Antibodies, antibody drug conjugates, their preparation and use

Abstract

The present disclosure provides antibodies, antibody drug conjugates that specifically target CD142, their preparation and uses. Antibodies or antigen-binding fragments thereof that bind to CD142 are covalently linked to a cytotoxic payload through a linker. Antibodies or antigen-binding fragments thereof that bind to CD142 and antibody drug conjugates exhibit cytotoxic effects on tumor cells.

Description

Technical Field

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

[0002] The statements in this section are provided only as background information in relation to this disclosure and do not necessarily constitute prior art.

[0003] Antibody-drug conjugates (ADCs) are carrier-based chemotherapeutic drugs that selectively deliver cytotoxic drugs to tumor / cancer cells (“Antibody-Drug Conjugates: The Last Decade,” Nicolas Joubert et al., Pharmaceuticals (Basel.), September 14, 2020, Vol. 13, No. 9: p. 245). Commercially available ADCs, trastuzumab and goxatuzumab, have shown excellent efficacy in treating tumors, particularly malignant tumors. Both trastuzumab and goxatuzumab use DNA topoisomerase inhibitors (camptothecin derivatives), which are more hydrophobic than microtubule inhibitors (such as MMAE and MMAF), as cytotoxic agents. Goxatuzumab uses MCC-triazole spacer-PEG7-lysine-PABC as a linker to break down and release camptothecin SN38 (US13 / 948,732) in lysosomes. The trastuzumab developed by AstraZeneca / Daiichi Sankyo Co., Ltd. uses a cathepsin B-activated GGFG (an amino acid sequence composed of glycine-glycine-phenylalanine-glycine linked by peptide bonds) tetrapeptide as a linker and introduces a self-cleaving structure to release the eczema derivative Dxd (Yusuke Ogitani et al., Clin Cancer Res (2016), Vol. 22, No. 20: pp. 5097–5108). However, the aforementioned cytotoxic drugs MMAE, SN38, and Dxd are all substrates of P-glycoprotein (P-gp) (Front Pharmacol, 2019, Vol. 10: p. 749) and may exhibit resistance to some tumors with high P-gp expression.

[0004] Tissue factor (TF), also known as CD142, is a transmembrane glycoprotein. In its complex with its ligand FVIIa, CD142 activates protease-activated receptor 2, thereby activating an intracellular signaling pathway that tumors can utilize to promote malignant cell survival, tumor growth, angiogenesis, and metastasis. In contrast to restricted surface expression in normal tissue cells, CD142 exhibits membrane-like expression in various solid tumor cells, including pancreatic cancer, lung cancer, cervical cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, and colon cancer. CD142 has been reported to be significantly expressed in tumor cells and the tumor vascular system and is associated with poor prognosis and increased metastasis. These characteristics suggest that CD142 is a potential target for ADCs (anti-cancer drug delivery systems). To date, ADCs targeting CD142 mainly include tisotumab vedotin, ICON-2 (XB002), and MRG004A.

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

[0006] ICON-2 is currently in Phase I clinical trials in the United States, with indications under development including adenocarcinoma, bladder cancer, fallopian tube cancer, and head and neck tumors. Its related patent is PCT / US2019 / 012427. The drug's connector-payload utilizes ZymeLink (ZLA), a patented technology from ZymeWorks. Similar to vetisol tumumab, severe or significant skin toxicity was observed in non-human primate (NHP) toxicity studies of ICON-2 (Thi-SauMigone et al., "ICON-2, a TissueFactor-Targeted Antibody-Drug Conjugate for the Treatment of Solid Tumors," proposed in 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 underway 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 that target CD142. Summary of the Invention

[0009] This disclosure provides a novel isolated antibody or antigen-binding fragment thereof that binds to CD142, an antibody-drug conjugate containing the antibody or antigen-binding fragment thereof, a method for its preparation and use, with the aim of reducing or minimizing serious adverse reactions and improving antitumor activity.

[0010] In one aspect, this disclosure provides a separated 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 HCDR 1, 2 and 3, and the VL comprises LCDR 1, 2 and 3; wherein the HCDR 1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR 2 comprises the amino acid sequence shown in IYPGX1GDX2 (SEQ ID NO:2), and the HCDR 3 comprises the amino acid sequence shown in SEQ ID NO:3; and the LCDR 1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR 2 comprises the amino acid sequence shown in LTS, and the LCDR 3 comprises the amino acid sequence shown in SEQ ID NO:5; X1 is D or Q, and X2 is S or A.

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

[0012] This disclosure provides a separated 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 HCDR 1, 2 and 3, and the VL comprises LCDR 1, 2 and 3; wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the HCDR2 comprises the amino acid sequence shown in IRNRAX3X4YTT (SEQ ID NO:21), and 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; X3 is N or Q, and X4 is G or A.

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

[0014] In one aspect, this disclosure provides nucleic acids encoding antibodies or antigen-binding fragments thereof that bind to the aforementioned CD142.

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

[0016] In one aspect, this disclosure provides a host cell, wherein the cell comprises the aforementioned nucleic acid or vector.

[0017] In one aspect, this 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 refers to the isolated antibody or its antigen-binding fragment that binds to CD142;

[0021] L is a connector covalently connected to Ab and D respectively;

[0022] D represents the payload;

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

[0024] In one aspect, this disclosure provides a method for preparing an antibody-drug conjugate of Formula I or an isomer, isotope variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, the method comprising the steps of: reducing a separated antibody or antigen-binding fragment bound to CD142 such 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 includes the steps of: reducing the antibody such that its disulfide bonds are at least partially reduced, and reacting with the 3-carbon atom of the maleimide-N-group of the linker-loaded type IV linker.

[0026]

[0027] in

[0028] In this connector-load, the carbonyl group in the ester group of the formula IV connector is attached to the amino group of the load;

[0029] In Formula IV, R1 and R2 are independently selected from hydrogen, methyl, and isopropyl.

[0030] R3 represents -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is hydrogen or benzyl, n 1 n represents an integer from 0 to 2. 2 Represents integers from 0 to 2; and

[0031] R4 represents methylamino or -(NCH3COCH2)n 3 -NCH3COCH3, and n 3 Represents integers from 1 to 20.

[0032] In one aspect, this disclosure provides a pharmaceutical composition comprising a separate antibody or antigen-binding fragment thereof that binds to CD142, or an antibody-drug conjugate of formula I, or an isomer, isotope variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient.

[0033] In one aspect, this disclosure provides a kit comprising a separated antibody or antigen-binding fragment thereof that binds to CD142, or the aforementioned antibody-drug conjugate.

[0034] In one aspect, this disclosure provides the use of isolated antibodies or antigen-binding fragments thereof that bind to CD142, antibody-drug conjugates of formula I, antibody-drug conjugates of formula I prepared by the methods described herein, the above-described pharmaceutical compositions, and kits in the preparation of therapeutic agents for the diagnosis, prevention, and treatment of cancer diseases.

[0035] In some implementations, the tumor includes solid tumors that express CD142.

[0036] In one aspect, this disclosure provides a method for reducing the number of cells expressing CD142, comprising administering a therapeutic dose of a therapeutic agent to a subject, wherein the therapeutic agent comprises a segregated antibody or an 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 method herein, a pharmaceutical composition, and the aforementioned kit.

[0037] The isolated antibodies or antigen-binding fragments thereof that bind to CD142, as well as antibody-drug conjugates of Formula I, described in this article, demonstrate improved or superior in vivo efficacy and safety, and HNSTD (not observed at the maximum dose with severe toxicity) can be achieved at 30 mg / kg. Furthermore, the isolated antibodies or antigen-binding fragments thereof that bind to CD142, as well as antibody-drug conjugates of Formula I, described in this article, have a weak effect on coagulation function, thus avoiding potential bleeding-related adverse reactions. Attached Figure Description

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

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

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

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

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

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

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

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

[0046] Figure 8 The flow cytometry diagrams show the endocytosis of BxPC3 cells with antibodies Mu01 prepared in Example 4 and Mu02 prepared in Example 5. MFI is an abbreviation for mean fluorescence intensity.

[0047] Figure 9 The cell viability-concentration curves of MDA-MB-231 cells against antibodies Mu01 prepared in Example 4 and Mu02 prepared in Example 5 are shown.

