far, ADCs targeting CD142 mainly include tisotumab vedotin, ICON-2 (XB002), and MRG004A.

[0005] Vetsotuxumab uses the linker MC-VC-PABA and the payload MMAE (PCT / EP2014 / 075326, PCT / EP2009 / 066755), and it was approved for marketing in the United States in September 2021. Vetsotuxumab is indicated for cervical cancer. According to the FDA (U.S. Food and Drug Administration), Vetsotuxumab is injected once every three weeks at a recommended dose of 2 mg / kg. The drug has serious adverse reactions such as skin toxicity, eye toxicity and bleeding, and the therapeutic window is only 3 mg / kg (BLA Multidisciplinary Review Evaluation {Biologics License Application (BLA) 761208} {Vetsotuxumab}).

[0006] ICON-2 is in Phase I clinical trials in the United States and is being developed for adenocarcinoma, bladder cancer, fallopian tube cancer, and head and neck tumors. Its related patent is PCT / US2019 / 012427. The linker-payload of the drug uses ZymeLink Auristatin (ZLA), a patented technology from ZymeWorks. Similar to viltisotuzumab, severe or significant skin toxicity was observed in the non-human primate (NHP) toxicity study of ICON-2 (Thi-SauMigone 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 expansion in Phase I / II clinical trials of MRG004A (related patent PCT / CN2017 / 087779) are currently being conducted in China and the United States to evaluate safety, tolerability, pharmacokinetic characteristics and preliminary efficacy. Preclinical studies have shown that MRG004A also inhibits coagulation (Oncotarget, 2017, Vol. 8 (Issue 35), pp. 59086-59102).

[0008] Therefore, there is still a need to develop new antibodies and antibody-drug conjugates targeting CD142. 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, a preparation method and use thereof, with the aim of reducing or minimizing severe 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), the VH comprising HCDRs 1, 2, and 3, and the VL comprising LCDRs 1, 2, and 3; wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 1. 1 GDX 2 The amino acid sequence shown in (SEQ ID NO: 2), the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 comprises the amino acid sequence shown in LTS, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 5; X 1 D or Q, X 2 It is S or A.

[0011] In some embodiments, X 1 is D, and X 2 is S. In some embodiments, X 1 is Q, and X 2 is S. In some embodiments, X 1 is Q, and X 2 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 HCDRs 1, 2, and 3, and the VL comprises LCDRs 1, 2, and 3; wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR2 comprises IRNRAX 3 X 4 The amino acid sequence of the present invention is as shown in YTS (SEQ ID NO: 21), the HCDR3 comprises the amino acid sequence of SEQ ID NO: 22; and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence of YTS, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24; X 3 N or Q, X 4 It is G or A.

[0013] In some embodiments, X 3 N, X 4 is G. In some embodiments, X 3 N, X 4 is A. In some embodiments, X 3 Q, X 4 For G.

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

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

[0016] In one aspect, the present disclosure provides a host cell, wherein the cell comprises the above-mentioned nucleic acid or vector.

[0017] In one aspect, the present disclosure provides an antibody drug conjugate of Formula I,

[0018] Ab-(LD)n(I)

[0019] or its isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates or combinations thereof; wherein

[0020] Ab is an isolated antibody or antigen-binding fragment thereof that binds to CD142;

[0021] L is a linker covalently linked to Ab and D respectively;

[0022] D is the effective load;

[0023] n is an integer of 1 to 10.

[0024] In one aspect, the present disclosure provides a method for preparing an antibody drug conjugate of Formula I or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate or a combination thereof, the method comprising the steps of reducing an isolated antibody or antigen-binding fragment thereof that binds to CD142 so that its 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.

[0025] In some embodiments, the method comprises the steps of reducing the antibody so that its disulfide bonds are at least partially reduced and reacting with the 3-carbon atom of the maleimide-N-group of the linker of Formula IV in the linker-payload,

[0026]

[0027] in

[0028] In the linker-payload, the carbonyl group of the ester group of the linker of formula IV is attached to the amino group of the payload;

[0029] In Formula IV, R 1 and R 2 independently selected from hydrogen, methyl and isopropyl;

[0030] R 3 Indicates -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or single bond, R 5 is hydrogen or benzyl, n 1 represents an integer from 0 to 2, n 2 represents an integer from 0 to 2; and

[0031] R 4 represents methylamino or -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , and n 3 Represents an integer from 1 to 20.

[0032] 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.

[0033] In one aspect, the present disclosure provides a kit comprising an isolated antibody or antigen-binding fragment thereof that binds to CD142, or the above-mentioned antibody-drug conjugate.

[0034] 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 the methods herein, the above-mentioned pharmaceutical compositions, and a kit in the preparation of a therapeutic agent for the diagnosis, prevention, and treatment of tumor diseases.

[0035] In some embodiments, the tumor comprises a solid tumor that expresses CD142.

[0036] In one aspect, the present disclosure provides a method for reducing the number of cells expressing CD142, 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, an antibody-drug conjugate of Formula I, an antibody-drug conjugate of Formula I prepared by the methods herein, a pharmaceutical composition, or the above-mentioned kit.

[0037] The isolated antibody or its antigen-binding fragment and the antibody drug conjugate of formula I provided herein that bind to CD142 have improved or excellent in vivo efficacy and safety, and HNSTD (not seen in the maximum dose of severe toxicity) can reach 30 mg / kg. In addition, the isolated antibody or its antigen-binding fragment and the antibody drug conjugate of formula I provided herein that bind to CD142 have a weak effect on coagulation function, thus avoiding potential adverse reactions related to bleeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The following is a brief description of the drawings, which are intended to illustrate exemplary embodiments disclosed herein and not to limit the disclosure.

[0039] Figure 1 A and Figure 1 B shows the detection graphs of size exclusion chromatography and hydrophobic interaction chromatography of the Hu01-L3H1 naked antibody prepared by Example 4, respectively; the Hu01-L3H1 naked antibody was used as a quality control substance.

[0040] Figure 2 A and Figure 2 B shows the detection graphs of size exclusion chromatography and hydrophobic interaction chromatography of the Hu02-L1H2 naked antibody prepared by Example 5, respectively; the Hu02-L1H2 naked antibody was used as a quality control substance.

[0041] Figure 3 A and Figure 3B shows the size exclusion chromatography and hydrophobic interaction chromatography detection diagrams of the antibody drug conjugate Hu01-L3H1-LP1-DAR8 prepared in Example 7, respectively.

[0042] Figure 4 A and Figure 4 B shows the size exclusion chromatography and hydrophobic interaction chromatography detection diagrams of the antibody drug conjugate Hu01-L3H1-LP1-DAR4 prepared in Example 8, respectively.

[0043] Figure 5 A and Figure 5 B shows the size exclusion chromatography and hydrophobic interaction chromatography detection diagrams of the antibody drug conjugate Hu02-L1H2-LP1-DAR8 prepared in Example 9, respectively.

[0044] Figure 6 A and Figure 6 B shows the size exclusion chromatography and hydrophobic interaction chromatography detection diagrams of the antibody drug conjugate HuIgG-LP1-DAR8 prepared in Comparative Example 1, respectively.

[0045] Figure 7 A and Figure 7 B shows the detection graphs of size exclusion chromatography and hydrophobic interaction chromatography of the reference ADC prepared in Comparative Example 2, respectively.

[0046] Figure 8 Flow cytometry graphs showing endocytosis of the antibody Mu01 prepared by Example 4 and the antibody Mu02 prepared by Example 5 by BxPC3 cells. MFI is an abbreviation for mean fluorescence intensity.

[0047] Fig. 9 The cell viability-concentration change curves of MDA-MB-231 cells to the antibody Mu01 prepared in Example 4 and the antibody Mu02 prepared in Example 5 are shown.

[0048] Fig.10 Shown are flow cytometry affinity curves of BxPC3 cells to humanized antibody candidate molecules L5H2, L5H1, L3H1, L3H2 and chimeric antibody Ch01 derived from Mu01.

[0049] Fig.11 Shown are flow cytometry affinity curves of BxPC3 cells for humanized antibody candidate molecules L1H1, L1H2, L1H3, L1H4, L1H5, L1H6, L2H1, L2H2, L2H3, L2H5, L2H6 and chimeric antibody Ch02 derived from Mu02.

[0050] Fig.12The curves of changes in the peak value of human plasma thrombin affected by the antibodies Hu01-L3H1 prepared in Example 4 and Hu02-L1H2 prepared in Example 5 are shown.

[0051] Fig.13 The killing assay of Hu01-L3H1-LP1-DAR8 prepared by Example 7 and Hu02-L1H2-LP1-DAR8 prepared by Example 9 on KYSE150 cells is shown.

[0052] Fig.14 The killing assay of Hu01-L3H1-LP1-DAR8 prepared by Example 7 and Hu02-L1H2-LP1-DAR8 prepared by Example 9 on 5637 cells is shown.

[0053] Fig.15 The killing assay of Hu01-L3H1-LP1-DAR8 prepared by Example 7 and Hu02-L1H2-LP1-DAR8 prepared by Example 9 on SW780 cells is shown.

[0054] Fig.16 The killing assay of Hu01-L3H1-LP1-DAR8 prepared by Example 7, Hu01-L3H1-LP1-DAR4 prepared by Example 8, and Hu02-L1H2-LP1-DAR8 prepared by Example 9 on Detroit 562 cells is shown.

[0055] Fig.17 The in vivo efficacy of the ADC in the NCI-H292 cell-derived xenograft mouse model is shown.

[0056] Fig.18 The body weight-time curve after treatment in the NCI-H292 cell-derived xenograft mouse model is shown.

[0057] Fig.19 The in vivo efficacy of the ADC in the NCI-H226 cell-derived xenograft mouse model is shown.

[0058] Fig. 20 The in vivo efficacy of the ADC in a cell-derived xenograft mouse model of IGROV1 was shown.

[0059] Fig.21 The in vivo efficacy of the ADC in a SW780 cell-derived xenograft mouse model is shown.

[0060] Fig. 22 The in vivo efficacy of the ADC in a patient-derived tumor xenograft mouse model established with human lung cancer tissue was shown.

[0061] Fig.23 The in vivo efficacy of the ADC in a patient-derived tumor xenograft mouse model established with human cervical cancer tissue was shown. DETAILED DESCRIPTION

[0062] The present disclosure will be explained in more detail below. This specification is not intended to be an exhaustive list of all the different ways in which the present invention can be implemented or all the features that can be added to the present invention. For example, features shown in relation to one embodiment may be incorporated into other embodiments, and features shown in relation to a particular embodiment may be deleted from that embodiment. In addition, many variations and additions to the various embodiments proposed herein will be apparent to those skilled in the art, based on the present disclosure, without departing from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although preferred materials and methods are described herein, any methods and materials similar or equivalent to the methods and materials described herein can be used to test the practice of the present disclosure. In describing and claiming the present disclosure, the following terms will be used.

[0064] Unless otherwise defined, all numbers used in this specification and claims to indicate content, concentration, ratio, mass, volume, time, temperature, thickness, technical effect, etc. should be understood to be modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the attached claims are approximate values. It may vary according to the desired properties and effects sought to be obtained by the present disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods, or as understood by those skilled in the art to those skilled in the art.

[0065] Although the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximate, the numerical values ​​set forth in the specific examples should be provided as precisely as possible. However, any numerical value inherently contains certain errors, which are necessarily caused by the standard deviation in their respective test measurements. Each numerical range given throughout the specification will include each narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written out herein.

[0066] [Antibody]

[0067] The present disclosure provides examples of isolated antibodies or antigen-binding fragments thereof that bind to CD142 (also known as tissue factor (TF)). Compared to limited surface expression in normal tissue cells, CD142 exhibits membranous CD142 expression on a variety of solid tumor cells and is associated with poor tumor prognosis and increased metastatic properties. Therefore, CD142 can be used as a target and / or biomarker for the treatment and diagnosis of target tumors.

[0068] The term "antibody" (which can be used interchangeably in the plural form) is an immunoglobulin molecule that can specifically bind to a target such as a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc. through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. A typical antibody molecule includes 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 where the amino acid composition and arrangement vary greatly. The site of specific binding, i.e., the antigen binding site, is used to determine the specificity of antibody recognition. The VH region and the VL region can be further subdivided into hypervariable regions, also referred to as "complementarity determining regions" (CDRs), interspersed with more conservative regions called "framework regions" (FRs). Each VH and VL is typically composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The scope of the framework region and CDR can be accurately identified using methods known in the art, such as by Kabat definition, Chothia definition, AbM definition, and / or Contact definition, all of which are well known in the art. See, e.g., Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1989) Nature, Vol. 342: 877; Chothia, C. et al., (1987) J. Mol. Biol., Vol. 196: 901-917; Al-lazikani et al., (1997) J. Molec. Biol., Vol. 273: 927-948; and Almagro, J. Mol. Recognit., Vol. 17: 132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. The antibody can be a complete (i.e., full-length) polyclonal or monoclonal antibody. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA or IgM (or its subclass), and antibodies do not need to be any particular class. According to the antibody amino acid sequence of its heavy chain constant domain, immunoglobulins can be divided into different classes. There are five main immunoglobulin classes: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the immunoglobulins of different classes are respectively referred to as α, δ, ε, γ and μ.The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0069] 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')2, Fv, single chain (scFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified constructs of immunoglobulin molecules comprising an antigen recognition site with the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0070] 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 the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 1. 1 GDX 2 The amino acid sequence of the present invention is as shown in (SEQ ID NO: 2), the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the LCDR2 comprises the amino acid sequence as shown in LTS (Leucine-Threonine-Serine), and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 5; X 1 D or Q, X 2 Either S or A. CDRs are defined / numbered by the IMGT system.

[0071] In some embodiments, X 1 is D, and X 2 is S. In some embodiments, X 1 is Q, and X 2 is S. In some embodiments, X 1 is Q, and X 2 A. Herein, amino acids are represented by single-letter codes, which are well known to those skilled in the art, for example, "D", "Q", "S" and "A" represent aspartic acid, glutamine, serine and alanine, respectively.