[0048] Figure 10 The flow cytometry affinity curves of BxPC3 cells for humanized antibody candidates L5H2, L5H1, L3H1, L3H2 and chimeric antibody Ch01 derived from Mu01 are shown.

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

[0050] Figure 12 The curves showing the changes in human plasma thrombin peaks under the influence of antibodies Hu01-L3H1 prepared in Example 4 and Hu02-L1H2 prepared in Example 5 are shown.

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

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

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

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

[0055] Figure 17 The in vivo efficacy of ADC in a cell-derived xenograft mouse model of NCI-H292 is demonstrated.

[0056] Figure 18 The body weight-time curves after treatment are shown in the NCI-H292 cell-derived xenograft mouse model.

[0057] Figure 19 The in vivo efficacy of ADC in a cell-derived xenograft mouse model of NCI-H226 is demonstrated.

[0058] Figure 20 The in vivo efficacy of ADC in a cell-derived xenograft mouse model of IGROV1 is demonstrated.

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

[0060] Figure 22 The in vivo efficacy of ADC in a mouse model of patient-derived tumor xenograft constructed from human lung cancer tissue is demonstrated.

[0061] Figure 23 The in vivo efficacy of ADC in a mouse model of patient-derived tumor xenograft constructed from human cervical cancer tissue is demonstrated. Detailed Implementation

[0062] This disclosure will now be explained in more detail. This specification is not intended to be a detailed list of all the different ways in which the invention may be practiced or all the features that may be added to the invention. For example, a feature shown with respect to one embodiment may be incorporated into other embodiments, and a feature shown with respect to a particular embodiment may be removed from that embodiment. Furthermore, based on this disclosure, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the 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 one of ordinary skill in the art to which this disclosure pertains. While preferred materials and methods are described herein, any methods and materials similar to or equivalent to those described and used herein may be used to test the practice of this disclosure. The following terms will be used in describing and claiming protection for this disclosure.

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

[0065] Although the numerical ranges and parameters described in this disclosure are approximate, the numerical values ​​illustrated in specific examples should be provided as precisely as possible. However, any numerical value inherently contains some error, which is necessarily caused by the standard deviation of their corresponding test measurements. Each numerical range given throughout the specification will include every narrower numerical range falling within such a wider range as if such narrower numerical range were explicitly stated herein.

[0066] [Antibody]

[0067] This 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 membrane-like 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 may be used interchangeably in the plural form) is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, or polypeptide through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Typical antibody molecules include 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 considerably. The specific binding site, or antigen-binding site, is used to determine the specificity of antibody recognition. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity-determining regions" (CDRs), which contain more conserved regions called "framework regions" (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methods known in the art, such as the Kabat definition, the Chothia definition, the AbM definition, and / or the Contact definition, all of which are well-known in the art. See, for example, Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDA Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, et al., (1989) Nature, Vol. 342: p. 877; Chothia, C. et al., (1987) J. Mol. Biol., Vol. 196: pp. 901-917; Allazikani et al., (1997) J. Molec. Biol., Vol. 273: pp. 927-948; and Almagro, J. Mol. Recognit., Vol. 17: pp. 132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. Antibodies can be complete (i.e., full-length) polyclonal or monoclonal antibodies. Antibodies include any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and antibodies do not need to be of any particular class. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively.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 containing antibody moieties, humanized antibodies, chimeric antibodies, biantibodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified constructs containing an immunoglobulin molecule with an antigen recognition site having the desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0070] This disclosure provides a separated 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, the HCDR2 comprises the amino acid sequence shown in IYPGX1GDX2 (SEQ ID NO:2), and 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 (leucine-threonine-serine), and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:5; X1 is D or Q, and X2 is S or A. CDRs are defined / numbered by the IMGT system.

[0071] In some embodiments, X1 is D and X2 is S. In some embodiments, X1 is Q and X2 is S. In some embodiments, X1 is Q and X2 is A. Throughout this document, amino acids are represented by single-letter codes, as is 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 of exemplary antibodies targeting CD142 are within the scope of this disclosure.

[0073] The antibody or the framework region of the antibody may have mutations that do not affect the binding of the antibody's variable region to the antigen, such mutations may increase the binding affinity of the antibody to the antigen or remain substantially unchanged. In some embodiments, the isolated antibody or its antigen-binding fragment that binds to CD142 also includes conserved modified variants, which include individual substitutions, deletions, or additions to the polypeptide sequence that result in the substitution of chemically similar amino acids. Conserved substitutions that provide functionally similar amino acids are well known in the art. Such conserved modified variants are complementary to, but not exclude from, polymorphic variants, interspecies homologs, and alleles. The following eight groups contain mutually conserved substituted amino acids: 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, for example, Creighton & Proteins, 1984). In some embodiments, the term "conserved sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of antibodies containing amino acid sequences.

[0074] In some embodiments of the isolated antibody or its antigen-binding fragment 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 its antigen-binding fragment 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 with the amino acid sequence shown in SEQ ID NO:6, and a VL having at least 70%, 79%, 80%, 87%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:7; (b-1) a VH having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:8, and a VL having at least 70%, 79%, 80%, 87%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:7; and a VL having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:8, ...70%, 79%, 80%, 87%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:7. The amino acid sequence shown in NO:15 has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with VL; (c-1) VH has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:8, and VL has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:17; (d-1) VH has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:9, and VL has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:15; and (e-1) VL has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:15; and (e-1) VL has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:15. VH, which has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in NO:9, and VL, which has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:17.

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

[0077] Besides the aforementioned percentage of sequence identity, another indicator that two polypeptides are substantially identical is the immunoreactivity between the polypeptide encoded by the first nucleic acid and antibodies generated against the polypeptide encoded by the second nucleic acid, as described below. Therefore, the polypeptide is generally substantially identical to the second polypeptide; for example, the two polypeptides differ only in conserved substitutions. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules or their complement hybridize to each other under stringent conditions. Yet another indicator that two nucleic acid sequences are substantially identical is that the same primers are available for amplifying the sequences.

[0078] In some embodiments, the VH of the isolated antibody or its antigen-binding fragment binding 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 its antigen-binding fragment binding to CD142 includes at least a heavy chain variable region (VH) and at least a light chain variable region (VL), wherein the VH includes HCDR 1, 2, and 3, and the VL includes LCDR 1, 2, and 3; wherein the HCDR1 contains the amino acid sequence shown in SEQ ID NO:20, the HCDR2 contains the amino acid sequence shown in INRAX3X4YTT (SEQ ID NO:21), and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; and the LCDR1 contains the amino acid sequence shown in SEQ ID NO:23, the LCDR2 contains the amino acid sequence shown in YTS (tyrosine-threonine-serine), and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:24; X3 is N or Q, and X4 is G or A. CDRs are defined by the IMGT system.

[0080] In some implementations, X3 is N and X4 is G. In some implementations, X3 is N and X4 is A. In some implementations, X3 is Q and X4 is G.

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

[0082] In some embodiments, the isolated antibody or antigen-binding fragment thereof binding to CD142 includes VH and VL selected from the following (a-2) to (l-2): (a-2) VH having at least 70%, 85%, 87%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:25, and VL having at least 70%, 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:26; (b-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:27, and VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:33; (c-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:27; (c-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:33; (d-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:27; (e ... The amino acid sequence shown in NO:28 has at least 95%, 96%, 97%, 98%, or 99% identity with VH, and the amino acid sequence shown in SEQ ID NO:33 has at least 95%, 96%, 97%, 98%, or 99% identity with VL; (d-2) The amino acid sequence shown in SEQ ID NO:29 has at least 95%, 96%, 97%, 98%, or 99% identity with VH, and the amino acid sequence shown in SEQ ID NO:33 has at least 95%, 96%, 97%, 98%, or 99% identity with VH; (e-2) The amino acid sequence shown in SEQ ID NO:30 has at least 95%, 96%, 97%, 98%, or 99% identity with VH, and the amino acid sequence shown in SEQ ID NO:33 has at least 95%, 96%, 97%, 98%, or 99% identity with VH; (f-2) The amino acid sequence shown in SEQ ID NO:29 has at least 95%, 96%, 97%, 98%, or 99% identity with VH; (f-2) The amino acid sequence shown in SEQ ID NO:3 ... The amino acid sequence shown in NO:31 has at least 95%, 96%, 97%, 98%, or 99% identity with VH, and the amino acid sequence shown in SEQ ID NO:33 has at least 95%, 96%, 97%, 98%, or 99% identity with VL; (g-2) the amino acid sequence shown in SEQ ID NO:32 has at least 95%, 96%, 97%, 98%, or 99% identity with VH, and the amino acid sequence shown in SEQ ID NO:33 has at least 95%, 96%, 97%, 98%, or 99% identity with VL; (h-2) the amino acid sequence shown in SEQ ID NO:27 has at least 95%, 96%, 97%, 98%, or 99% identity with VH, and the amino acid sequence shown in SEQ ID NO:34 has at least 95%, 96%, 97%, 98%, or 99% identity with VH.(i-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:28, and VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34; (j-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:29, and VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34; (k-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:31, and VL having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34; and (l-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:31; and (l-2) VH having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34; and (j ...4; and VH, which has at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:32, and VL, which has at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34.

[0083] In some embodiments, the VH of the isolated antibody or its antigen-binding fragment binding 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 shown in SEQ ID NO:27, and VL comprises the amino acid sequence shown in SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:28, and VL comprises the amino acid sequence shown in SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:29, and VL comprises the amino acid sequence shown in SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:31, and VL comprises the amino acid sequence shown in SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO:32, and VL comprises the amino acid sequence shown in SEQ ID NO:34.

[0084] The isolated antibody or antigen-binding fragment thereof provided in this disclosure can bind to the CD142 protein in mammals (e.g., human or mouse). In some embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to human CD142. In some embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to mouse CD142.

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

[0086] In some embodiments, the heavy chain of any isolated antibody or antigen-binding fragment thereof that binds to CD142, as described herein, may further include a heavy chain constant region (CH) or a portion thereof, and the light chain may further include a light chain constant region (CL) or a portion thereof. The constant region may be from any suitable source, such as human, mouse, rat, or rabbit. Antibody heavy and light chain constant regions are well known in the art, for example, those available 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 contains the amino acid sequence shown in SEQ ID NO:35, and the light chain constant region contains the amino acid sequence shown in SEQ ID NO:36.

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

[0089] The isolated antibody or its antigen-binding fragment that binds to CD142 described in this article may be a human antibody, a humanized antibody, or a chimeric antibody.

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

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

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

[0093] The term "humanized antibody" refers to a form of non-human (e.g., mouse) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), where residues from the receptor complementarity-determining region (CDR) are derived from non-human species, such as mouse, rat, or rabbit CDRs (donor antibodies), with the desired specificity, affinity, and ability to replace residues. In some cases, Fv frame region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not present in the receptor antibody or in the introduced CDR or frame sequence, but these residues are included to further improve and optimize antibody performance. Typically, humanized antibodies will contain at least one, usually two, substantially the entire variable region, where all or substantially all of the CDR region corresponds to the CDR region of the non-human immunoglobulin, and all or substantially all of the FR region is the FR region of the human immunoglobulin common sequence. Humanized antibodies will also preferably contain at least a portion of an immunoglobulin constant region or structural domain (Fc), typically a human immunoglobulin constant region or structural 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) altered relative to the original antibody, also referred to as “derived from” one or more CDRs derived from one or more CDRs of the original antibody.

[0094] In some embodiments, the isolated antibody or antigen-binding fragment thereof that binds to CD142 described herein is a chimeric antibody, which may include constant regions of the heavy and light chains derived from human antibodies. A chimeric antibody is an antibody having a variable region or a portion thereof derived from a first species and a constant region derived from a second species. Typically, in these chimeric antibodies, the variable regions of the light and heavy chains mimic the variable regions derived from antibodies of one mammal (e.g., non-human mammals such as mice, rabbits, and rats), while the constant regions are sequence homologous to those derived from antibodies of another mammal such as humans. In some embodiments, amino acid modifications may be made in the variable and / or constant regions.

[0095] The isolated antibody or its antigen-binding fragment 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,” ColdSpring Harbor Laboratory, New York.

[0096] According to methods commonly practiced in the art, isolated antibodies or antigen-binding fragments of CD142 can be obtained by immunizing animals with CD142 or any polypeptide selected from the amino acid sequence of CD142, collecting and purifying the antibodies produced in vivo. In this case, antibodies applicable to human diseases can be selected by examining the cross-reactivity of the antibody bound to the heterologous CD142 with human CD142. Alternatively, it can be obtained by fusing antibody-producing cells against CD142 with myeloma cells to establish a hybridoma, and obtaining monoclonal antibodies from the hybridoma, according to 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)). CD142 used as an antigen can be obtained by expressing the CD142 gene in host cells using genetic engineering.

[0097] Hybridomas can be engineered to obtain chimeric antibodies, such as antibodies in which the variable region of a mouse or rat-derived antibody is linked to a constant region of a human-derived antibody (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 human-derived antibodies (see Nature (1986), Vol. 321, pp. 522-525) and antibodies obtained by CDR transplantation, such as antibodies obtained by transplanting partial amino acid residues of the framework other than the CDR sequence (WO 90 / 07861). Human antibodies can be obtained by generating mice using human antibodies containing human chromosome segments with heavy and light chain genes for 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 (edited by Kitagawa, Y., Matsuda, T., and Iijima, S.), Kluwer Academic Publishers, 1999; Tomizuka, K. (edited), Proc. Natl. Acad. Sci. USA (2000), Vol. 97, pp. 722–727, etc.).

[0099] The isolated antibody or its antigen-binding fragment provided herein specifically binds to CD142 and exhibits good affinity for CD142-expressing cells. The isolated antibody or its antigen-binding fragment also demonstrates significant cytotoxic activity against tumor cells. In some embodiments, the isolated antibody or its antigen-binding fragment provided herein that binds to CD142 can reduce / eliminate diseased cells, such as CD142. + Tumor cells, thereby treating and / or diagnosing CD142-targeted tumors.

[0100] The isolated antibodies or antigen-binding fragments thereof provided herein have reduced effects on coagulation function and reduced coagulation toxicity. In some embodiments, compared with prior art CD142-targeting antibodies such as vetixatumumab, the isolated antibodies or antigen-binding fragments thereof provided herein have reduced effects on clotting time and effectively avoid bleeding side effects.

[0101] This disclosure provides nucleic acids encoding the isolated antibody or its antigen-binding fragment that binds to CD142 as described above, wherein the nucleic acid encodes VH and / or VL. In some embodiments, the nucleic acid encoding VH comprises the nucleotide sequence shown in SEQ ID NO:18; 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.

[0102] This disclosure also provides vectors containing the aforementioned nucleic acids. Additionally, this disclosure provides host cells containing the aforementioned nucleic acids or vectors.

[0103] Antibody-drug conjugates

[0104] This disclosure provides antibody-drug conjugates having the structure of Formula I, or isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates, or combinations thereof.

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

[0106] Where Ab is the isolated antibody or its antigen-binding fragment that binds to CD142;

[0107] L is a connector covalently connected to Ab and D respectively;

[0108] D represents the payload;

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

[0110] The term "antibody-drug conjugate," also known as "ADC," refers to a conjugate in which an antibody or its antigen-conjugate that binds to CD142 as described herein is covalently linked to a payload. Typically, an antibody-drug conjugate may include an antibody, a payload, and optionally a linker between the antibody and the payload. ADCs deliver the payload to CD142, which is targeted by the antibody. + Cells, especially CD142 + Tumor cells are used to deliver the therapeutic effect. Antibody-drug conjugates can be prepared using various methods known to those skilled in the art.

[0111] The term "connector" refers to the linker structure that connects an antibody and its payload. The molecular design and properties of the connector are key determinants of ADC efficacy in terms of pharmacokinetic (PK) / pharmacodynamic (PD) and therapeutic window. For optimal efficacy, an ideal connector should possess the following properties: (1) The connector needs to be sufficiently stable in plasma so that the ADC can circulate in the bloodstream and localize to the tumor site without premature cleavage. Connector instability leads to premature release of the toxic payload and undesirable damage to non-target healthy cells, resulting in systemic toxicity and side effects. (2) The connector needs to be able to be rapidly cleaved once the ADC is internalized into the target tumor cells, releasing the free and toxic payload. (3) Another property to consider in connector design is hydrophobicity. Hydrophobic connectors coupled to hydrophobic payloads generally promote ADC aggregation. This molecule is not conducive to the search for therapeutically useful ADCs and may cause hepatotoxicity or undesirable immune responses (Kyoji Tsuchikama et al., “Antibody-drug conjugates: recent advances inconjugation and linker chemistrie”, Protein Cell., January 2018; Vol. 9, No. 1: pp. 33-46).