[0072] Antibodies having the same light / heavy chain CDR1, CDR2, and CDR3 regions as those exemplary antibodies targeting CD142 are within the scope of the present disclosure.

[0073] The antibody or the framework region of the antibody has a mutation that does not affect the variable region of the antibody and the antigen binding, and the mutation can increase the binding affinity of the antibody to the antigen or remain substantially unchanged. In some embodiments, the separated antibody or its antigen-binding fragment bound to CD142 also includes conservatively modified variants, which include a single substitution, deletion or addition to the polypeptide sequence, which causes the amino acid to be replaced by a chemically similar amino acid. Providing a conservative substitution table of functionally similar amino acids is well known in the art. Such conservatively modified variants are supplements to polymorphic variants, interspecies homologs and alleles and do not exclude them. The following eight groups contain amino acids that are conservative substitutions for each other: 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 modification" is used to refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence.

[0074] In some embodiments of the isolated antibody or antigen-binding fragment thereof that binds to CD142, VH comprises the amino acid sequence shown in SEQ ID NO:8, 9, 10, 11 or 12, and VL comprises the amino acid sequence shown in SEQ ID NO:13, 14, 15, 16 or 17.

[0075] 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 a VL having at least 70%, 79%, 80%, 87%, 88%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:7; (b-1) a VH having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:8, and a VL having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:9. NO:15 has a VL that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:15; (c-1) a VH that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:8, and a VL that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:17; (d-1) a VH that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:9, and a VL that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:15; and (e-1) a VH that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:8 The amino acid sequence shown in NO:9 has a VH that is at least 90%, 95%, 96%, 97%, 98% or 99% identical, and the VL that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:17.

[0076] In the present disclosure, in the context of two or more nucleic acids or peptide sequences, the term "identity" refers to the degree to which two or more sequences or subsequences are identical. If two sequences have identical amino acids or nucleotide sequences in the region being compared, they are "identical". As measured using one of the following sequence comparison algorithms or by manual comparison and visual inspection, when comparing and comparing the maximum correspondence in a comparison window or a specified region, if two sequences have identical amino acid residues or nucleotides of a specified percentage (that is, 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity in the entire sequence in the specified region or when not specified), then two sequences are "substantially identical". Optionally, identity is present in a region having a length of at least about 30 nucleotides (or 10 amino acids), or more preferably in a region having a length of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res., Vol. 25: 3389-3402, 1997; and Altschul et al., J. Mol. Biol., Vol. 215: 403-410, 1990, respectively.

[0077] In addition to the above sequence identity percentages, another indication that two polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., the two polypeptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0078] In some embodiments, the VH of the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises the amino acid sequence shown in SEQ ID NO: 6, and the VL comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 8, and the VL comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 8, and the VL comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 9, and the VL comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 9, and the VL comprises the amino acid sequence shown in SEQ ID NO: 17.

[0079] In some other 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), the VH comprising HCDRs 1, 2, and 3, and the VL comprising LCDRs 1, 2, and 3; wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR2 comprises IRNRAX 3 X 4 The amino acid sequence of the nucleotide sequence of YTS (Tyrosine-Threonine-Serine) is shown in Figure 24, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 25, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 26, the LCDR2 comprises the amino acid sequence of YTS (Tyrosine-Threonine-Serine), and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 27; 3 N or Q, X 4 Either G or A. CDR is defined by the IMGT system.

[0080] In some embodiments, X 3 N, X 4 is G. In some embodiments, X 3 N, X 4 is A. In some embodiments, X 3 Q, X 4 For G.

[0081] In some embodiments, the VH of the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises the amino acid sequence shown in SEQ ID NO: 27, 28, 29, 30, 31 or 32, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33 or 34.

[0082] 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 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 27, and a VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 33; (c-2) a VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: NO:28 has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:28, and a VL has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:33; (d-2) a VH has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:29, and a VL has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:33; (e-2) a VH has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:30, and a VL has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:33; (f-2) a VH has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:31, and a VL has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:32; NO:31 has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:31, and has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:33. (g-2) has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:32, and has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:33. VH; (h-2) has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:27, and has at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:34. VL;(i-2) a VH having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO:28, and a VL having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO:34; (j-2) a VH having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO:29, and a VL having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO:34; (k-2) a VH having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO:31, and a VL having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO:34; and (l-2) a VH having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO: A VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:32, and a VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:34. ;

[0083] In some embodiments, the VH of the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises the amino acid sequence shown in SEQ ID NO: 25, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 29, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO:27, and VL comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO:28, and VL comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO:29, and VL comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO:31, and VL comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO:32, and VL comprises the amino acid sequence set forth in SEQ ID NO:34.

[0084] The isolated antibody or its antigen binding fragment that is bound 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 its antigen binding fragment specifically binds to human CD142. In some embodiments, the isolated antibody or its antigen binding fragment specifically binds to mouse CD142.

[0085] The isolated antibody or antigen-binding fragment thereof that binds to CD142 provided herein includes the constant region of heavy chain and light chain, and the constant chain is derived from immunoglobulin IgM, IgG, IgA, IgD, IgE class or its subclass. In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds to CD142 is IgG class, optionally, IgG1, IgG2, IgG3 or IgG4 subclass. In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds to CD142 is human IgG1 subclass.

[0086] In some embodiments, the heavy chain of any isolated antibody or antigen-binding fragment thereof bound to CD142 as 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 (CL) or a portion thereof. The constant region may be of any suitable origin, such as 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 vbase2.org, both of which are incorporated herein by reference.

[0087] In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:35, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:36.

[0088] When desired, the isolated antibody or antigen-binding fragment thereof bound to CD142 as described herein may include a modified constant region. For example, it may include a modified constant region that is immunologically inert, for example, does not trigger complement-mediated cleavage, or does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity can be assessed using the method disclosed in U.S. Patent No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol., (1999) Vol. 29: pp. 2613-2624; PCT Application No. PCT / GB99 / 01441; and / or UK Patent Application No. 9809951.8.

[0089] 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.

[0090] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein that binds to CD142 is a human antibody.

[0091] The term "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing a human antibody library or human antibody coding sequence (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0092] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein that binds to CD142 is a humanized antibody.

[0093] The term "humanized antibody" refers to a form of non-human (e.g., mouse) antibody, which is a specific chimeric immunoglobulin, immunoglobulin chain, or its antigen-binding fragment containing a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the receptor complementary determining region (CDR) are from non-human species, such as mouse, rat, or rabbit CDR (donor antibody) with desired specificity, affinity, and ability residue substitutions. In some cases, the Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may contain residues that are neither present in the receptor antibody nor in the CDR or framework sequence introduced, but these residues are included in order to further improve and optimize antibody performance. Typically, a humanized antibody will contain at least one, usually two, substantially entire variable region, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin consensus sequences. Humanized antibodies will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically a human immunoglobulin constant region or domain. The antibody may have a modified Fc region 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 from which the one or more CDRs are derived.

[0094] In some embodiments, the isolated antibody or antigen-binding fragment thereof described herein that binds to CD142 is a chimeric antibody that may include heavy and light chain constant regions from human antibodies. A chimeric antibody refers to an antibody having a variable region or part 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 the light and heavy chains simulate the variable regions of antibodies derived from a mammal (e.g., non-human mammals such as mice, rabbits, and rats), while the constant portion is homologous to the sequence in antibodies derived from another mammal such as a human. In some embodiments, amino acid modifications may be performed in the variable region and / or constant region.

[0095] An isolated antibody or antigen-binding fragment thereof that binds to CD142 as described herein can be prepared by any method known in the art. For example, Harlow and Lane, (1998) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York.

[0096] According to the methods commonly implemented in the art, by immunizing animals with CD142 or any polypeptide selected from the amino acid sequence of CD142, collecting and purifying the antibodies produced in vivo, it is possible to obtain an isolated antibody or an antigen-binding fragment thereof that binds to CD142. In this case, by checking the cross-reactivity of the antibody obtained by binding to the heterogeneous CD142 and human CD142, an antibody that can be applied to human diseases can be selected. Alternatively, it can be obtained by the following known methods (e.g., Kohler and Milstein, Nature (1975) Vol. 256, pp. 495-497; Kennet, R. ed., "Monoclonal Antibodies", pp. 365-367, Plenum Press, NY (1980)), producing antibody-producing cells for CD142 and fusing them with myeloma cells to establish hybridomas, and obtaining monoclonal antibodies from hybridomas. CD142 used as an antigen can be obtained by expressing the CD142 gene in a host cell using genetic engineering.

[0097] Hybridomas can be engineered to obtain chimeric antibodies, such as antibodies in which the variable regions of an antibody of mouse or rat origin are linked to constant regions of human origin (see Proc. Natl. Acad. Sci. USA, Vol. 81, pp. 6851-6855 (1984)).

[0098] Examples of humanized antibodies include antibodies obtained by incorporating only the complementarity determining region (CDR) into an antibody derived from a human (see Nature (1986) Vol. 321, pp. 522-525) and antibodies obtained by CDR transplantation, for example, antibodies obtained by transplanting part of the amino acid residues of the framework excluding the sequence of the CDR (WO 90 / 07861). Human antibodies can be obtained by a method using human antibody-producing mice having human chromosome fragments comprising heavy and light chain genes of human antibodies (see Tomizuka, K. et al., Nature Genetics (1997) Vol. 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res., (1998) Vol. 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. ed., Proc. Natl. Acad. Sci. USA (2000) Vol. 97, pp. 722-727, etc.).

[0099] The isolated antibodies or antigen-binding fragments thereof provided herein specifically bind to CD142 and show good affinity for CD142-expressing cells. The isolated antibodies or antigen-binding fragments thereof have a significant killing effect on tumor cells. In some embodiments, the isolated antibodies or antigen-binding fragments thereof provided herein that bind to CD142 can reduce / eliminate diseased cells, such as CD142. + Tumor cells, thereby treating and / or diagnosing CD142 target tumors.

[0100] The separated antibody or its antigen-binding fragment provided herein has a reduced effect on coagulation function, and coagulopathy is reduced. In some embodiments, compared with antibodies targeting CD142 in the prior art such as vetisoltuzumab, the separated antibody or its antigen-binding fragment provided herein has a reduced effect on coagulation time, and effectively avoids bleeding side effects.

[0101] The present disclosure provides nucleic acids encoding the above-mentioned isolated antibodies or antigen-binding fragments thereof that bind to CD142, wherein the nucleic acids encode VH and / or VL. In some embodiments, the nucleic acid encoding VH includes the nucleotide sequence shown in SEQ ID NO: 18; the nucleic acid encoding VL includes the nucleotide sequence shown in SEQ ID NO: 19; or the nucleic acid encoding VH includes the nucleotide sequence shown in SEQ ID NO: 37; the nucleic acid encoding VL includes the nucleotide sequence shown in SEQ ID NO: 38.

[0102] The present disclosure also provides a vector comprising the above nucleic acid. At the same time, the present disclosure provides a host cell comprising the above nucleic acid or vector.

[0103] [Antibody Drug Conjugates]

[0104] The present disclosure provides an antibody drug conjugate having a structure of Formula I, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof,

[0105] Ab-(LD)n(I)

[0106] wherein Ab is an isolated antibody or antigen-binding fragment thereof that binds to CD142;

[0107] L is a linker covalently linked to Ab and D respectively;

[0108] D is the effective load;

[0109] n is an integer of 1 to 10.

[0110] The term "antibody drug conjugate", also referred to as "ADC", refers to a conjugate of an antibody or antigen-binding substance thereof that binds to CD142 as described herein and covalently linked to a payload. Generally, an antibody drug conjugate may include an antibody, a payload, and optionally a linker between the antibody and the payload. ADCs can be administered by delivering a payload to CD142 targeted by the antibody. + cells, especially CD142 + Antibody drug conjugates can be prepared by various methods known to those skilled in the art.

[0111] The term "linker" refers to the connecting structure that connects the antibody and the payload. The molecular design and properties of the linker are key determinants of the efficacy of ADC in terms of pharmacokinetics (PK) / pharmacodynamics (PD) and therapeutic window. In order to achieve optimal efficacy, the ideal linker should have the following properties: (1) The linker needs to have sufficient stability in plasma so that the ADC can circulate in the bloodstream and localize to the tumor site without premature cleavage. The instability of the linker leads to the premature release of the toxic payload and undesirable damage to non-target healthy cells, resulting in systemic toxicity and side effects. (2) The linker needs to have the ability to be rapidly cleaved and release free and toxic payloads once the ADC is internalized into the target tumor cells. (3) Another property to consider in linker design is hydrophobicity. Hydrophobic linkers coupled to hydrophobic payloads generally promote aggregation of ADCs. Such molecules are not conducive to the search for therapeutically useful ADCs and may cause liver toxicity or elicit undesirable immune responses (Kyoji Tsuchikama et al., "Antibody-drug conjugates: recent advances in conjugation and linker chemistrie", Protein Cell., January 2018; Vol. 9, No. 1: pp. 33-46).

[0112] The term "isomer" refers to a compound with the same molecular formula but different structures, which is also called a structural isomer, generally including structural isomers and stereoisomers. Structural isomers refer to isomers produced by differences in the order of connection of atoms in a molecule or different bonding properties, preferably including tautomers. Tautomers refer to functional group isomers produced by the rapid movement of atoms in two positions in a molecule. Stereoisomers refer to isomers produced by atoms or groups of atoms in a molecule being connected to each other in the same order and bond but with different spatial arrangements, preferably including optical isomers. Optical isomers refer to stereoisomers with different optical properties due to the absence of anti-axis symmetry in the molecule, such as enantiomers, diastereomers, racemates and mesomers.