[0112] The term "isomer" refers to compounds with the same molecular formula but different structures; these are also called structural isomers and typically include structural isomers and stereoisomers. Structural isomers are those arising from differences in the order of atomic connection or bonding properties within a molecule, preferably including tautomers. Tautomers are functional group isomers arising from the rapid movement of atoms at two positions within a molecule. Stereoisomers are those arising from atoms or groups of atoms in a molecule connected by bonds in the same order but with different spatial arrangements, preferably including optical isomers. Optical isomers are stereoisomers that possess different optical properties due to the absence of antiaxial symmetry in the molecule, such as enantiomers, diastereomers, racemates, and mesomates.

[0113] The term "prodrug" refers to a compound obtained by modifying the chemical structure of a drug, which is inactive or has low activity in vitro, and exerts its pharmacological effect in vivo by releasing the active drug through enzymatic or non-enzymatic conversion. In this disclosure, a prodrug may be an ADC molecule or a payload.

[0114] In some implementations, the connector is either a cuttable connector or a non-cuttable connector.

[0115] In some implementations, the connector includes a cleavable peptide.

[0116] In some implementations, the cleavable peptide can be cleaved by an enzyme.

[0117] In some implementations, the enzyme includes cathepsin B.

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

[0119] In some implementations, the amino acid unit includes a dipeptide, tripeptide, tetrapeptide, or 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 represented by 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 implementations, L includes at least one spacer that provides the distance between the payload and the antibody.

[0122] In some implementations, the spacer comprises a self-degrading spacer.

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

[0124] Self-degrading spacers can be defined as bifunctional chemical moieties that can covalently link two spaced-apart chemical moieties together to form a normally stable three-part molecule from which one spaced-apart chemical moieties can be released, for example, by enzyme cleavage, and after cleavage (e.g., enzyme cleavage), another spaced-apart chemical moieties can be spontaneously cleaved from the rest of the molecule to release the other spaced-apart chemical moieties.

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

[0126] In some embodiments, the spacer contains -NH-(CH2)n 4 The structure shown is -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 can independently represent a C1–C6 alkyl group, or -(CH2)n. a -NH2、-(CH2)n b-COOH or -(CH2)n c -OH,n 4 n represents an integer from 0 to 6. a n b and n c Each represents an integer from 1 to 4 independently, but when n a When R is 0, 2 and R 3 They are not the same, and Lc represents -C(=O)-.

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

[0128] In some embodiments, the connector comprises the structure shown in -L1-L2-L3-, where L1 represents -(succinimide-3-yl-N)-(CH2)m 1 -C(=O)-, -CH2-C(=O)-NH-(CH2)m 2 -C(=O)- or -C(=O)-(CH2)m 3 -C(=O)-, where m 1 m represents an integer from 2 to 8. 2 Represents integers from 1 to 8, and m 3 L1 represents integers from 1 to 8; L2 represents amino acid units; L3 represents self-degrading spacers.

[0129] In some implementations, m 1 This represents 2, 3, 4, 5, 6, 7, or 8. In some implementations, m 2 This represents 1, 2, 3, 4, 5, 6, 7, or 8. In some implementations, m 3 It represents 1, 2, 3, 4, 5, 6, 7 or 8.

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

[0131] -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-PABC-;

[0132] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-;

[0133] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-PABC-;

[0134] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O-GGFG-PABC-;

[0135] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O-GGFG-PABC-;

[0136] -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-PABC-;

[0137] -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-;

[0138] -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O-);

[0139] -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O-);

[0140] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O-);

[0141] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O-);

[0142] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-;-(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-;

[0143] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O-);

[0144] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-;

[0145] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-;

[0146] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-;

[0147] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-;

[0148] -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-;

[0149] -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-;

[0150] -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-PABC-;

[0151] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-PABC-;

[0152] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-PABC-;

[0153] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2o-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-PABC-;

[0154] -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-PABC-;

[0155] -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-PABC-;

[0156] -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O-);

[0157] -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O-);

[0158] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O-);

[0159] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O-);

[0160] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2CH2CH2-C(=O-);

[0161] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2-O-CH2-C(=O-);

[0162] -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-O-CH2-C(=O-);

[0163] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-;

[0164] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-;

[0165] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-;

[0166] -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-;

[0167] -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; and

[0168] -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-.

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

[0170] In some implementations, the connector includes type II.

[0171]

[0172] In formula II, R1 and R2 are independently selected from hydrogen, methyl, and isopropyl; R3 represents -(CR5HCONH)n 1 -(CH2CONH)n 2 - or single bond; R5 is selected from hydrogen or benzyl, n 1 Represents integers from 0 to 2, and n 2 R4 represents an integer from 0 to 2; R4 represents methylamino or -(NCH3COCH2)n 3 -NCH3COCH3, and n 3 Represents integers from 1 to 20.

[0173] In some implementations, in the connector of Formula II, R4 represents -(NCH3COCH2)n 3 -NCH3COCH3,n 3n represents an integer from 1 to 20. 3 It can be selected from any integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0174] In some implementations, in the connector of Formula II, R4 represents -(NCH3COCH2)n 3 -NCH3COCH3,n 3 Represents an integer from 8 to 15. In some implementations, in the connector of Formula II, R4 represents -(NCH3COCH2)n 3 -NCH3COCH3,n 3 Represents integers from 10 to 12.

[0175] In some implementations, R4 in the connector of Formula II represents methylamino.

[0176] In this disclosure, the introduction of R4 containing a hydrophilic amino group (e.g., polysarcosine or methylamino) is beneficial for increasing the hydrophilicity of antibody-drug conjugates, especially when a hydrophobic payload is coupled to the antibody-drug conjugate. Increased ADC hydrophilicity helps reduce ADC aggregation during preparation, thereby improving the stability, uniformity, and purity of the ADC.

[0177] In some implementations, R3 represents a single bond in the connector of Formula II.

[0178] In some implementations, in the connector of Formula II, R3 represents -(CR5HCONH)n 1 -(CH2CONH)n 2 -, R5 is benzyl, n 1 n represents an integer that is either 1 or 2. 2 An integer representing 1 or 2.

[0179] In some embodiments, in the connector of Formula II, R3 represents -CR5HCONH-, -CH2CONH-, -CR5HCONH-CH2CONH-, -(CR5HCONH)2-CH2CONH-, -CR5HCONH-(CH2CONH)2- or -(CR5HCONH)2-(CH2CONH)2-; R5 is benzyl.

[0180] In some embodiments, R1 in the connector of Formula II is hydrogen. In some embodiments, R1 in the connector of Formula II is isopropyl.

[0181] In some embodiments, R2 is hydrogen in the connector of Formula II. In some embodiments, R2 is methyl in the connector of Formula II.

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

[0183]

[0184] In this disclosure, in the antibody-drug conjugate, the succinimide group of the linker of Formula II is covalently linked to the antibody. In some embodiments, the terminal succinimide group of the linker of Formula II forms a thioether bond with a thiol group obtained by reducing the interchain disulfide bond of the antibody. The succinimide group is... It forms a thioether bond with the thiol moiety obtained through the interchain disulfide bond between the carbon atom at position 3 and the reducing antibody. In the structural formula... The bond indicated represents a chemical bond that connects the bond to other groups.

[0185] In this 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 into thiol groups and then bonded to the linker. The amino acids in the antibody chemically bonded to the succinimide 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 an antibody-drug conjugate; compounds that serve as detection markers or have cytotoxic effects; radiolabeled radionuclides or peptides; fluorophores, chromophores, contrast agents, and / or metal ions; and compounds, nucleic acids, peptides or proteins, enzymes, hormones, or nucleic acids that can modulate immune activity in vivo (including activation or inhibition).