[0113] 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 releases the active drug through enzymatic or non-enzymatic conversion in vivo to exert a pharmacological effect. In the present disclosure, the prodrug can be an ADC molecule or a payload.

[0114] In some embodiments, the linker is a cleavable linker or a non-cleavable linker.

[0115] In some embodiments, the linker comprises a cleavable peptide.

[0116] In some embodiments, the cleavable peptide is cleavable by an enzyme.

[0117] In some embodiments, the enzyme comprises cathepsin B.

[0118] In some embodiments, the cleavable peptide or L comprises amino acid units.

[0119] In some embodiments, the amino acid unit comprises a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide.

[0120] 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 shown in three-letter codes are well known to those skilled 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.

[0121] In some embodiments, L includes at least one spacer that can provide distance between the payload and the antibody.

[0122] In some embodiments, the spacer comprises a self-immolative spacer.

[0123] In some embodiments, the self-immolative spacer comprises a p-aminophenoxycarbonyl group (PABC) or a p-aminobenzyl group (PAB).

[0124] A self-degradable spacer can be defined as a bifunctional chemical moiety that is capable of covalently linking two spaced-apart chemical moieties together to form a normally stable three-part molecule, from which one spaced-apart chemical moiety can be released, for example, by enzymatic cleavage, and after cleavage (e.g., enzymatic cleavage), can be spontaneously cleaved from the remainder of the molecule to release the other spaced-apart chemical moiety.

[0125] In some embodiments, the cleavable peptide is spliced ​​directly to the spacer.

[0126] In some embodiments, the spacer comprises -NH-(CH 2 ) 4 -La-Lb-Lc-, where La represents -O- or a single bond; Lb represents -CR 2 (-CR 3 )- or a single bond, where R 2 and R 3 Each independently represents C 1 ~C 6 Alkyl, -(CH2 ) a -NH 2 、-(CH 2 ) b -COOH or -(CH 2 ) c -OH, n 4 represents an integer from 0 to 6, n a 、n b and n c Each independently represents an integer from 1 to 4, but when n a When R is 0, 2 and R 3 are not the same, and Lc represents -C(=O)-.

[0127] In some embodiments, the spacer comprises -NH-(CH 2 ) 3 -C(=O)-, -NH-CH 2 -O-CH 2 -C(=O)- or -NH-(CH2) 2 -O-CH 2 -C(=O)-.

[0128] In some embodiments, the linker comprises -L 1 -L 2 -L 3 -The structure shown, where L 1 represents -(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 -C(=O)-, where m 1 represents an integer from 2 to 8, m 2 represents an integer from 1 to 8, and m 3 represents an integer from 1 to 8; L 2 represents an amino acid unit; L 3 represents a self-degradable spacer.

[0129] 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 Means 1, 2, 3, 4, 5, 6, 7, or 8.

[0130] In some embodiments, L is selected from the group consisting of:

[0131] -(Succinimidyl-3-yl-N)-CH 2 CH 2 -C(=O)-GGFG-PABC-;

[0132] -(Succinimidyl-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-PABC-;

[0133] -(Succinimidyl-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-PABC-;

[0134] -(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-;

[0135] -(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-;

[0136] -CH 2 -C(=O)-NH-CH 2 CH 2 -C(=O)-GGFG-PABC-;

[0137] -C(=O)-CH 2 CH2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-PABC-;

[0138] -(Succinimidyl-3-yl-N)-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 -C(=O)-;

[0139] -(Succinimidyl-3-yl-N)-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-;

[0140] -(Succinimidyl-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 -C(=O)-;

[0141] -(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)-;

[0142] -(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 2CH 2 -C(=O)-GGFG-NH-CH 2 -O-CH 2 -C(=O)-;

[0143] -(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)-;

[0144] -(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)-;

[0145] -(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)-;

[0146] -(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 2CH 2 CH 2 -C(=O)-;

[0147] -(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)-;

[0148] -CH 2 -C(=O)-NH-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-;

[0149] -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)-;

[0150] -(Succinimidyl-3-yl-N)-CH 2 CH 2 -C(=O)-VA-PABC-;

[0151] -(Succinimidyl-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-VA-PABC-;

[0152] -(Succinimidyl-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH2 -C(=O)-VA-NH-PABC-;

[0153] -(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-;

[0154] -CH 2 -C(=O)-NH-CH 2 CH 2 -C(=O)-VA-PABC-;

[0155] -C(=O)-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-VA-PABC-;

[0156] -(Succinimidyl-3-yl-N)-CH 2 CH 2 -C(=O)-VA-NH-CH 2 CH 2 -C(=O)-;

[0157] -(Succinimidyl-3-yl-N)-CH 2 CH 2 -C(=O)-VA-NH-CH 2 CH 2 CH 2 -C(=O)-;

[0158] -(Succinimidyl-3-yl-N)-CH 2 CH 2 CH2 CH 2 CH 2 -C(=O)-VA-NH-CH 2 CH 2 -C(=O)-;

[0159] -(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)-;

[0160] -(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)-;

[0161] -(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)-;

[0162] -(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)-;

[0163] -(Succinimidyl-3-yl-N)-CH 2 CH 2 -C(=O)-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH2 CH 2 -C(=O)-VA-NH-CH 2 CH 2 CH 2 -C(=O)-;

[0164] -(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)-;

[0165] -(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)-;

[0166] -(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)-;

[0167] -CH 2 -C(=O)-NH-CH 2 CH 2-C(=O)-VA-NH-CH 2 CH 2 CH 2 -C(=O)-; and

[0168] -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)-.

[0169] In some embodiments, the p-aminophenoxycarbonyl (PABC) or p-aminobenzyl (PAB) group includes a polysarcosine (poly-N-methylglycine) residue or a methylamino group.

[0170] In some embodiments, the linker comprises Formula II,

[0171]

[0172] In Formula II, R 1 and R 2 R is independently selected from hydrogen, methyl and isopropyl; 3 Indicates -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or single bond; R 5 is selected from hydrogen or benzyl, n 1 represents an integer from 0 to 2, and n 2 represents an integer from 0 to 2; R 4 represents methylamino or -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , and n 3 Represents an integer from 1 to 20.

[0173] In some embodiments, in the linker of Formula II, R 4 Indicates -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , n 3 represents an integer from 1 to 20, n 3Any integer can be selected from, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0174] In some embodiments, in the linker of Formula II, R 4 Indicates -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , n 3 represents an integer from 8 to 15. In some embodiments, in the linker of formula II, R 4 Indicates -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , n 3 Represents an integer from 10 to 12.

[0175] In some embodiments, in the linker of Formula II, R 4 It represents methylamino.

[0176] In the present disclosure, R containing a hydrophilic amino group (e.g., a polysarcosine group or a methylamino group) is introduced. 4 It is beneficial to increase the hydrophilicity of antibody drug conjugates, especially when hydrophobic payloads are conjugated to antibody drug conjugates. The increase in ADC hydrophilicity helps to reduce ADC aggregation during the preparation process, thereby improving the stability, uniformity and purity of ADC.

[0177] In some embodiments, in the linker of Formula II, R 3 Indicates a single bond.

[0178] In some embodiments, in the linker of Formula II, R 3 Indicates -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 -, R 5 is benzyl, n 1 An integer representing 1 or 2, n 2 An integer representing 1 or 2.

[0179] In some embodiments, in the linker of Formula II, R 3 Indicates -CR 5 HCONH-、-CH 2 CONH-, -CR 5 HCONH-CH 2CONH-、-(CR 5 HCONH) 2 -CH 2 CONH-, -CR 5 HCONH-(CH 2 CONH) 2 -or-(CR 5 HCONH) 2 -(CH 2 CONH) 2 -; R 5 It is benzyl.

[0180] In some embodiments, in the linker of Formula II, R 1 In some embodiments, in the linker of Formula II, R 1 It is isopropyl.

[0181] In some embodiments, in the linker of Formula II, R 2 In some embodiments, in the linker of Formula II, R 2 It is methyl.

[0182] In some embodiments, the linker of the antibody drug conjugate is selected from the group consisting of:

[0183]

[0184] In the present disclosure, in the antibody drug conjugate, the succinimidyl group of the linker of Formula II is connected to the antibody via a covalent bond. In some embodiments, the terminal succinimidyl group of the linker of Formula II forms a thioether bond with the sulfhydryl group obtained by reducing the interchain disulfide bonds of the antibody. It forms a thioether bond with the sulfhydryl moiety obtained by the carbon atom at position 3 and the interchain disulfide bond of the reduced antibody. The bonds represented represent chemical bonds to other groups.

[0185] In the present disclosure, the disulfide bonds of the antibody include interchain disulfide bonds and intrachain disulfide bonds, preferably the interchain disulfide bonds are treated, for example, activated to sulfhydryl groups and then bonded to the linker. The amino acids in the antibody chemically bonded to the succinimidyl group include one or a combination of lysine, histidine, tyrosine and cysteine, preferably cysteine.

[0186] The term "payload" includes compounds that are cytotoxic or capable of killing cells after release from the antibody drug conjugate; compounds that serve as detection labels or have cell-killing effects, radioactively labeled radionuclides or polypeptides, fluorophores, chromophores, imaging agents and / or metal ions; and compounds, nucleic acids, polypeptides or proteins, enzymes, hormones or nucleic acids that can modulate immune activity in vivo (including activation or inhibition).

[0187] Under some ideal conditions, in an antibody drug conjugate, the coupled payload has little cytotoxicity, or its cytotoxicity is so low that administration of a therapeutically effective dose of ADC will not cause systemic toxicity in a subject due to the coupled payload. The payload can be a clinically validated drug for the treatment of a specific disease, or a compound, radionuclide, nucleic acid, protein or peptide with acceptable pharmacological activity under clinical use conditions.

[0188] In some embodiments, the payload in the antibody drug conjugate is a label containing a radioactive label, a fluorophore, a chromophore, a developer 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. Wherein the radioactive label refers to a labeled compound, wherein one or more atoms of the compound molecule are replaced by a radionuclide, so that the compound can be identified and used as a tracer, and the radioactive label includes amino acids, polypeptides, proteins, carbohydrates, nucleotides, nucleosides, purines, pyrimidines, steroids, lipid compounds, and tumor antigens, hormones, receptors, vitamins and drugs for medical research. Radionuclides are generally nuclides that can spontaneously emit radiation, including but not limited to tritium, iodine 125, iodine 131, sulfur 35, phosphorus 32 and carbon 14. Fluorophores are generally groups including conjugated double bonds, and when the molecule returns to the ground state from an excited state, the fluorophore emits fluorescence. Chromophores refer to unsaturated groups and their associated chemical bonds contained in a molecule, which can absorb light radiation and have transitions. In nuclear medicine, contrast agents generally refer to radioactive drugs that, when introduced into the body, are able to image organs, tissues, or molecules.

[0189] In some embodiments, the payload in the antibody drug conjugate is a nucleic acid, which can be RNA and / or DNA.

[0190] In some embodiments, the payload in the antibody drug conjugate is a hormone, a growth factor, a coagulation factor, and a fibrinolytic enzyme (eg, a prodrug convertase, a ribonuclease capable of converting a prodrug into an active drug).

[0191] In some embodiments, the payload in the antibody drug conjugate is an immunomodulator (including cytokines and chemokines that can affect immunity), or an agonistic or antagonistic antibody with biological activity.

[0192] In some embodiments, the useful load in the antibody drug conjugate is a cytotoxic compound. In some embodiments, the useful load in the antibody drug conjugate has anti-tumor activity or is an anti-tumor drug. The useful load 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.

[0193] The term "topoisomerase inhibitor" generally refers to a compound that inhibits the activity of a topoisomerase. 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.

[0194] The term "tubulin inhibitors" generally refers to compounds that inhibit the microtubule system of eukaryotic cells, interfere with cell division and inhibit cell proliferation.

[0195] In some embodiments, the effective load is camptothecin or its derivatives with topoisomerase inhibition. The term "derivative" refers to a compound formed by replacing an atom or a group of atoms in a parent compound molecule with other atoms or groups of atoms, and is referred to as 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 etirinotecan, irinotecan, topotecan, lortotecan, silatecan, etirinotecan pegol, TAS103, 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methylenedioxycamptothecin, 9-amino-10,11-methylenedioxycamptothecin, 9-chloro-10,11-methylenedioxycamptothecin, (7-(4-methylpiperazinylmethylene)-10,11-ethylenedioxy-20(S)-camptothecin, 7-(4-methylpiperazinylmethylene)-10,11-methylenedioxy-20(S)-camptothecin and 7-(2-N-isopropylamino)ethyl)-(20S)-camptothecin, as well as stereoisomers, salts and esters thereof. Methods for the synthesis of camptothecin and camptothecin analogs or derivatives thereof are known and are summarized and described in US Pat. No. 5,244,903, which is incorporated herein by reference in its entirety.

[0196] In some embodiments, the payload is auristatin or a derivative thereof, maytansine or a derivative thereof having tubulin inhibition. The term "auritin or a derivative thereof" generally includes auristatin F and auristatin F derivative. 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.

[0197] In some embodiments, the payload is ixotecan, a camptothecin derivative that acts as a topoisomerase inhibitor, which can act throughout the cell cycle and has strong penetration and good therapeutic effects on slow-growing solid tumors. In addition, the number of intracellular targets is much less than that of tubulin inhibitors, so when the ADC molecule carries the same amount of payload into the cell, it can achieve a better killing effect. At the same time, the ixotecan molecule is not a substrate of P-gp, which is conducive to reducing or alleviating drug resistance problems.

[0198] In some embodiments, the payload is a camptothecin of Formula III or a pharmaceutically acceptable salt thereof, which is connected to the linker via the nitrogen atom of the amino group on its cyclohexane ring,

[0199]

[0200] The molecular structure of exotecan is rigid and has poor hydrophilicity. Therefore, when it is connected to the GGFG tetrapeptide linker commonly used in the prior art to prepare ADC, it is easy to cause aggregation between ADC molecules, which does not meet the development requirements of ADC drugs (Bioorg. MedChem. Lett. Vol. 26 (2016) pp. 1542-1545). Therefore, the selection and matching of the linker and the payload have an impact on the safety and stability of ADC drugs.

[0201] Without being bound by any theory, due to the multiple hydrophilic groups in the linker of the antibody drug conjugate, the hydrophilicity of the linker-payload structure is improved and the aggregation and precipitation of the ADC caused by the hydrophobic payload can be reduced.

[0202] After the ADC molecule is endocytosed into the cell, the linker is degraded to release the payload or the linker (or a portion of the linker)-payload structure compound. In some embodiments, the amino group on the cyclohexane ring of the isotecan of Formula III is bonded to the carbonyl group in the ester group of the linker of Formula I, and a linker-payload structure comprising a carbamate is formed. Without being bound by any theory, after the ADC molecule is endocytosed into the cell, the linker is cleaved by a cathepsin (e.g., cathepsin B) to form an intermediate or active metabolite shown in the following formula V,

[0203]

[0204] R 4 represents methylamino or -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , and n 3 Represents an integer from 1 to 20.

[0205] Then, the PABC group in the intermediate or active metabolite of Formula V undergoes 1,6-elimination to release exotecan. The 1,6-elimination mechanism of PABC is described in detail in the literature Angew.Chem.Int.Ed., 2015, Vol. 54: pp. 7492-7509. Therefore, the linker-payload structure in the ADC provided by the present disclosure has good in vivo stability and biological activity.

[0206] Without being bound by any theory, the cleavage site in the linker-payload structure can be an amide bond in the linker, e.g., R 2 The carbon atom where the substituent is located is 3 represents an amide bond between the groups, or R 3 The amide bond in the group represented by .

[0207] In the antibody drug conjugate, n, i.e., the ratio of the number of molecules of coupled payload to each molecule of antibody (DAR) is 1: 10. In some embodiments, n is 1-10, 1-2, 2-4, 4-6, 2-8, 4-8, 4-10, 6-10, 7-10, or 8-10, and exemplary n is 4.66, 7.67, or 7.83.

[0208] The term "DAR (drug to antibody ratio)" represents the average value of the number of coupled payload or drug molecules per antibody molecule, i.e., the average number of coupled drug molecules. In the antibody drug conjugate, the number of coupled payload molecules per antibody molecule is a key factor affecting its efficacy and safety. The preparation of the antibody drug conjugate is carried out by specifying reaction conditions such as the amount of starting materials and reagents used for the reaction, so as to obtain a constant number of coupled payload molecules. When preparing an antibody drug conjugate, a mixture containing different numbers of coupled payload molecules is generally obtained. Unless otherwise specified, in the present disclosure, the number of coupled payload or drug molecules per antibody molecule is defined as the average value, i.e., the average number of coupled payload or drug molecules.

[0209] In some embodiments, ADCs with the above linkers are chemically coupled to antibodies targeting CD142, resulting in higher DAR values ​​(eg, DAR8).

[0210] In some embodiments, the antibody drug conjugate comprises any of the following structures:

[0211]

[0212]

[0213] Ab represents an isolated antibody or antigen-binding fragment thereof that binds to CD142; n equals the ratio of the number of molecules of payload to the number of antibodies per molecule, or DAR.

[0214] The antibody in the antibody drug conjugate is CD142-specifically targeted, and the antibody forms a reactive thiol group through a disulfide bond and is then connected to a linker. In some embodiments, a disulfide bond in the hinge region of the antibody forms a reactive thiol group and is then connected to a linker.

[0215] 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 as shown in SEQ ID NO: 8 and a VL sequence as shown in SEQ ID NO: 15. In some embodiments, the antibody or antigen binding fragment thereof in the antibody drug conjugate comprises a VH sequence as shown in SEQ ID NO: 28 and a VL sequence as shown in SEQ ID NO: 33.

[0216] Improved hydrophilicity of the antibody drug conjugate can also increase homogeneity, and thus, the antibody drug conjugate with improved hydrophilicity and / or homogeneity can improve the cytotoxic effect on target cells, and improve or maintain biological activity, safety, and other pharmaceutical properties.

[0217] The antibody drug conjugate of the present disclosure has improved HNSTD (maximum dose without severe toxicity). In addition, it shows a significant therapeutic effect on tumor suppression, and it may not cause obvious digestive system toxicity. It should be noted that the antibody drug conjugate of the present disclosure can absorb moisture, retain the adsorbed moisture, or become a hydrate due to being left in the atmosphere or recrystallized, and such water-containing compounds and salts thereof are also included in the present disclosure. In addition, compounds of isotope variants labeled with various radioactive or non-radioactive isotopes are also included in the present disclosure. More than one atom constituting the antibody drug conjugate of the present disclosure may also contain atomic isotopes in non-natural proportions. As atomic isotopes, for example, deuterium (2H), tritium (3H), iodine-125 (125I) or carbon-14 (14C) can be cited. In addition, the compounds of the present disclosure can be radiolabeled with radioactive isotopes such as tritium (3H), iodine-125 (125I) or carbon-14 (14C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents such as test reagents, and diagnostic agents, for example, in vivo imaging diagnostics.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.

[0218] [Drug composition]

[0219] The present disclosure provides a pharmaceutical composition comprising the above-mentioned isolated antibody or antigen-binding fragment thereof that binds to CD142, and a pharmaceutically acceptable excipient.

[0220] The present disclosure provides a pharmaceutical composition, which comprises the above-mentioned antibody-drug conjugate, or its isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates or combinations thereof, and a pharmaceutically acceptable excipient.

[0221] According to the specific application form, physicochemical properties, etc. of pharmaceutically acceptable excipients, the pharmaceutical composition can be applied in a suitable manner. In some embodiments, the pharmaceutical composition can be formulated into a freeze-dried preparation or a liquid preparation, which may contain a suitable formulation additive in the art. For example, the above-mentioned pharmaceutical composition usually contains more than one pharmaceutical carrier, such as sterile liquids such as water and oil (including petroleum, oils of animal origin, plant origin or synthetic origin (for example, peanut oil, soybean oil, mineral oil and sesame oil, etc.)). In the case of intravenous administration of the above-mentioned pharmaceutical composition, water is a more representative carrier. In addition, saline solution, aqueous glucose solution and glycerol solution can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients are known in the art. The above-mentioned pharmaceutical composition may also contain trace amounts of wetting agents, emulsifiers or pH buffers as needed. The mode of administration of the pharmaceutical composition is usually parenteral administration, which may be intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous injection, but is not limited thereto, for example, the pharmaceutical composition can be administered by infusion or push injection. See, e.g., the Handbook of Pharmaceutical Excipients, 3rd edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated herein by reference in its entirety. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated herein by reference in its entirety.

[0222] The pharmaceutical composition may contain an active agent, that is, an isolated antibody or its antigen-binding fragment or the above-mentioned antibody-drug conjugate that binds to CD142, and a second therapeutic agent (e.g., a cancer therapeutic agent). In some embodiments, the isolated antibody or its antigen-binding fragment 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. Other anti-cancer agents 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.

[0223] With regard to the present disclosure, an active agent or a pharmaceutical composition comprising the active agent can be administered to a subject via any suitable route of administration. For example, the active agent can be administered to a subject parenterally, nasally, orally, pulmonary, topically, vaginally or rectally. The following discussion of routes of administration is intended only to illustrate various embodiments and should not be construed as limiting the scope in any way.

[0224] The present disclosure also provides a kit, which includes an isolated antibody or an antigen-binding fragment thereof that binds to CD142 or the above-mentioned antibody-drug conjugate, which can be used to detect CD142 or cells expressing CD142.

[0225] [Preparation method]

[0226] The present disclosure provides a method for preparing an antibody-drug conjugate, the preparation method comprising the following steps:

[0227] reducing the antibody or antigen-binding fragment thereof so that its disulfide bonds are at least partially reduced and reacting with the reactive groups of the linker in the linker-payload to obtain an antibody drug conjugate having a structure of Formula I,

[0228] Ab-(LD)n(I)

[0229] or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof;

[0230] Wherein Ab is the above-mentioned isolated antibody or antigen-binding fragment thereof that binds to CD142; L is a linker covalently connected to Ab and D respectively; D is a payload; and n is an integer from 1 to 10.

[0231] The reactive group is not particularly limited as long as the reactive group contains a portion capable of reacting with the resulting thiol group of the antibody.

[0232] In some embodiments, the carbon atom at position 3 of the maleimide-N-group in LD is reacted with a reduced antibody and covalently linked to prepare an ADC.

[0233] In some embodiments, the method comprises the steps of reducing the antibody so that its interchain disulfide bonds are at least partially reduced, and reacting with the carbon atom at position 3 of the maleimide-N-group of the linker of formula IV,

[0234]

[0235] The carbonyl group in the ester group of the linker of formula IV is connected to the amino group of the payload in the antibody drug conjugate;

[0236] In Formula IV, R 1 and R 2 are each independently selected from hydrogen, methyl and isopropyl;

[0237] R 3 Indicates -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or single bond, R5 is selected from hydrogen or benzyl, n 1 represents an integer from 0 to 2, and n 2 represents an integer from 0 to 2;

[0238] R 4 represents methylamino or -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , and n 3 Represents an integer from 1 to 20.

[0239] In antibody drug conjugates, in many practical cases, the linker of the above formula IV structure with a payload is connected to the same antibody molecule with a reactive thiol group. In some embodiments, the antibody reacts with a reducing agent such as dithiothreitol (DTT), 2-mercaptoethanol or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to cause the disulfide bonds of the antibody chain to form reactive thiol groups. The amount of the reducing agent can be 0.3-10 times the antibody molar equivalent, for example 1-10, 3-10, 5-10, 7-10 times the antibody molar equivalent.

[0240] In some embodiments, the method further comprises: reacting the antibody with a reducing agent in a buffer solution containing a chelating agent, and then adding a linker-payload solution to carry out the reaction. The linker-payload is particularly a compound formed by bonding a linker of formula IV to the payload, wherein the amino group (primary amino group) in the payload is connected to the carbonyl group in the ester group of the linker of formula IV. The term "chelating agent" refers to a complex that can form a complex having a cyclic structure by coordination bonding with a metal atom or ion.

[0241] 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 concentration of the chelating agent is 1mM to 20mM, for example, 2mM to 20mM, 5mM to 20mM, 8mM to 20mM, 1mM to 15mM or 1mM to 10mM. The 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.

[0242] The reaction of the antibody with the reducing agent is carried out at an adjusted 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 acidic or alkaline chemicals, and exemplary acidic or alkaline chemicals include acetic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sodium bicarbonate, sodium carbonate, sodium hydroxide, and triethylamine.

[0243] The reaction of the antibody and the reducing agent is carried out at an adjusted temperature, and exemplary reaction temperatures are -10°C to 40°C, -5°C to 40°C, 0°C to 40°C, 5°C to 40°C, 25°C to 40°C, 30°C to 40°C, 35°C to 38°C, for example about 37°C.

[0244] 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.

[0245] In some embodiments, the linker-payload solution is added to the antibody buffer solution that has been reduced or has reactive thiol groups in an amount of 1% to 20% by volume based on the volume of the antibody buffer solution. In some embodiments, the volume ratio of the linker-payload solution added 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 antibody buffer solution.

[0246] In some embodiments, the DAR is 4-20, optionally 8-20. In some embodiments, the DAR is 10-20, 14-20, 16-20, or 18-20.

[0247] In some embodiments, the temperature of the antibody and linker-payload reaction is -10°C to 40°C, -5°C to 40°C, 0°C to 40°C, 5°C to 40°C, 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, 0°C to 37°C. In some embodiments, the reaction temperature is 5°C to 37°C, 10°C to 37°C, 10°C to 25°C, or 15°C to 30°C.

[0248] In some embodiments, the time for the antibody to react with the linker-payload is 0.5 hours to 2 hours. In some embodiments, the time for the antibody to react with the linker-payload is 0.5 hours to 1.75 hours, 0.5 hours to 1.5 hours, 0.5 hours to 1.25 hours, 0.75 hours to 2 hours, or 1 hour to 2 hours.

[0249] The reaction can be terminated by inactivating unreacted linker-payload with a thiol-containing reagent. Thiol-containing reagents include, but are not limited to, cysteine ​​or N-acetyl-(L)-cysteine ​​(NAC). More specifically, a thiol-containing reagent having a molar equivalent of 1-2 times the linker-payload is added to the reaction solution and incubated at room temperature (10° C.-25° C.) for 10-30 minutes, thereby terminating the reaction.

[0250] In the case where the antibody has a sulfhydryl group, the antibody drug conjugate can also be obtained by reacting the compound using a known method (e.g., obtainable by the method described in patent publication US2016 / 297890 (e.g., obtainable by the method 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)).

[0251] The antibody drug conjugate provided by the present disclosure can be obtained by the above-mentioned preparation method. In some embodiments, the prepared antibody drug conjugate is subjected to a purification process, which includes but is not limited to gel filtration, such as purification using a gel column.

[0252] [How to use]

[0253] The present disclosure also provides use of the above-mentioned antibody, the above-mentioned antibody-drug conjugate, the above-mentioned pharmaceutical composition, or the antibody-drug conjugate prepared by the above-mentioned method, or the above-mentioned kit in preparing a therapeutic agent for diagnosing, preventing and treating tumor diseases, wherein the tumor diseases include benign tumors and malignant tumors (e.g., cancer).

[0254] The present disclosure provides a method for diagnosing, preventing and treating tumor diseases, which comprises administering a therapeutically effective amount of the above-mentioned antibody, antibody-drug conjugate, antibody-drug conjugate prepared by the above-mentioned method, pharmaceutical composition or kit to a subject in need thereof.

[0255] Tumor diseases are not limited to the above diseases, as long as the cells in the lesion site express proteins that can be recognized by CD142 targeting antibodies. In some embodiments, tumor diseases include ovarian cancer, gastric cancer, esophageal cancer, cervical cancer, prostate cancer, pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer, bladder cancer, melanoma and kidney cancer.