[0187] Under ideal conditions, in antibody-drug conjugates (ADCs), the conjugated payload has very low cytotoxicity, or its cytotoxicity is so low that administration of a therapeutically effective dose of the ADC will not cause systemic toxicity in subjects due to the conjugated 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 conditions of use.

[0188] In some embodiments, the payload in the antibody-drug conjugate is a label containing a radioactive marker, a fluorophore, a chromophore, a contrast agent, and / or a metal ion as a detection marker. The marker includes, but is not limited to, chemically synthesized organic compounds, radionuclides, metal complexes, or peptides. The radioactive marker refers to a labeled compound in which one or more atoms of the compound molecule are replaced by a radioactive nuclide, making the compound identifiable and usable as a tracer. This radioactive marker includes amino acids, peptides, proteins, carbohydrates, nucleotides, nucleosides, purines, pyrimidines, steroids, lipid compounds, and tumor antigens, hormones, receptors, vitamins, and drugs used in medical research. The radionuclide is typically a nuclide capable of spontaneously emitting radiation, including but not limited to tritium, iodine-125, iodine-131, sulfur-35, phosphorus-32, and carbon-14. The fluorophore typically comprises a group containing conjugated double bonds and emits fluorescence when the molecule returns from an excited state to its ground state. The chromophore is an unsaturated group contained in a molecule and its associated chemical bonds, capable of absorbing light radiation and undergoing transitions. In nuclear medicine, contrast agents are generally radiopharmaceuticals that, when introduced into the body, can image organs, tissues, or molecules.

[0189] In some implementations, the payload in the antibody-drug conjugate is a nucleic acid, which may be ribonucleic acid and / or deoxyribonucleic acid.

[0190] In some implementations, the payload in the antibody-drug conjugate is a hormone, growth factor, coagulation factor, and plasminogen activator (e.g., a prodrug-converting enzyme or ribonuclease capable of converting a prodrug into an active drug).

[0191] In some implementations, the payload in the antibody-drug conjugate is an immunomodulator (including cytokines and chemokines that can affect immunity) or a biologically active agonist or antagonist antibody.

[0192] In some embodiments, the payload in the antibody-drug conjugate is a cytotoxic compound. In some embodiments, the payload in the antibody-drug conjugate has antitumor activity or is an antitumor drug. The payload is selected from DNA topoisomerase inhibitors or tubulin inhibitors. The DNA topoisomerase inhibitor can be a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0193] The term "topoisomerase inhibitor" generally refers to compounds that inhibit the activity of topoisomerases. Compounds called 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 called topoisomerase I / II inhibitors.

[0194] The term "microtubule inhibitor" 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 payload is camptothecin or a derivative thereof, which has topoisomerase inhibitory activity. The term "derivative" refers to a compound formed by replacing atoms or groups 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 action by irreversibly inhibiting topoisomerase I. Camptothecin derivatives include icertena, irinotecan, topotecan, letopotecan, 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 their stereoisomers, salts and esters. Methods for synthesizing camptothecin and its analogues or derivatives are known and are summarized and described in U.S. Patent No. 5,244,903, the entire contents of which are incorporated herein by reference.

[0196] In some embodiments, the payload is auristatin or a derivative thereof, or maytansine or a derivative thereof, which have microtubule-inhibiting activity. The term "aurestatin or a derivative thereof" generally includes auristatin F and auristatin F derivatives. Auristatin F derivatives include monomethylaurestatin E (MMAE) and monomethylaurestatin F (MMAF). The term "matansine or a derivative thereof" generally includes maytansine and maytansine derivatives. Maytansine derivatives include maytansine DM1, maytansine DM2, and maytansine DM4.

[0197] In some implementations, the payload is eczema, a camptothecin derivative that acts as a topoisomerase inhibitor, which functions throughout the cell cycle and exhibits strong penetration and good therapeutic efficacy against slowly growing solid tumors. Furthermore, the number of intracellular targets is far less than that of microtubule inhibitors, thus the ADC molecule carrying the same payload can achieve better killing effects when it enters the cell. Simultaneously, eczema is not a substrate of P-gp, which helps reduce or alleviate drug resistance issues.

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

[0199]

[0200] Ecinotecan has a rigid molecular structure and poor hydrophilicity. Therefore, when it is linked to the GGFG tetrapeptide linker commonly used in existing technologies to prepare ADCs, it easily causes polymerization between ADC molecules, which does not meet the requirements for ADC drug development (Bioorg. MedChem. Lett. Vol. 26 (2016) pp. 1542-1545). Therefore, the selection and matching of linkers and payloads have an impact on the safety and stability of ADC drugs.

[0201] Unbound by any theory, the hydrophilicity of the linker-payload structure is improved due to the multiple hydrophilic groups in the linker of the antibody-drug conjugate, and the aggregation and precipitation of ADCs caused by hydrophobic payloads can be reduced.

[0202] After ADC molecules are endocytosed into cells, the linker is degraded to release the payload or a linker (or a portion thereof)-payload structure. In some embodiments, the amino group on the eczetane ring of Formula III bonds to the carbonyl group in the ester group of the linker of Formula I, forming a linker-payload structure comprising a carbamate. Unbound by any theory, after ADC molecules are endocytosed into cells, the linker is cleaved by cathepsins (e.g., cathepsin B) to form an intermediate or active metabolite as shown in Formula V.

[0203]

[0204] R4 represents methylamino or -(NCH3COCH2)n 3 -NCH3COCH3, and n 3 Represents integers from 1 to 20.

[0205] Then, the PABC group in the intermediate or active metabolite of Formula V undergoes 1,6-elimination to release eczema. The 1,6-elimination mechanism of PABC is described in detail in Angew. Chem. Int. Ed., 2015, Vol. 54: 7492-7509. Therefore, the linker-loaded structure in the ADC provided in this disclosure exhibits good in vivo stability and bioactivity.

[0206] Unbound by any theory, the cleavage site in the joint-load structure can be an amide bond in the joint, for example, an amide bond between the carbon atom where the substituent represented by R2 is located and the group represented by R3, or an amide bond in the group represented by R3.

[0207] In antibody-drug conjugates, n, the ratio of the number of conjugated payload molecules to the antibody molecule (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, with exemplary n being 4.66, 7.67, or 7.83.

[0208] The term "DAR (Drug to Antibody Ratio)" refers to the average number of conjugated payload molecules or drug molecules per antibody molecule, i.e., the average number of conjugated drug molecules. In this antibody-drug conjugate, the number of conjugated payload molecules per antibody molecule is a key factor affecting its efficacy and safety. The preparation of this antibody-drug conjugate is carried out by specifying reaction conditions, such as the amounts of starting materials and reagents used in the reaction, to obtain a constant number of conjugated payload molecules. When preparing antibody-drug conjugates, mixtures containing varying numbers of conjugated payload molecules are typically obtained. Unless otherwise specified, in this disclosure, the number of conjugated payload molecules or drug molecules per antibody molecule is defined as an average, i.e., the average number of conjugated payload molecules or drug molecules.

[0209] In some implementations, the ADC with the above-described connector is chemically conjugated to an antibody targeting CD142, resulting in a higher DAR value (e.g., DAR8).

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

[0211]

[0212]

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

[0214] The antibody in the antibody-drug conjugate is CD142-specifically targeted, and the antibody forms a reactive thiol group via a disulfide bond, which then binds to the adapter. In some embodiments, the disulfide bond in the antibody hinge region forms a reactive thiol group, which then binds to the adapter.

[0215] In some embodiments, the antibody in the antibody-drug conjugate is the antibody or its antigen-binding fragment provided above. In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate includes the VH sequence as shown in SEQ ID NO:8 and the VL sequence as shown in SEQ ID NO:15. In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate includes the VH sequence as shown in SEQ ID NO:28 and the VL sequence as shown in SEQ ID NO:33.

[0216] The improved hydrophilicity of antibody-drug conjugates can also increase homogeneity. Therefore, antibody-drug conjugates with improved hydrophilicity and / or homogeneity can improve cytotoxicity to target cells and improve or maintain biological activity, safety and other pharmaceutical properties.

[0217] The antibody-drug conjugate of this disclosure has an improved HNSTD (maximum dose without serious toxicity). Furthermore, it demonstrates significant efficacy in tumor suppression and is unlikely to cause significant gastrointestinal toxicity. It should be noted that the antibody-drug conjugate of this disclosure can absorb water, retain the adsorbed water, or become hydrated due to exposure to the atmosphere or recrystallization, and such hydrated compounds and their salts are also included in this disclosure. In addition, compounds labeled with various radioactive or non-radioactive isotope variants are also included in this disclosure. More than one atom constituting the antibody-drug conjugate of this disclosure may also contain atomic isotopes in non-natural proportions. Examples of atomic isotopes include, for example, deuterium (2H), tritium (3H), iodine-125 (125I), or carbon-14 (14C). Furthermore, the compounds of this disclosure can be radiolabeled with radioactive isotopes such as tritium (3H), iodine-125 (125I), or carbon-14 (14C). Radiolabeled compounds can be used as therapeutic or preventative agents, research reagents such as test reagents, and diagnostic reagents, such as in vivo imaging diagnostics. All isotopic variants of the antibody-drug conjugates disclosed herein, whether radioactive or not, are included within the scope of this disclosure.

[0218] [Pharmaceutical Composition]

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

[0220] This disclosure provides a pharmaceutical composition comprising the above-described antibody-drug conjugate, or an isomer, isotope variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient.

[0221] The pharmaceutical composition may be administered in a suitable manner depending on the specific application form, physicochemical properties, etc., of the pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition may be formulated as a lyophilized or liquid formulation, which may contain suitable formulation additives in the art. For example, the pharmaceutical composition typically contains more than one drug carrier, such as sterile liquids such as water and oils (including petroleum, animal-derived, plant-derived, or synthetically derived oils (e.g., peanut oil, soybean oil, mineral oil, and sesame oil)). In the case of intravenous administration of the pharmaceutical composition, water is a more representative carrier. In addition, saline solutions, glucose solutions, and glycerol solutions may also be used as liquid carriers, especially for injectable solutions. Suitable drug excipients are known in the art. The pharmaceutical composition may also contain trace amounts of wetting agents, emulsifiers, or pH buffers as needed. The pharmaceutical composition is typically administered parenterally, and may be administered intradermally, intramuscularly, intraperitoneally, intravenously, or subcutaneously, but is not limited to these methods; for example, the pharmaceutical composition may be administered by infusion or bolus injection. See, for example, the Handbook of Pharmaceutical Excipients, 3rd edition, AHKibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated herein by reference in its entirety. Remington's Pharmaceutical Sciences, 16th edition, EWMartin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated herein by reference in its entirety.

[0222] The pharmaceutical composition may contain an active agent, namely, a separable antibody or antigen-binding fragment thereof that binds to CD142, or the aforementioned antibody-drug conjugate, and a second therapeutic agent (e.g., a cancer therapeutic agent). In some embodiments, the separable antibody or antigen-binding fragment thereof that binds to CD142 of this disclosure may be administered together with other cancer therapeutic agents to enhance the anticancer effect. In some embodiments, the antibody-drug conjugate of this disclosure may be administered together with other cancer therapeutic agents to enhance the anticancer effect. Other anticancer agents for this purpose may be administered to an individual simultaneously, separately, or sequentially with the antibody-drug conjugate of this disclosure, or may be administered at different intervals. Exemplary other cancer therapeutic agents may be, for example, paclitaxel, cisplatin, vincristine, etc., but are not limited thereto, as long as they have antitumor activity.

[0223] With respect to this disclosure, the active agent or pharmaceutical composition comprising the active agent may be administered to a subject via any suitable route of administration. For example, the active agent may be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion of routes of administration is for illustrative purposes only and should not be construed as limiting the scope in any way.

[0224] This disclosure also provides a kit comprising a separable antibody or antigen-binding fragment thereof that binds to CD142, or an antibody-drug conjugate thereof, which can be used to detect CD142 or cells expressing CD142.

[0225] [Preparation Method]

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

[0227] The antibody or its antigen-binding fragment is reduced such that its disulfide bonds are at least partially reduced, and then reacted with the reactive group of the linker in the linker-payload to obtain an antibody-drug conjugate having the structure of Formula I.

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

[0229] Or its isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates, or combinations thereof;

[0230] Where Ab is the isolated antibody or its antigen-binding fragment that binds to CD142 as described above; L is the linker covalently linked to Ab and D respectively; D is the payload; and n is an integer from 1 to 10.

[0231] There are no particular restrictions on the reactive group, as long as the reactive group contains a portion that can react with the thiol group obtained from the antibody.

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

[0233] In some embodiments, the method includes the steps of: reducing the antibody such that its interchain disulfide bonds are at least partially reduced, and reacting it with the maleimide-N-group of the linker shown in Formula IV at the 3-carbon position.

[0234]

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

[0236] In Formula IV, R1 and R2 are each independently selected from hydrogen, methyl, and isopropyl;

[0237] R3 represents -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is selected from hydrogen or benzyl, n 1 Represents integers from 0 to 2, and n 2 Represents integers from 0 to 2;

[0238] R4 represents methylamino or -(NCH3COCH2)n 3 -NCH3COCH3, and n 3 Represents integers from 1 to 20.

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

[0240] In some embodiments, the method further includes reacting the antibody with a reducing agent in a buffer solution containing a chelating agent, followed by the addition of a linker-loador solution to carry out the reaction. The linker-loador is particularly a compound formed by bonding a linker of formula IV to the load, wherein the amino group (primary amino group) in the load is linked to a carbonyl group in the ester group of the linker of formula IV. The term "chelating agent" refers to a complex capable of forming a cyclic complex by coordination bonding with metal atoms or ions.

[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 used is 1 mM to 20 mM, for example, 2 mM to 20 mM, 5 mM to 20 mM, 8 mM to 20 mM, 1 mM to 15 mM, or 1 mM to 10 mM. 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 between the antibody and the reducing agent is carried out at a regulated pH. In some embodiments, the pH of the solution is 5–9, optionally 6–8, 6–7, 6.5–7.5, or 7–8, when the antibody reacts with the reducing agent. 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 basic chemicals, and exemplary acidic or basic chemicals include acetic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sodium bicarbonate, sodium carbonate, sodium hydroxide, and triethylamine.

[0243] The reaction between the antibody and the reducing agent is carried out at a regulated 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 connector-payload can be dissolved in an organic solvent selected from dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP) or combinations thereof.

[0245] In some embodiments, the adapter-loador solution is added to an antibody buffer solution that has been reduced or has reactive thiol groups at a volume ratio of 1% to 20% of the antibody buffer solution volume. In some embodiments, the volume ratio of the added adapter-loador solution to the antibody buffer solution is 1%–20%, 2%–20%, 5%–20%, 10%–20%, 15%–20%, 1%–18%, 1%–15%, 1%–13%, 1%–10%, or 5%–15% of the antibody buffer solution volume.

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

[0247] In some embodiments, the reaction temperature between the antibody and the adapter-payload 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, or 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 reaction time between the antibody and the adapter-payload is 0.5 hours to 2 hours. In some embodiments, the reaction time between the antibody and the adapter-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 the unreacted linker-load using a thiol-containing reagent. Thiol-containing reagents include, but are not limited to, cysteine ​​or N-acetyl-(L)-cysteine ​​(NAC). More specifically, the reaction is terminated by adding a thiol-containing reagent in a molar equivalent of 1-2 times the linker-load to the reaction solution and incubating at room temperature (10°C-25°C) for 10-30 minutes.

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

[0336] to

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

[0251] The antibody-drug conjugates provided in this disclosure can be obtained by the preparation method described above. In some embodiments, the prepared antibody-drug conjugates are 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] This disclosure also provides the use of the above-described antibody, antibody-drug conjugate, pharmaceutical composition, or antibody-drug conjugate prepared by the above method, or the above-described kit, in the preparation of therapeutic agents for the diagnosis, prevention, and treatment of tumor diseases, including benign tumors and malignant tumors (e.g., cancer).

[0254] This disclosure provides a method for diagnosing, preventing, and treating cancer, the method comprising administering a therapeutically effective amount of the antibody, antibody-drug conjugate, antibody-drug conjugate prepared by the above method, pharmaceutical composition, or kit to a subject in need.