[0256] In some embodiments, the tumor disease is a CD142-related disease, such as a solid tumor expressing CD 142. In some embodiments, the CD142-related disease includes pancreatic cancer, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer and cervical cancer.

[0257] The present disclosure provides use of the above-mentioned antibody, the above-mentioned antibody-drug conjugate, the above-mentioned pharmaceutical composition or the antibody-drug conjugate prepared by the above-mentioned method in preparing a therapeutic agent targeting CD142.

[0258] The present disclosure provides a method for reducing the number of cells expressing CD142, comprising administering to a subject in need thereof a therapeutically effective amount of the above-mentioned antibody, the above-mentioned antibody-drug conjugate, the above-mentioned pharmaceutical composition, or the above-mentioned antibody-drug conjugate prepared by the above-mentioned method. Elimination of cells expressing CD142 in a subject is beneficial for treating CD142-related diseases.

[0259] As used herein, the subject can be a non-human mammal and a human. Non-human mammals include, but are not limited to, farm animals, sports animals, pets, primates, horses, dogs, cats, mice, and rats. In some embodiments, the subject is a human. The human subject in need of treatment can be a human subject suffering from, at risk of suffering from, or suspected of suffering from a target disease / disorder associated with a tumor or a CD142-associated tumor.

[0260] A subject with a target disease or condition can be identified by routine medical examinations (e.g., laboratory tests, organ function tests, CT scans, or ultrasound). A subject suspected of having any such target disease / condition may exhibit one or more symptoms of the disease / condition. A subject at risk of having the disease / condition may be a subject with one or more risk factors for the disease / condition.

[0261] The term "therapeutically effective amount" refers to the amount of each active agent required to bring about a therapeutic effect on a subject alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect refers to decreased CD142 activity or decreased CD142 + Cell activity. It will be apparent to those skilled in the art to determine whether the amount of the antibody or ADC comprising the antibody achieves a therapeutic effect. As will be appreciated by those skilled in the art, the effective amount will depend on the specific condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, constitution, sex and weight), duration of treatment, nature of concomitant treatment (if any), specific route of administration, and the knowledge and expertise of health practitioners and similar factors. These factors are factors well known to those of ordinary skill in the art and can be explained only by routine experiments. It is generally preferred to use the maximum amount of a single component or a combination thereof, i.e., the highest safe amount according to reasonable medical judgment.

[0262] In some embodiments, the dosage of an antibody or its antigen-binding fragment, or an antibody drug conjugate can be determined empirically in an individual who has received one or more administrations of the antibody. In some embodiments, the acceptable therapeutic dose of an antibody drug conjugate is 0.1 mg / kg to 30 mg / kg, 0.5 mg / kg to 30 mg / kg, 1 mg / kg to 30 mg / kg, 1 mg / kg to 25 mg / kg, 0.1 mg / kg to 25 mg / kg, 0.1 mg / kg to 20 mg / kg, 1 mg / kg to 20 mg / kg, or 0.5 mg / kg to 20 mg / kg. In some embodiments, the frequency of administration is once every 12 hours, once a day, once a week, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks; or once a month, once every 2 months, or once every 3 months or longer. The therapeutic dose and frequency of administration may vary with the treatment regimen.

[0263] The various embodiments and preferences of the present disclosure may be combined with each other as long as they are not inherently inconsistent with each other, and the various embodiments formed by the combination are considered to be part of the disclosure of the present application.

[0264] The technical solution of the present disclosure will be described more clearly and specifically with reference to the embodiments by way of example. It should be understood that these embodiments are only for illustrative purposes and are by no means intended to limit the scope of protection of the present disclosure. The scope of protection of the present disclosure is limited only by the claims.

[0265] Example

[0266] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0267] Example 1: Preparation of Compound LP-1

[0268]

[0269] Step 1: Synthesis of Intermediate 11-1

[0270] At room temperature (20°C to 30°C), 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). The reaction solution was stirred at room temperature for 24 hours, and the solvent was removed therefrom in vacuo. Then, the crude residue was further purified by flash chromatography to obtain compound 11-1 (3.9 g, 71%). LC-MS (ESI, m / z): 868.49 (M+H).

[0271] Step 2: Synthesis of Intermediate 11-2

[0272] 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 at 0°C under argon atmosphere. 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 was dried and concentrated. The residue was purified by silica gel chromatography to obtain compound 11-2 (1.1 g, 76%). LC-MS (ESI, m / z): 630.31 (M+H).

[0273] Step 3: Synthesis of Intermediate 11-3

[0274] 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 under an argon environment and room temperature. Then, the reaction mixture was stirred overnight at ambient temperature. The solvent in the reaction mixture was removed by concentration, and the product thus obtained was precipitated using MTBE (methyl tert-butyl ether). The yellow solid was collected by filtration, washed with ether, and dried to obtain compound 11-3. LC-MS (ESI, m / z): 795.41 (M+H).

[0275] Step 4: Synthesis of Intermediate 11-4

[0276] Compound 11-3 (300 mg, 0.44 mmol) was dissolved in anhydrous DMF (4 mL), and dry pyridine (1 mL) was added thereto, followed by addition of exotecan mesylate (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd. MedChemExpressShanghai; 234 mg, 0.44 mmol) and HOBt (1-hydroxybenzotriazole; 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 obtain intermediate 11-4 (230 mg, 48%). LC-MS (ESI, m / z): 1091.53 (M+H).

[0277] Step 5: Synthesis of Intermediate 11-5

[0278] 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 was removed therein by concentration to obtain the TFA salt of intermediate 11-5, which was used in the next step without further purification. LC-MS (ESI, m / z): 991.47 (M+H).

[0279] Step 6: Synthesis of compound LP-1

[0280] Compound 11-5 (120 mg, 0.109 mmol) was dissolved in 1 mL of anhydrous DMF, and Ac-Sar10-COOH (N-acetyl decasarcosine; 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 at room temperature overnight, and the solvent was removed therefrom by concentration. The crude product was purified by preparative HPLC to obtain compound LP-1 (74 mg, 38%). LC-MS (ESI, m / z): 1743.85 (M+H).

[0281] Example 2: Preparation of Compound LP-2

[0282]

[0283] The synthesis of compound LP-2 was carried out according to the synthesis steps of compound LP-1. Mc-GGFG-OH (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) was used to replace the starting material 11-1A to obtain compound LP-2, which was a beige amorphous solid. LC-MS (ESI, m / z): 1891.90 (M+H).

[0284] Example 3: Preparation of Compound LP-3

[0285]

[0286] Compound LP-3 is an intermediate of LP-1. By removing step 6, intermediate 11-5 will be LP-3.

[0287] Example 4: Preparation of monoclonal antibodies Mu01 and Hu01

[0288] Balb / c mice (8-12 weeks old) were immunized with human CD142 protein antigen extracellular region fragment (purchased from ACROBiosystems, product number TF3-H52H5), and their serum titers were monitored to determine the number of immunizations. After the initial immunization, mice were boosted three or four times, and serum was collected for titer detection. Mice with qualified titers were boosted once, and the whole spleen and 1 / 2 lymph node were harvested and fused with myeloma SP2 / 0 cell line for PEG fusion. The fused cells were cultured in a plate. Supernatant was collected and antigens were screened by ELISA. Positive cells were transferred to 96-well plates for further cultivation. After 7 days, supernatant was collected, and the reaction with antigen was determined using ELISA. The antigen binding affinity of positive cells was further tested at different dilutions. 20 parent clones with the highest immunogenic affinity entered subclones. Monoclonal hybridoma cells were obtained by limiting dilution and ELISA screening. Hybridoma cells were injected into the abdomen of mice to prepare ascites. After collection and purification, a monoclonal antibody named Mu01 was obtained. The variable region and CDR of Mu01 were sequenced as shown in Table 1. The amino acid sequence of the CDR is underlined.

[0289] Table 1 Hybridoma sequences

[0290]

[0291] The DNA sequence encoding VH is shown in SEQ ID NO:18, and the DNA sequence encoding VL is shown in SEQ ID NO:19.

[0292] As shown in Table 2, humanized 01 variable regions (called Hu01) were obtained by modifying the VH and VL of the hybridoma sequences. H1, H2, H3, H4 and H5 represent the sequence codes of VH, respectively. L1, L2, L3, L4 and L5 represent the sequence codes of VL, respectively. L3H1 (or Hu01-L3H1") represents a humanized antibody comprising a light chain variable region encoding L3 and a heavy chain variable region encoding H1. L3H2 represents a humanized antibody comprising a light chain variable region encoding L3 and a heavy chain variable region encoding H2. L5H1 represents a humanized antibody comprising a light chain variable region encoding L5 and a heavy chain variable region encoding H1. L5H2 represents a humanized antibody comprising a light chain variable region encoding L5 and a heavy chain variable region encoding H2.

[0293] Table 2 Variable region sequences of Hu01

[0294]

[0295]

[0296] HCDR 2 is underlined and amino acid substitutions are in bold italics.

[0297] The CDRs of Mu01 and Hu01 were defined and numbered according to the IMGT system.

[0298] 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 Hu01 antibody, wherein the heavy chain constant region and the light chain constant region are shown as SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

[0299] Example 5: Preparation of monoclonal antibodies Mu02 and Hu02

[0300] Balb / c mice (8-12 weeks old) were immunized with human CD142 protein antigen extracellular region fragment (purchased from ACROBiosystems, product number TF3-H52H5), and their serum titers were monitored to determine the number of immunizations. After the initial immunization, mice were boosted three or four times, and serum was collected for titer detection. Mice with qualified titers were boosted once, and the whole spleen and 1 / 2 lymph node were harvested and fused with myeloma SP2 / 0 cell line for PEG fusion. The fused cells were cultured in a plate. Supernatant was collected and antigens were screened by ELISA. Positive cells were transferred to 96-well plates for further cultivation. After 7 days, supernatant was collected, and the reaction with antigen was determined using ELISA. The antigen binding affinity of positive cells was further tested at different dilutions. 20 parent clones with the highest immunogenic affinity entered subclones. Monoclonal hybridoma cells were obtained by limiting dilution and ELISA screening. Hybridoma cells were injected into the abdomen of mice to prepare ascites. After collection and purification, a monoclonal antibody named Mu02 was obtained. The amino acid sequence of Mu02 and its variable regions and CDRs were determined as shown in Table 3. CDRs are underlined.

[0301] Table 3 Hybridoma sequences

[0302]

[0303]

[0304] The DNA sequence encoding VH is shown in SEQ ID NO:37, and the DNA sequence encoding VL is shown in SEQ ID NO:38.

[0305] As shown in Table 4, humanized 02 variable regions (referred to as "Hu02") were obtained by modifying the VH and VL of the hybridoma sequences. H1, H2, H3, H4, H5 and H6 represent the sequence codes of VH, respectively. L1 and L2 represent the sequence codes of 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 represents a humanized antibody comprising a light chain variable region encoding L1 and a heavy chain variable region encoding H6. L2H1 represents a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H1. L2H2 represents a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H2. L2H3 represents a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H3. L2H4 represents a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H4. L2H5 represents a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H5. L2H6 represents a humanized antibody comprising a light chain variable region encoding L2 and a heavy chain variable region encoding H6.

[0306] Table 4 Variable region of Hu02

[0307]

[0308]

[0309] HCDR 2 is underlined and amino acid substitutions are in bold italics.

[0310] The CDRs of Mu02 and Hu02 are defined and numbered according to the IMGT system.

[0311] For the purpose of preparation and / or detection in the examples, the heavy chain constant region sequence and the light chain constant region sequence of the Hu02 antibody are the same as the heavy chain constant region sequence and the light chain constant region sequence of the humanized antibody in Example 4.

[0312] Example 6: Detection method of antibody drug conjugate

[0313] According to the following method, the antibody drug conjugate is concentrated, the culture medium is exchanged, and the antibody drug conjugate is purified, the concentration of the antibody is measured, and the average number of drug molecules carried by each antibody is calculated to identify the antibody drug conjugate.

[0314] Procedure A: Concentration of Antibody or Antibody Drug Conjugate

[0315] Take out an ultrafiltration tube (Amicon Ultra, 50000 MWCO, Millipore Corporation), add the antibody or antibody drug conjugate solution to be concentrated, centrifuge the ultrafiltration tube until the antibody or antibody drug conjugate solution therein reaches the desired volume, and then take it out.

[0316] Procedure B: Measurement of antibody concentration

[0317] The absorbance of the antibody was measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific) according to the method defined by the manufacturer. The concentration of the antibody is the ratio of the absorbance value of the antibody at the detection wavelength to the absorption coefficient.

[0318] Procedure C: Medium exchange for antibodies

[0319] According to the instructions provided by the manufacturer (Thermo Fisher Scientific), Zeba centrifugal desalting columns (5 mL, 40K MWCO) were pre-equilibrated with phosphate buffered saline (referred to as "PBS7.0 / EDTA", 50 mM, pH 7.0) containing sodium chloride (50 mM) and EDTA (2 mM). 2 mL of sample was loaded onto each Zeba centrifugal desalting column and centrifuged (1000 g, 4 minutes). Then, the flow-through fractions were collected and concentrated by operation A, the antibody concentration was determined by operation B, and the antibody concentration was adjusted with PBS7.0 / EDTA.

[0320] Procedure D: Purification of Antibody Drug Conjugates

[0321] According to the specification provided by manufacturer (Thermo Fisher Scientific), Zeba centrifugal desalting column (5mL, 40K MWCO) is pre-balanced with storage buffer. Histidine-acetate buffer (20mM histidine, pH 5.5) containing 150mM NaCl or phosphate buffer (50mM, pH 7.0) containing 50mM NaCl is used as storage buffer. The reaction solution (about 2mL) containing antibody drug conjugate is applied to Zeba centrifugal desalting column (5mL), and centrifuged (1000g, 4 minutes). Then, flow through component (about 2mL) is collected, and elution process is repeated twice to remove uncombined joint-payload and low molecular weight compounds including reducing agent.