[0255] Tumor diseases are not limited to those mentioned above, as long as the cells at the lesion site express proteins that can be recognized by CD142-targeting antibodies. In some implementations, 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 implementations, the oncological disease is a CD142-related disease, such as a solid tumor expressing CD142. In some implementations, CD142-related diseases include pancreatic cancer, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, and cervical cancer.

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

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

[0259] As used herein, subjects can be non-human mammals and humans. Non-human mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, mice, and rats. In some implementations, subjects are humans. Human subjects requiring treatment can be human subjects who have, are at risk of having, or are suspected of having the target disease / condition associated with a tumor or CD142-related tumor.

[0260] Subjects suspected of having a target disease or condition can be identified through routine medical examinations (e.g., laboratory tests, organ function tests, CT scans, or ultrasound). Subjects suspected of having any such target disease / condition may exhibit one or more symptoms of that disease / condition. Subjects at risk of having that disease / condition may be those who have one or more risk factors for that disease / condition.

[0261] The term "therapeutic effective amount" refers to the amount of each active agent required, alone or in combination with one or more other active agents, to produce a therapeutic effect in a subject. In some embodiments, therapeutic effect refers to reduced CD142 activity or reduced CD142 + Cellular activity. Determining whether the amount of antibody or antibody-containing ADC achieves a therapeutic effect will be apparent to those skilled in the art. 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, the nature of concomitant treatments (if any), the specific route of administration, and the knowledge and expertise of the healthcare practitioner, as well as similar factors. These factors are well known to those skilled in the art and can be explained simply through routine experiments. Generally, the maximum amount of a single component or combination thereof is preferred, i.e., the highest safe amount based on reasonable medical judgment.

[0262] In some implementations, the dosage of the antibody or its antigen-binding fragment, or the antibody-drug conjugate, can be determined empirically in individuals who have received one or more antibody administrations. In some implementations, acceptable therapeutic doses of the antibody-drug conjugate are 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 implementations, the administration frequency is once every 12 hours, once daily, once weekly, 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 monthly, once every 2 months, or once every 3 months, or for longer periods. The therapeutic dose and administration frequency may vary depending on the treatment regimen.

[0263] Various embodiments and preferences of this disclosure may be combined with each other, provided that they are not inherently inconsistent with each other, and the various embodiments formed by combination are considered to be part of the disclosure of this application.

[0264] The technical solutions of this disclosure will be described more clearly and specifically below with reference to illustrative embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this disclosure. The scope of protection of this disclosure is defined only by the claims.

[0265] Example

[0266] The present invention is further described in the following embodiments, which do not limit the scope of the invention as 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℃~30℃), DCM (dichloromethane):MeOH (methanol) (v:v=2:1, 90mL) and EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; 1.86g, 7.55mmol) were added to a mixed solution of compound 11-1A (Mc-Val-Ala-OH, purchased from MedChemExpress Shanghai; 2.4g, 6.29mmol) and compound 11-1B (3.18g, 6.29mmol). The reaction solution was stirred at room temperature for 24 hours, and the solvent was removed under vacuum. The crude residue was then further purified by rapid chromatography to obtain compound 11-1 (3.9g, 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 to it under argon atmosphere at 0 °C. The reaction mixture was then stirred at 0 °C for 2 hours. The reaction was quenched by adding water. The resulting mixture was extracted with DCM, dried, and concentrated. The residue was purified by silica gel chromatography to give 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). DIPEA (N,N-diisopropylethylamine; 0.39 mL, 2.23 mmol) and 4,4'-dinitrodiphenyl carbonate (406 mg, 1.33 mmol) were added under argon atmosphere and at room temperature. The reaction mixture was then stirred overnight at ambient temperature. The solvent in the reaction mixture was removed by concentration, and the product obtained was precipitated using MTBE (methyl tert-butyl ether). The yellow solid was collected by filtration, washed with diethyl ether, and dried to give compound 11-3. LC-MS (ESI, m / z): 795.41 (M+H).

[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 dried pyridine (1 mL) was added, followed by eczemab mesylate (purchased from 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 resulting product was purified by preparative HPLC (preparative high performance liquid chromatography) to give intermediate 11-4 (230 mg, 48%). LC-MS (ESI, m / z): 1091.53 (M+H).

[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 at 0 °C. The reaction mixture was stirred at room temperature for 30 min, and the solvent was removed by concentration to give the TFA salt of intermediate 11-5, which could be used in the next step without further purification. LC-MS (ESI, m / z): 991.47 (M+H).

[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-acetyldecasarcosine; 84 mg, 0.109 mmol) was added, followed by HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; 50 mg, 0.130 mmol) and DIPEA (38 μL, 0.22 mmol). The reaction mixture was stirred overnight at room temperature, and the solvent was removed by concentration. The crude product was purified by preparative HPLC to give compound LP-1 (74 mg, 38%). LC-MS (ESI, m / z): 1743.85 (M+H).

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

[0282]

[0283] The synthesis of compound LP-2 was performed following the same steps as that of compound LP-1. Starting material 11-1A was replaced with Mc-GGFG-OH (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd.) to obtain compound LP-2, 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. Excluding 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 an extracellular fragment of the human CD142 protein antigen (purchased from ACROBiosystems, product number TF3-H52H5), and their serum titers were monitored to determine the number of immunizations. After initial immunization, mice were given three or four booster immunizations, and serum was collected for titer detection. Mice with acceptable titers received a single booster immunization, and the entire spleen and half a lymph node were harvested and fused with myeloma SP2 / 0 cell lines for PEG fusion. The fused cells were cultured in plates. The supernatant was collected and antigen was screened by ELISA. Positive cells were transferred to 96-well plates for further culture. After 7 days, the supernatant was collected, and the reaction with the antigen was measured by ELISA. The antigen-binding affinity of positive cells was further tested at different dilutions. The 20 parental clones with the highest immunogen affinity were selected for subcloning. Monoclonal hybridoma cells were obtained through limiting dilution and ELISA screening. The 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, the humanized O1 variable region (referred to as Hu01) is obtained by modifying the VH and VL regions of the hybridoma sequence. 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 including the light chain variable region encoding L3 and the heavy chain variable region encoding H1. L3H2 represents a humanized antibody including the light chain variable region encoding L3 and the heavy chain variable region encoding H2. L5H1 represents a humanized antibody including the light chain variable region encoding L5 and the heavy chain variable region encoding H1. L5H2 represents a humanized antibody including the light chain variable region encoding L5 and the heavy chain variable region encoding H2.

[0293] Table 2 Variable Region Sequence of Hu01

[0294]

[0295]

[0296]

[0297] HCDR 2 is underlined, and amino acid substitutions are indicated in bold italics.

[0298] The CDRs for Mu01 and Hu01 are defined and numbered by the IMGT system.

[0299] For the purposes of preparation and / or detection in the examples, the constant region of human IgG1 was selected as the constant region of the above-mentioned Hu01 antibody, 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.

[0300] Example 5: Preparation of monoclonal antibodies Mu02 and Hu02

[0301] Balb / c mice (8-12 weeks old) were immunized with an extracellular fragment of the human CD142 protein antigen (purchased from ACROBiosystems, product number TF3-H52H5), and their serum titers were monitored to determine the number of immunizations. After initial immunization, mice were given three or four booster immunizations, and serum was collected for titer detection. Mice with acceptable titers received a single booster immunization, and the entire spleen and half a lymph node were harvested and fused with myeloma SP2 / 0 cell lines for PEG fusion. The fused cells were cultured in plates. The supernatant was collected and antigen was screened by ELISA. Positive cells were transferred to 96-well plates for further culture. After 7 days, the supernatant was collected, and the reaction with the antigen was determined by ELISA. The antigen-binding affinity of positive cells was further tested at different dilutions. The 20 parental clones with the highest immunogen affinity were selected for subcloning. Monoclonal hybridoma cells were obtained through limiting dilution and ELISA screening. The 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, its variable region, and CDR were determined, as shown in Table 3. The CDR is underlined.