[0322] Procedure E: Measurement of antibody concentration in antibody-drug conjugates and the average number of drug molecules attached to each antibody (DAR value) - (1)

[0323] The concentration of the drug coupled to the antibody-drug conjugate can be obtained by measuring the UV absorption value of the antibody-drug conjugate aqueous solution at 280 nm and 370 nm and calculating by the following formula.

[0324] At any given wavelength, the total absorbance of a 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 antibody and drug conjugation, the concentrations of the antibody and drug in the antibody-drug conjugate can be expressed by the following formula.

[0325] A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Formula (1)

[0326] A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Formula (2)

[0327] A 280 A represents the total absorbance of the antibody-drug conjugate aqueous solution at 280 nm. 370 A represents the total absorbance of the antibody-drug conjugate aqueous solution at 370 nm. A,280 A represents the absorbance of the antibody at 280 nm. A,370 A represents the absorbance of the antibody at 370 nm. D,280 Indicates the absorbance value of drug molecules at 280nm, A D,370 represents the absorbance value of drug molecules at 370nm, ε A,280 represents the molar extinction coefficient of the antibody at 280 nm, ε A,370 represents the molar extinction coefficient of the antibody at 370 nm, ε D,280 represents the molar extinction coefficient of the drug molecule at 280nm, ε D,370 represents the molar extinction coefficient of the drug molecule at 370nm, C A represents the antibody concentration in the antibody drug conjugate, and C D Represents the concentration of drug molecules in antibody drug conjugates.

[0328] In this case, ε A,280 , ε A,370 , ε D,280 and ε D,370are all known values ​​(calculated from the antibody sequence or measured by UV absorption of the compound). For example, ε A,280 It can be calculated from the amino acid sequence of the antibody using a known method (Protein Science, 1995, Vol. 4, pp. 2411-2423). Antibodies generally have no absorbance at 370 nm, so ε A,370 Usually 0. ε D,280 and ε D,370 The value of C can be calculated by measuring the absorbance of drug molecules at 280 nm and 370 nm as a function of concentration, respectively, and using the Lambert-Beer law (absorbance = molar concentration × molar extinction coefficient × optical path length). A and C D The absorbance of the antibody-drug conjugate at 280 nm and 370 nm can be measured. 280 and A 370 , and then solve the simultaneous equations (1) and (2) to obtain. In addition, the average number of drug molecules attached to each antibody (DAR value) can be obtained by using C D Divide by C A get.

[0329] Operation F: Average number of drug molecules attached to each antibody (DAR value) - (2)

[0330] In addition to the above-mentioned "Operation E", the average number of drug molecules conjugated to each antibody molecule in the antibody drug conjugate can be determined using the hydrophobic interaction chromatography (HIC) analysis method described below.

[0331] The antibody drug conjugate is eluted on the hydrophobic interaction chromatography column based on the difference in salt ion concentration in the eluent. As the salt ion concentration decreases, the number of small molecule drugs in the eluted antibody drug conjugate increases, that is, the antibody drug conjugate with a low DAR value is preferentially eluted. The peak order of each component is D0 (antibody not coupled to any linker-payload), D2 (antibody coupled to an average of about 2 linkers-payloads), D4 (antibody coupled to an average of about 4 linkers-payloads), D6 (antibody coupled to an average of about 6 linkers-payloads), D8 (antibody coupled to an average of about 8 linkers-payloads). The content percentage of each component can be obtained by measuring the peak area ratio of each peak. The HIC-DAR of the corresponding sample is then calculated as follows:

[0332] Average number of coupled 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

[0333] Procedure G: Measurement of Aggregates in Antibody Drug Conjugates

[0334] Aggregates in antibody drug conjugates were detected using size exclusion chromatography in HPLC. The method is as follows.

[0335] High performance liquid chromatography system: Agilent 1260 Infinity II HPLC system

[0336] Detector: UV absorption spectrometer (detection wavelength: 280nm)

[0337] Column type: TOSOH TSKgel G3000SWXL (7.8×300mm, 5μm)

[0338] Mobile phase: 200mmol / L KHPO 4 , 150mmol / L NaCl, 15% (v / v) isopropanol, pH 7.0

[0339] Flow rate: 0.75mL / min

[0340] Analysis time: 18 minutes

[0341] Column temperature: room temperature

[0342] Injection volume: 50 μg

[0343] Data Analysis: Matter

[0344] The size exclusion chromatogram of the quality control material (QC, Hu01-L3H1 naked antibody, i.e., L3H1 encoded by the humanized antibody derived from Mu01 without linker-payload conjugation) is shown in Figure 1 A; the size exclusion chromatogram of the quality control material (QC, Hu02-L1H2 naked antibody, i.e., the antibody-encoded L1H2 derived from antibody Mu02 is not coupled to the linker-payload) is shown in Figure 2 A. The retention time of the main peak (single peak) of the 150 kDa mass control substance is 9.5 minutes to 10.5 minutes. The retention time of the aggregate should be earlier than the retention time of the above monomer.

[0345] Procedure H: Comparison of the hydrophobicity of antibody drug conjugates

[0346] The hydrophobicity of antibody drug conjugates was analyzed using HPLC hydrophobic interaction chromatography (HIC). The method is as follows:

[0347] High performance liquid chromatography system: Agilent 1260 Infinity II HPLC system

[0348] Detector: UV absorption spectrometer (detection wavelength: 280nm)

[0349] Column type: TOSOH TSKgel Butyl-NPR (4.6 mm inner diameter × 3.5 cm, 2.5 μm)

[0350] Mobile phase A: mol / L (NH 4 ) 2 SO 4 , 50mmol / L KHPO 4 , pH 7.0

[0351] Mobile phase B: 50mmol / L KHPO 4 , 25% (v / v) isopropanol, pH 7.0

[0352] Analysis time: 25 minutes

[0353] Column temperature: room temperature

[0354] Elution steps (B%): 0%-25% (0 min-1 min), 25% (1 min-3 min), 25%-80% (3 min-13 min), 80% (13 min-17 min), 80%-0% (17 min-17.10 min), 0% (17.10 min-25 min)

[0355] Injection volume: 10 μL

[0356] Data analysis: The hydrophobic interaction chromatogram of the quality control material (QC, Hu01-L3H1 naked antibody) is shown in Figure 1 B. The hydrophobic chromatogram of the quality control material (QC, Hu02-L1H2 naked antibody) is shown in Figure 2 In B. Samples with shorter retention times are less hydrophobic. The antibody drug conjugate is more hydrophobic than the unconjugated naked antibody, so its retention time is longer.

[0357] Example 7: Preparation of Antibody Drug Conjugate Hu01-L3H1-LP1-DAR8

[0358] Antibody reduction: by operation B in Example 6 (the extinction coefficient of the antibody at 280 nm is 1.658 mL mg - 1 cm -1 ) and operation C, the medium of Hu01-L3H1 antibody was exchanged with PBS7.0 / EDTA, and the antibody concentration after the medium exchange was 7.353 mg / mL. 19.51 μL of 5mM TCEP solution (equivalent to 7 times the antibody content) was added to 284.24 μL of Hu01-L3H1 antibody aqueous solution and 76 μL of 50mM phosphate buffer (PBS7.0), and 0.26 μL of ultrapure water was added thereto at the same time. The mixed solution was placed in an environment of 37°C for reaction for 2 hours.

[0359] Coupling of antibody and 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 (equivalent to 15 times 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.

[0360] Purification of antibody drug conjugate: The reaction solution was purified by the method of operation D in Example 6 to obtain antibody drug conjugate Hu01-L3H1-LP1-DAR8.

[0361] Characterization of Antibody Drug Conjugates: Using the procedure E (ε D,280 =6384 and ε D,370 =16180), operation F, operation G and operation H to characterize the obtained antibody drug conjugate.

[0362] The concentration of the antibody drug conjugate measured and calculated by Procedure E was 4.93 mg / mL, and the average number of payloads per antibody conjugate measured and calculated by Procedure E was 7.67. Figure 3 A shows a detection diagram of aggregates, and the content of aggregates in the antibody drug conjugate Hu01-L3H1-LP1-DAR8 measured by operation G was 1.33%. Figure 3 B shows a detection chart of the hydrophobic interaction chromatography of the antibody drug conjugate Hu01-L3H1-LP1-DAR8, and the retention time of the antibody drug conjugate measured by operation H is 7.199 minutes.

[0363] Example 8: Preparation of Antibody Drug Conjugate Hu01-L3H1-LP1-DAR4

[0364] Antibody reduction: by operation B in Example 6 (the extinction coefficient of the antibody at 280 nm is 1.658 mL mg - 1 cm -1 ) and operation C, the medium of Hu01-L3H1 antibody was exchanged with PBS7.0 / EDTA, and the antibody concentration after the medium exchange was 22.51 mg / mL. 40 μL of 5mM TCEP solution (equivalent to 2.5 times the antibody content) was added to 533.10 μL of Hu01-L3H1 antibody aqueous solution, and 320 μL of 50mM phosphate buffer (PBS7.0) and 706.90 μL of ultrapure water were added thereto at the same time. The mixed solution was placed in an environment of 37°C for reaction for 2 hours.

[0365] Coupling of antibody and 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 then 64 μL (equivalent to 8 times 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.

[0366] Purification of antibody drug conjugate: The reaction solution was purified by the method of operation D in Example 6 to obtain antibody drug conjugate Hu01-L3H1-LP1-DAR4.

[0367] Characterization of Antibody Drug Conjugates: Using the procedure E (ε D,280 =6384 and ε D,370 =16180), operation F, operation G and operation H to characterize the obtained antibody drug conjugate.

[0368] The concentration of the antibody drug conjugate measured and calculated by Operation E was 7.19 mg / mL, and the average number of payloads per antibody conjugate measured and calculated by Operation E was 4.66. Figure 4 A shows a detection diagram of aggregates, and the content of aggregates in the antibody drug conjugate Hu01-L3H1-LP1-DAR4 measured by operation G was 1.16%. Figure 4 B shows the detection graph of the hydrophobic interaction chromatography of the retention time distribution of the antibody drug conjugate measured by operation H.

[0369] Example 9: Preparation of Antibody Drug Conjugate Hu02-L1H2-LP1-DAR8

[0370] Antibody reduction: by operation B in Example 6 (the extinction coefficient of the antibody at 280 nm is 1.382 mL mg - 1 cm -1 ) and operation C, the medium of Hu02-L1H2 antibody was exchanged with PBS7.0 / EDTA, and the antibody concentration after the medium exchange was 11.98 mg / mL. 106.67 μL of 5mM TCEP solution (equivalent to 10 times the antibody content) was added to 667.78 μL of Hu02-L1H2 antibody aqueous solution, and 320 μL of 50mM phosphate buffer (PBS7.0) and 505.50 μL of ultrapure water were added thereto at the same time. The mixed solution was placed in an environment of 37°C for reaction for 2 hours.

[0371] Coupling of antibody and 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 then 80 μL (equivalent to 15 times 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.

[0372] Purification of antibody drug conjugate: The reaction solution was purified by the method of operation D in Example 6 to obtain antibody drug conjugate Hu02-L1H2-LP1-DAR8.

[0373] Characterization of Antibody Drug Conjugates: Using the procedure E (ε D,280 =6384 and ε D,370 =16180), operation F, operation G and operation H to characterize the obtained antibody drug conjugate.

[0374] The concentration of the antibody drug conjugate measured and calculated by Procedure E was 5.03 mg / mL, and the average number of payloads per antibody conjugate measured and calculated by Procedure E was 7.83. Figure 5 A shows a detection diagram of aggregates, and the content of aggregates in the antibody drug conjugate Hu02-L1H2-LP1-DAR8 measured by operation G was 1.31%. Figure 5 B shows a detection graph of the measured hydrophobic interaction chromatogram of the antibody drug conjugate, and the retention time of the antibody drug conjugate measured by operation H is 6.768 minutes.

[0375] Comparative Example 1: Preparation of Antibody Drug Conjugate HuIgG-LP1-DAR8

[0376] Antibody reduction: by operation B in Example 6 (the extinction coefficient of the antibody at 280 nm is 1.35 mL mg - 1 cm -1 ) and operation C, the culture medium of human IgG antibody is exchanged with PBS7.0 / EDTA, and the antibody concentration after the culture medium exchange is 10mg / mL. 0.267mL 5mM TCEP solution (equivalent to 10 times the equivalent of the antibody content) is added to 1.33mL human IgG protein aqueous solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number SP001), and 0.4mL 50mM PBS7.0 is added thereto. After confirming that the pH of the solution is 7.0±0.1, the mixture is placed in an environment of 37°C for reaction for 2 hours.

[0377] Coupling of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload LP-1 was dissolved in DMA, and then 0.2 mL (equivalent to 15 times 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.

[0378] Purification of antibody drug conjugate: The reaction solution was purified by the method of operation D in Example 6 to obtain antibody drug conjugate HuIgG-LP1-DAR8.

[0379] Characterization of Antibody Drug Conjugates: Using the procedure E (ε D,280 =6384 and ε D,370 =16180), operation G and operation H to characterize the resulting antibody drug conjugate.

[0380] The concentration of the antibody drug conjugate measured and calculated by Operation E was 11.08 mg / mL, and the average number of payloads per antibody conjugate measured and calculated by Operation E was 9.15. Figure 6 A shows a detection diagram of aggregates, and the content of aggregates in the antibody drug conjugate HuIgG-LP1-DAR8 measured by operation G was 4.38%. Figure 6 B shows a detection diagram of the hydrophobic interaction chromatography of the antibody drug conjugate HuIgG-LP1-DAR8, and the retention time of the antibody drug conjugate human IgG-LP1 measured by operation H is 6.213 minutes.

[0381] Comparative Example 2: Preparation of Antibody Drug Conjugate Tisomab-MMAE (Referred to as: Reference ADC)

[0382] The sequence of the antibody tesomab (referred to as: reference antibody) is recorded in PCT / EP2009 / 066755 (VH: SEQ ID NO: 9; VL: SEQ ID NO: 65), wherein the antibody is named TF-11.

[0383] Antibody reduction: by operation B in Example 6 (the extinction coefficient of the antibody at 280 nm is 1.508 mL mg - 1 cm -1 ) and operation C, the medium of the antibody was exchanged with PBS7.0 / EDTA, and the antibody concentration after the medium exchange was 7.318 mg / mL. 23.21 μL of 5mM TCEP solution (equivalent to 2.04 times the antibody content) was added to 1127.36 μL of reference antibody aqueous solution, and 300 μL of 50mM PBS7.0 and 49.43 μL of ultrapure water were added thereto. The mixture was placed in an environment of 37°C for reaction for 2 hours.

[0384] Coupling of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload vc-MMAE (DC Chemicals, DC50025) was dissolved in DMA, and then 79.66 μL (equivalent to 7 times 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.

[0385] Purification of antibody drug conjugate: The reaction solution was purified by the method of operation D in Example 6 to obtain an antibody drug conjugate reference ADC.

[0386] Characterization of antibody drug conjugates: The obtained antibody drug conjugates were characterized using Procedures B, F, G and H in Example 6.

[0387] The concentration of the antibody drug conjugate measured and calculated by Run B was 5.01 mg / mL. The average number of payloads per antibody conjugate in the reference ADC measured and calculated by Runs F and G was 3.73. Figure 7 A shows a detection graph of aggregates, and the content of aggregates in the reference ADC measured by operation G was 0.92%. Figure 7 B shows the detection profile of the hydrophobic interaction chromatogram of the reference ADC measured by operation H. The HNSTD (maximum dose without severe toxicity) of the reference ADC in cynomolgus monkeys was 3 mg / kg.

[0388] Experimental Example 1: Determination of the affinity of Mu01 and Mu02 for BXPC3 cells

[0389] BxPC3 cells (human pancreatic cancer cells, obtained from the cell bank of the Chinese Academy of Sciences) were digested with trypsin (purchased from Gibco, product number 25200072) in a culture dish. The digested cells were centrifuged at 1000 rpm for 5 minutes and blocked with 10% goat serum (purchased from Nanjing Senbeijia Biotechnology Co., Ltd., product number SRJ-SE-GO0012) for 30 minutes. Then, the Mu01 and Mu02 antibodies prepared in Example 4 and Example 5 were added at a final concentration of 20 μg / mL and incubated for 1 hour. After washing twice with phosphate buffered saline (PBS, PH 7.2-7.4), FITC-labeled goat anti-mouse antibody (purchased from Jackson ImmunoResearch, number 115-545-003) was added at 1:500 and incubated for 1 hour. After washing twice with PBS, the resulting product was analyzed by flow cytometry.

[0390] like Figure 8 As shown, both Mu01 and Mu02 showed good affinity for CD142-positive BXPC3 cells.

[0391] Experimental Example 2: In vitro killing assay of MDA-MB-231 cells by Mu01 and Mu02

[0392] MDA-MB-231 (human breast cancer, from Zhejiang Meisen Cell Technology Co., Ltd.) cells were cultured to a cell density of 80%. The cells were collected and added to a 96-well plate, and the cell density was adjusted to 2-5×10 4 / mL. 100μl was added to each well. Mu01 prepared in Example 4, Mu02 prepared in Example 5 or negative control IgG (mouse anti-IgG, purchased from Abimate Pharmaceutical Technology (Shanghai) Co., Ltd., product number M070890) was diluted 3 times with an initial concentration of 30nM and then added to the cell culture medium. At the same time, 2μg / mL of goat anti-mouse antibody coupled to MMAE (goat anti-mouse antibody purchased from Jackson, catalog number 115-545-003) was added. The cells were cultured for 3 days. Cell survival was observed regularly during this period. After 3 days, 15μL of CCK-8 kit stock solution (purchased from Yacoin Biotechnology Co., Ltd., catalog number ATUOC1303) was directly added to a 96-well plate. After reacting in an incubator at 37°C for 0.5 to 2 hours, the absorbance at 450nm was detected using an enzyme reader, and a cell survival curve was drawn based on the absorbance value and the antibody dilution gradient.

[0393] The method of coupling goat anti-mouse antibody with MMAE was similar to that in Comparative Example 2.

[0394] like Fig. 9 As shown, in the human breast cancer cell line MDA-MB-231, the cell survival rate continued to decrease with the increase of Mu01 and Mu02 concentrations, indicating that antibodies Mu01 and Mu02 have a significant killing effect on tumor cells.

[0395] Experimental Example 3: Determination of affinity of Hu01 candidate molecule for BXPC3 cells

[0396] BxPC3 cells (human pancreatic cancer cells, obtained from the cell bank of the Chinese Academy of Sciences) were digested with trypsin (purchased from Gibco, product number 25200072) in a culture dish. The digested cells were centrifuged at 1000rpm for 5 minutes and blocked with 10% goat serum (purchased from Nanjing Senbeijia Biotechnology Co., Ltd., product number SRJ-SE-GO0012) for 30 minutes. Then the Hu01 antibodies L5H2, L5H1, L3H1, and L3H2 prepared in Example 4 were diluted with a 3-fold gradient at an initial concentration of 500nM. Afterwards, the Hu01 antibody was 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. The results were analyzed by flow cytometry after washing twice with PBS.

[0397] The Ch01 antibody is a chimeric antibody whose variable region is identical to that of Mu01 prepared in Example 4 and whose constant region is identical to that of human IgG1 described in Example 4.

[0398] like Fig.10 As shown, the four candidate molecules of Hu01 antibody all showed good affinity to BXPC3 cells, and L3H1 showed the highest affinity.

[0399] Experimental Example 4: Determination of affinity of Hu02 candidate molecule for BXPC3 cells

[0400] BxPC3 cells (human pancreatic cancer cells, obtained from the Chinese Academy of Sciences Cell Bank) were digested with trypsin (purchased from Gibco, product number 25200072) in a culture dish. The digested cells were centrifuged at 1000rpm for 5 minutes and blocked with 10% goat serum (purchased from Nanjing Senbeijia Biotechnology Co., Ltd., product number SRJ-SE-GO0012) for 30 minutes. Then the Hu02 antibodies L1H1, L1H2, L1H3, L1H4, L1H5, L1H6, L2H1, L2H2, L2H3, L2H5 and L2H6 prepared in Example 5 were diluted 3 times with an initial concentration of 500nM. Afterwards, the Hu02 antibody was 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. The results were analyzed by flow cytometry (BD Biosciences, product number BD Accuri C6Plus) after washing twice with PBS.

[0401] The Ch02 antibody is a chimeric antibody whose variable region is identical to that of Mu02 prepared in Example 5 and whose constant region is identical to that of human IgG1 described in Example 4.

[0402] like Fig.11 As shown in Table 5, the 11 candidate molecules of Hu02 antibody all showed good affinity to BXPC3 cells, among which L2H6 showed the highest affinity, and L1H2 showed good affinity.

[0403] Table 5 Affinity determination of Hu02 antibody for BXPC3 cells

[0404]

[0405] Experimental Example 5: Effects of Hu01-L3H1, Hu02-L1H2 and Tesomab on blood coagulation

[0406] The effects of Hu01-L3H1, Hu02-L1H2 and a reference antibody (tesozumab) on coagulation were examined by thrombin generation assay (TGA).

[0407] 30 μL 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 the diluted concentration of 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 mixed solution was added to the reaction cup, and then the tissue factor-dependent thrombin formation reaction was initiated by adding 30 μl FluCa reagent (containing calcium and thrombin substrate, purchased from Stago, product number TS50.00). TC TechnoClone instrument was used to detect and automatically derive parameters.

[0408] like Fig.12 As shown, the inhibition rate of Hu01-L3H1 and Hu02-L1H2 on the thrombin peak was lower than that of the reference antibody on the thrombin peak. The results showed that Hu01-L3H1 and Hu02-L1H2 had less effect on coagulation function and lower coagulopathy than the reference antibody in vitro.

[0409] Experimental Example 6: In vitro killing assay of ADC Hu01-L3H1-LP1-DAR8, Hu01-L3H1-LP1-DAR4 and Hu02-L1H2-LP1-DAR8 against KYSE150, 5637, SW780 and Detroit562 cells

[0410] KYSE150 (human esophageal cancer cells, obtained from the Cell Bank of the Chinese Academy of Sciences), 5637 (human bladder cancer cells, obtained from the Cell Bank of the Chinese Academy of Sciences), SW780 (human bladder cancer, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) and Detroit 562 (human pancreatic cancer cells, obtained from the Cell Bank of the Chinese Academy of Sciences) were cultured to a cell density of 80%. The cells were collected and seeded in 96-well plates, and the cell density was adjusted to 2-5×10 4 / ml. 100 μL of cells were inoculated in each well. The ADC molecule was diluted 3 times with an initial concentration of 300 nM, then added to the cell culture medium and incubated for 5 days. Cell apoptosis was observed regularly during this period. After 5 days, 15 μL of CCK-8 kit stock solution (purchased from Yacoin Biotechnology Co., Ltd., product number ATUOC1303) was directly added to the 96-well plate and reacted in an incubator at 37 ° C for 0.5 hours to 2 hours. The absorbance at 450 nm was detected with an enzyme reader, and the cell survival curve was drawn based on the absorbance value and the antibody dilution gradient.

[0411] 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 isotype control HuIgG-LP1-DAR8 prepared in Comparative Example 1 were selected as ADC molecules.

[0412] Fig.13 , Fig.14 and Fig.15 The cell survival curves of Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 on KYSE150, 5637 and SW780 cells are shown respectively. The results show that Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 have obvious cytotoxic effects on these cell lines in vitro, and Hu01-L3H1-LP1-DAR8 has better cytotoxic effects.

[0413] Fig.16 The cell survival curves of Hu01-L3H1-LP1-DAR8, Hu01-L3H1-LP1-DAR4 and Hu02-L1H2-LP-DAR8 on Detroit 562 cells in vitro are shown. The results show that the cytotoxicity of ADC is very obvious in vitro, and the cytotoxicity is Hu01-L3H1-LP1-DAR8>Hu01-L3H1-LP1-DAR4>Hu02-L1H2-LP1-DAR8 from high to low.

[0414] Experimental Example 7: Tumor Inhibitory Effects of Hu01-L3H1-LP1-DAR8, Hu02-L1H2-LP1-DAR8 and Reference ADC in the CDX Mouse Model of NCI-H292

[0415] Six-week-old Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. 7 NCI-H292 (human lung cancer cells, obtained from the Cell Bank of the Chinese Academy of Sciences) cells were subcutaneously inoculated into each mouse to construct a CDX (cell-derived xenograft) mouse model. When the average tumor volume was about 100 mm 3 At 2.5 mpk (mg / kg), 5 mpk, and 10 mpk of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 10 mpk of Hu02-L1H2-LP1-DAR8 prepared in Example 9, 3 mpk of reference ADC prepared in Comparative Example 2, and 10 mpk of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were intravenously injected, respectively. After administration, the tumor volume was measured twice a week with a vernier caliper and calculated according to the following formula: TV = (length × width) 2 / 2.

[0416] Fig.17 The in vivo tumor inhibition effects of Hu01-L3H1-LP1-DAR8, Hu02-L1H2-LP1-DAR8 and the reference ADC in the CDX mouse model of NCI-H292 are shown. It shows that Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 have similar tumor inhibition efficacy as the reference ADC. In addition, according to the tumor inhibition curves of different doses, the inhibitory effect of Hu01-L3H1-LP1-DAR8 on tumors is dose-dependent.

[0417] Fig.18 The effects of Hu01-L3H1-LP1-DAR8, Hu02-L1H2-LP1-DAR8 and reference ADC on mouse body weight are shown. The results show that the above ADC molecules have little effect on mouse body weight, indicating that no obvious digestive system toxicity will be caused at this dose.

[0418] Experimental Example 8: Tumor Inhibitory Effects of Hu01-L3H1-LP1-DAR8 and Reference ADC in the CDX Model of NCI-H226

[0419] 6-week-old Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. 6NCI-H226 (human lung cancer cells, obtained from the Cell Bank of the Chinese Academy of Sciences) cells were subcutaneously inoculated into each mouse to construct the CDX mouse model. 3 At 3 pm, 5 mg / kg and 10 mg / kg of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 3 mg / kg of reference ADC prepared in Comparative Example 2, and 10 mg / kg of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were intravenously injected, respectively. The tumor volume was measured and calculated according to the method in Experimental Example 7.

[0420] Fig.19 The in vivo tumor inhibition effect of Hu01-L3H1-LP1-DAR8 and the reference ADC on the CDX mouse model of NCI-H226 is shown. The results show that the tumor inhibition effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg is better than that of the reference ADC at a dose of 3 mg / kg. The tumor inhibition effect of Hu01-L3H1-LP1-DAR8 at a dose of 5 mg / kg is similar to that of the reference ADC at a dose of 3 mg / kg.

[0421] Experimental Example 9: Tumor inhibitory effects of Hu01-L3H1-LP1-DAR8 and reference ADC on mouse models constructed with IGROV1 cells

[0422] Six-week-old Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. 7 IGROV1 cells (human ovarian cancer, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) were subcutaneously inoculated into each mouse to construct the CDX mouse model. When the average tumor volume was about 100 mm 3 At 10 mpk of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 3 mpk of reference ADC prepared in Comparative Example 2, and 10 mpk of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were intravenously injected. The tumor volume was measured and calculated according to the method in Experimental Example 7.

[0423] Fig. 20 The in vivo tumor inhibition effect of Hu01-L3H1-LP1-DAR8 and the reference ADC on the CDX mouse model constructed with IGROV1 cells is shown. The results show that the tumor inhibition effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg is better than that of the reference ADC at a dose of 3 mg / kg.

[0424] Experimental Example 10: Tumor inhibitory effects of Hu01-L3H1-LP1-DAR8 and reference ADC on a mouse model constructed with SW780 cells

[0425] 6-week-old Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. 6 SW780 cells (human bladder cancer, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) were subcutaneously inoculated into each mouse to construct the CDX mouse model. When the average tumor volume was about 100 mm 3 At 10 mg / kg of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 3 mg / kg of reference ADC prepared in Comparative Example 2, and 10 mg / kg of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were intravenously injected. The tumor volume was measured and calculated according to the method in Experimental Example 7.

[0426] Fig.21 The in vivo tumor inhibition effect of Hu01-L3H1-LP1-DAR8 and the reference ADC on the CDX mouse model constructed with SW780 cells is shown. The results show that the tumor inhibition effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg is better than that of the reference ADC at a dose of 3 mg / kg.

[0427] Experimental Example 11: Tumor inhibitory effect of Hu01-L3H1-LP1-DAR8 on the PDX1 mouse model constructed with human lung cancer tissue

[0428] 6-week-old NU / NU female nude mice weighing 18 g to 21 g were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Each mouse was subcutaneously inoculated with human lung cancer tissue (obtained from Shanghai Lidi Biotechnology Co., Ltd.) to construct a patient-derived tumor xenograft model (PDX). When the average tumor volume was about 160 mm 3 At 10 mg / kg of Hu01-L3H1-LP1-DAR8 prepared in Example 7 and PBS were intravenously injected. The tumor volume was measured and calculated according to the method in Experimental Example 7.

[0429] Fig. 22 The in vivo tumor inhibition effect of Hu01-L3H1-LP1-DAR8 and PBS on the PDX1 mouse model is shown. The results show that Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg has a significant tumor inhibition effect compared with PBS. In addition, on day 22, tumors in 5 mice were completely eliminated, indicating that Hu01-L3H1-LP1-DAR8 has a significant therapeutic effect on tumor inhibition.

[0430] Experimental Example 12: Tumor inhibitory effect of Hu01-L3H1-LP1-DAR4 on the PDX2 mouse model constructed with human cervical cancer tissue

[0431] 6-week-old NU / NU female nude mice weighing 18 g to 21 g were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Each mouse was subcutaneously inoculated with human lung cancer tissue (obtained from Shanghai Lidi Biotechnology Co., Ltd.) to construct a PDX model. When the average tumor volume was about 160 mm 3 At 10 mg / kg of Hu01-L3H1-LP1-DAR4 prepared in Example 8, 3 mg / kg of reference ADC prepared in Comparative Example 2, and PBS were intravenously injected, respectively. The tumor volume was measured and calculated according to the method in Experimental Example 7.

[0432] Fig.23 The in vivo tumor inhibition effect of Hu01-L3H1-LP1-DAR4 and the reference ADC on the PDX2 mouse model is shown. The results show that the 10 mg / kg dose of Hu01-L3H1-LP1-DAR4 has a significant tumor inhibition effect compared with the reference ADC, indicating that Hu01-L3H1-LP1-DAR4 has a significant therapeutic effect on tumor inhibition.

[0433] Experimental Example 13: Toxicology experiment of Hu01-L3H1-LP1-DAR8 on cynomolgus monkeys

[0434] Toxicology experiments of Hu01-L3H1-LP1-DAR8 were performed by repeated administration to cynomolgus monkeys.

[0435] Six cynomolgus monkeys (purchased from Guangxi Frontier Biotechnology Co., Ltd.) were randomly divided into three groups, and each group had one male and one female. The vehicle, 10 mg / kg Hu01-L3H1-LP1-DAR8 and 30 mg / kg Hu01-L3H1-LP1-DAR8 were intravenously infused on the 1st, 22nd and 43rd days, respectively. During the experiment, any abnormality of the animals was observed, and their blood samples were collected for analysis of blood routine and blood biochemical indicators. On the 50th day, the animals were euthanized and samples were taken for pathological analysis. As shown in Table 6, in this experiment, all doses of the test substances did not cause animal death. The target organs associated with the test substance are bone marrow, intestine, thymus and spleen. The HNSTD (maximum dose without severe toxicity) of Hu01-L3H1-LP1-DAR8 is 30 mg / kg, indicating that Hu01-L3H1-LP1-DAR8 is safe.

[0436] Table 6 Toxicology test results in cynomolgus monkeys

[0437]

[0438]

[0439] ADCs with the new linkers provided herein have better safety profiles than ADCs with payload MMAE analogs, which are microtubule inhibitor toxins. Specifically, ADCs linked to VC-MMAE (e.g., vesotuzumab) have a HNSTD of 3 mpk (data from the U.S. Food and Drug Administration) and are compatible with ZymeLink TM Auristatin-linked ADCs (e.g., XB002 / ICON-2) have HNSTDs 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 and has a higher therapeutic window. At the same time, the ADCs provided herein have superior in vivo anti-tumor efficacy.

[0440] Although specific embodiments have been described, the applicant or those skilled in the art may think of substitutions, modifications, changes, improvements, and substantial equivalents that are not currently foreseeable or may not be foreseeable. Therefore, the appended claims submitted and their possible amendments are intended to include all such substitutions, modifications, changes, improvements, and substantial equivalents.

Claims

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 HCDRs 1, 2, and 3, and the VL comprises LCDRs 1, 2, and 3; wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

1. 1 GDX 2 The amino acid sequence shown in (SEQ ID NO: 2), the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 comprises the amino acid sequence shown in LTS, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 5; X 1 D or Q, X 2 It is S or A.

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

3. The isolated antibody or antigen-binding fragment thereof that binds to CD142 according to 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 shown in 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 shown in SEQ ID NO:7; (b-1) the VH having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:8, and the VL having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:15; (c-1) the VH having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:8, and the VL having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:17; (d-1) the VH having at least 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:9, and the VL having at least 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:15; and (e-1) the VH having at least 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:9, and the VL having at least 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:

17.

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

17.

5. 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 HCDRs 1, 2, and 3, and the VL comprises LCDRs 1, 2, and 3; wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR2 comprises IRNRAX 3 X 4 YTT (SEQ ID NO: 21), the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence shown in YTS, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; X 3 N or Q, X 4 It is G or A.

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

7. The isolated antibody or antigen-binding fragment thereof that binds to CD142 according to 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 to the amino acid sequence shown in SEQ ID NO:25, and the VL having at least 70%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:26; (b-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 27, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 33; (c-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:28, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:33; (d-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:29, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:33; (e-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:30, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:33; (f-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:31, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:33; (g-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:32, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:33; (h-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:27, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:34; (i-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:28, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:34; (j-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:29, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:34; (k-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:31, and the VL having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:34; and (l-2) the VH having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:32, and the VL having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:

34.

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

34. 9 . The 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. The isolated antibody or antigen-binding fragment thereof that binds to CD142 according to any one of claims 1 to 9, wherein the isolated antibody or antigen-binding fragment thereof that binds to CD142 comprises the 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. The isolated antibody or antigen-binding fragment thereof that binds to CD142 according to any one of claims 1 to 10, comprising a heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 35 and a light chain constant region comprising the amino acid sequence shown in SEQ ID NO:

36.

12. The 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 the antibody or antigen-binding fragment thereof that binds to CD142 according to any one of claims 1 to 12, wherein the nucleic acid encodes VH and / or VL.

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

38.

15. A vector, wherein the vector comprises the nucleic acid of claim 13 or 14.

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

17. An antibody drug conjugate of formula I, Ab-(LD)n(I) or its isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates or combinations 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 linked to Ab and D respectively; D is the effective load; n is an integer of 1 to 10. The antibody drug conjugate according to claim 17 , wherein said L is a cleavable linker or a non-cleavable linker.

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

20. The antibody drug conjugate of claim 19, wherein the cleavable peptide is capable of being cleaved by an enzyme.

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

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

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

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

26. The antibody drug conjugate of claim 25, wherein the self-immolative spacer is p-aminophenoxycarbonyl (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 antibody drug conjugate of any one of claims 25 to 27, wherein the spacer comprises -NH-(CH 2 ) 4 -La-Lb-Lc-, where La represents -O- or a single bond; Lb represents -CR 2 (-CR 3 )- or a single bond, where R 2 and R 3 Each independently represents C 1 -C 6 Alkyl, -(CH 2 ) a -NH 2 、-(CH 2 ) b -COOH or -(CH 2 ) c -OH, n 4 represents an integer from 0 to 6, n a 、n b and n c Each independently represents an integer from 1 to 4, but when n a When R is 0, 2 and R 3 are not the same, and Lc represents -C(=O)-.

29. The antibody drug conjugate of claim 28, wherein the spacer comprises -NH-(CH 2 ) 3 -C(=O)-, -NH-CH 2 -O-CH 2 -C(=O)- or -NH-(CH 2 ) 2 -O-CH 2 -C(=O)-.

30. The antibody drug conjugate according to any one of claims 17 to 29, wherein L comprises -L 1 -L 2 -L 3 -, L 1 represents -(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 -C(=O)-, where m 1 represents an integer from 2 to 8, m 2 represents an integer from 2 to 8, and m 3 represents an integer from 2 to 8; L2 represents an amino acid unit; and L3 represents a self-degradable spacer.

31. The antibody drug conjugate according to 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-aminophenoxycarbonyl (PABC) or p-aminobenzyl (PAB) comprises a polysarcosine (poly-N-methylglycine) residue.

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

34. The antibody drug conjugate according to claim 33, wherein R 4 Indicates -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , and n 3 Indicates an integer from 8 to 15.

35. The antibody drug conjugate according to claim 33 or 34, wherein R 4 Indicates -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , and n 3 Represents an integer from 10 to 12.

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

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

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

39. The antibody drug conjugate according to 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 of 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 via 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 agonist antibody, an antagonist antibody and fragments thereof, a fusion protein, or a derivative thereof.

43. The antibody drug conjugate of claim 42, wherein the cytotoxic agent comprises a tubulin inhibitor and / or a topoisomerase inhibitor; the tubulin inhibitor comprises auristatin or a derivative thereof, maytansine or a derivative thereof; the topoisomerase inhibitor comprises 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 the carbonyl group in the ester group of the linker is linked to the amino group of the payload.

46. ​​The antibody drug conjugate according to any one of claims 17 to 45, wherein the antibody drug conjugate is n represents a DAR value of 1 to 10 or 4 to 10.

47. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof that binds to CD142 as described in any one of claims 1 to 12, or the antibody-drug conjugate as 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, wherein the method The following steps are involved: reducing the isolated antibody or antigen-binding fragment thereof bound to CD142 so that its disulfide bonds are at least partially reduced and react with the reactive groups of the linker in the linker-payload to obtain 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 linked to Ab and D respectively; D is the effective load; n is an integer of 1 to 10.

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

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

51. The method of claim 50, wherein the cleavable peptide is capable of being cleaved by an enzyme.

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

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

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

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

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

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

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

60. The method of claim 59, wherein the spacer comprises -NH-(CH 2 ) 3 -C(=O)-, -NH-CH 2 -O-CH 2 -C(=O)- or -NH-(CH 2 ) 2 -O-CH 2 -C(=O)-.

61. The method of any one of claims 48 to 60, wherein L comprises -L 1 -L 2 -L 3 -, L 1 represents -(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 -C(=O)-, where m 1 represents an integer from 2 to 8, m 2 represents an integer from 2 to 8, and m 3 represents an integer from 2 to 8; L2 represents an amino acid unit; and L3 represents a self-degradable 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. The method of any one of claims 57 to 62, wherein the p-aminophenoxycarbonyl (PABC) or p-aminobenzyl (PAB) comprises a polysarcosine (poly-N-methylglycine) residue.

64. The method according to any one of claims 48 to 63, The following steps are involved: The antibody is reduced so that its disulfide bonds are at least partially reduced and reacted with the carbon atom at position 3 of the maleimide-N-group of the linker of formula IV in the linker-payload, wherein In the linker-payload, the carbonyl group in the ester group of the linker of formula IV is connected to the amino group of the payload; In Formula IV, R 1 and R 2 independently selected from hydrogen, methyl and isopropyl; R 3 Indicates -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or single bond, R 5 is hydrogen or benzyl, n 1 represents an integer from 0 to 2, n 2 represents an integer from 0 to 2; and R 4 represents methylamino or -(NCH 3 COCH 2 ) 3 -NCH 3 COCH 3 , and n 3 Represents an integer from 1 to 20.

65. The method according to claim 64, further comprising: include: The antibody is reacted with a reducing agent in a buffer solution containing a chelating agent, and then a solution of the linker-payload as indicated by LD is added, and the pH of the reaction solution is adjusted.

66. The method of claim 64 or 65, wherein the payload is as shown in formula III, which is connected to the carbonyl group of the ester group in the linker of formula IV through the nitrogen atom of the amino group on its cyclohexane ring, 67. A kit comprising the isolated antibody or antigen-binding fragment thereof that binds to CD142 of any one of claims 1 to 12 or the antibody drug conjugate of any one of claims 17 to 46.

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

69. The use according to claim 68, comprising use in the preparation of a therapeutic agent targeting CD142.

70. The use according to claim 68 or 69, wherein the tumor comprises a solid tumor expressing CD142.

71. The use according to any one of claims 68 to 70, wherein the tumor diseases include 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, the method comprising administering a therapeutic dose of a therapeutic agent to a subject, wherein the therapeutic agent comprises an isolated antibody or antigen-binding fragment thereof that binds to CD142 as described in any one of claims 1 to 12, an antibody-drug conjugate as described in any one of claims 17 to 46, a pharmaceutical composition as described in claim 47, an antibody-drug conjugate prepared by the method as described in any one of claims 48 to 66, or a kit as described in claim 68.

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