[0302] Table 3 Hybridoma Sequences

[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, the humanized O2 variable region (referred to as "Hu02") is obtained by modifying the VH and VL regions of the hybridoma sequence. 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 including the light chain variable region encoding L1 and the heavy chain variable region encoding H1. L1H2 (or Hu02-L1H2) represents a humanized antibody including the light chain variable region encoding L1 and the heavy chain variable region encoding H2. L1H3 represents a humanized antibody including the light chain variable region encoding L1 and the heavy chain variable region encoding H3. L1H4 represents a humanized antibody including the light chain variable region encoding L1 and the heavy chain variable region encoding H4. L1H5 represents a humanized antibody including the light chain variable region encoding L1 and the heavy chain variable region encoding H5. L1H6 represents a humanized antibody including the light chain variable region encoding L1 and the 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 Zones of Hu02

[0307]

[0308]

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

[0310] The CDRs for Mu02 and Hu02 are defined and numbered by the IMGT system.

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

[0312] Example 6: Detection method for antibody-drug conjugates

[0313] The antibody-drug conjugates were identified by concentrating, exchanging, and purifying the culture medium, measuring the antibody concentration, and calculating the average number of drug molecules carried by each antibody.

[0314] Operation A: Concentration of antibody or antibody-drug conjugate

[0315] Remove the ultrafiltration tube (Amicon Ultra, 50000MWCO, Millipore Corporation) and add the antibody or antibody-drug conjugate solution to be concentrated. Centrifuge the ultrafiltration tube until the antibody or antibody-drug conjugate solution reaches the desired volume, then remove it.

[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 manufacturer's defined method. The antibody concentration is the ratio of the absorbance value to the absorption coefficient at the detection wavelength.

[0318] Operation C: Antibody culture medium exchange

[0319] Following the manufacturer's (Thermo Fisher Scientific) instructions, the Zeba centrifugal desalting column (5 mL, 40 K MWCO) was pre-equilibrated with phosphate-buffered saline (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 min). The flow-through fraction was then collected and concentrated via procedure A, and the antibody concentration was determined via procedure B, adjusting the antibody concentration with PBS7.0 / EDTA.

[0320] Operation D: Purification of antibody-drug conjugates

[0321] According to the manufacturer's (Thermo Fisher Scientific) instructions, the Zeba centrifugal desalting column (5 mL, 40 K MWCO) is pre-equilibrated with storage buffer. Histidine-acetate buffer (20 mM histidine, pH 5.5) containing 150 mM NaCl or phosphate buffer (50 mM, pH 7.0) containing 50 mM NaCl is used as the storage buffer. Approximately 2 mL of the reaction solution containing the antibody-drug conjugate is applied to the Zeba centrifugal desalting column (5 mL) and centrifuged (1000 g, 4 min). The flow-through fraction (approximately 2 mL) is then collected, and the elution process is repeated twice to remove unbound linker-loaded compounds and low molecular weight compounds, including reducing agents.

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

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

[0324] At any given wavelength, the total absorbance of the system is equal to the sum of the absorbances of all the photosensitive chemicals present in the system (additivity of absorbance). Therefore, assuming that the molar absorptivity of the antibody and drug remains unchanged before and after antibody-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 an aqueous solution of an antibody-drug conjugate at 280 nm. 370 This represents the total absorbance of the antibody-drug conjugate aqueous solution at 370 nm. (A) 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 A represents the absorbance of a drug molecule at 280 nm. D,370 ε represents the absorbance of a drug molecule at 370 nm. A,280 ε represents the molar extinction coefficient of the antibody at 280 nm. A,370 ε represents the molar extinction coefficient of the antibody at 370 nm. D,280 ε represents the molar extinction coefficient of a drug molecule at 280 nm. D,370 C represents the molar extinction coefficient of a drug molecule at 370 nm. A This indicates the antibody concentration in the antibody-drug conjugate, and C D This indicates the concentration of drug molecules in the antibody-drug conjugate.

[0328] In this case, ε A,280 ε A,370 ε D,280 and ε D,370These are all known values ​​(calculated from the antibody sequence or measured by the UV absorbance of the compound). For example, ε A,280 The ε can be calculated from the amino acid sequence of the antibody using known methods (Protein Science, 1995, Vol. 4, pp. 2411-2423). Antibodies typically have no absorbance at 370 nm, therefore ε... A,370 It is usually 0. ε D,280 and ε D,370 The value can be calculated by measuring the absorbance of the drug molecule at 280 nm and 370 nm as a function of concentration, and using the Lambert-Beer law (absorbance = molar concentration × molar extinction coefficient × optical path length). A and C D The absorbance values ​​of antibody-drug conjugates at 280 nm and 370 nm can be measured. 280 and A 370 Then, the simultaneous equations (1) and (2) are solved to obtain the result. Furthermore, the average number of drug molecules linked 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 linked to each antibody (DAR value) - (2)

[0330] In addition to "Operation E" above, 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) method described below.

[0331] Antibody-drug conjugates (ADCs) are eluted on hydrophobically interacting columns based on differences in salt ion concentration in the eluent. As the salt ion concentration decreases, the amount of small molecule drug in the eluted ADC increases; that is, ADCs with low DAR values ​​are preferentially eluted. The peak order of each component is D0 (antibody not conjugated with any linker-load), D2 (antibody conjugated with an average of approximately 2 linker-loads), D4 (antibody conjugated with an average of approximately 4 linker-loads), D6 (antibody conjugated with an average of approximately 6 linker-loads), and D8 (antibody conjugated with an average of approximately 8 linker-loads). The percentage content 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] The average number of conjugated drug molecules = D0 peak area ratio × 0 + D2 peak area ratio × 2 + D4 peak area ratio × 4 + D6 peak area ratio × 6 + D8 peak area ratio × 8

[0333] Operation G: Measurement of aggregates in antibody-drug conjugates

[0334] Detection of aggregates in antibody-drug conjugates using size exclusion chromatography in high performance liquid chromatography. The method is as follows.

[0335] High Performance Liquid Chromatography System: Agilent 1260 Infinity II HPLC System

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

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

[0338] Mobile phase: 200 mmol / L KHPO4, 150 mmol / L NaCl, 15% (v / v) isopropanol, pH 7.0

[0339] Flow rate: 0.75 mL / min

[0340] Analysis time: 18 minutes

[0341] Column temperature: room temperature

[0342] Injection volume: 50μg

[0343] Data Analysis: Matter

[0344] Size exclusion chromatogram of the quality control substance (QC, Hu01-L3H1 naked antibody, i.e., L3H1 encoded by a humanized antibody derived from Mu01, not coupled with the adapter-payload) is shown in [image / image]. Figure 1 In A; the size exclusion chromatogram of the quality control substance (QC, Hu02-L1H2 naked antibody, i.e., the L1H2 encoded by the antibody Mu02, not coupled to the linker-payload) is shown in [image / image]. Figure 2 In A, the retention time of the main peak (single peak) of the 150 kDa mass control substance is 9.5 to 10.5 minutes. The retention time of the aggregates should be earlier than that of the monomers.

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

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

[0347] High Performance Liquid Chromatography System: Agilent 1260 Infinity II HPLC System

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

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

[0350] Mobile phase A: mol / L (NH₄)₂SO₄, 50 mmol / L KHPO₄, pH 7.0

[0351] Mobile phase B: 50 mmol / L KHPO4, 25% (v / v) isopropanol, pH 7.0

[0352] Analysis time: 25 minutes

[0353] Column temperature: room temperature

[0354] Washing 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: Chromatogram of the hydrophobic interaction of the quality control substance (QC, Hu01-L3H1 naked antibody) is shown in [the image]. Figure 1 In section B, the hydrophobic chromatogram of the quality control substance (QC, Hu02-L1H2 naked antibody) is shown in [image / image]. Figure 2 In sample B, the sample with the shorter retention time has lower hydrophobicity. Antibody-drug conjugates are more hydrophobic than unconjugated naked antibodies, and therefore have a longer retention time.

[0357] Example 7: Preparation of antibody-drug conjugate Hu01-L3H1-LP1-DAR8

[0358] Antibody reduction: Performed according to operation B in Example 6 (the extinction coefficient of the antibody at 280 nm was 1.658 mL mg). - 1 cm -1 Following procedure C, the culture medium for the Hu01-L3H1 antibody was exchanged with PBS 7.0 / EDTA, resulting in an antibody concentration of 7.353 mg / mL. 19.51 μL of 5 mM TCEP solution (equivalent to 7 times the antibody concentration) was added to 284.24 μL of Hu01-L3H1 antibody aqueous solution and 76 μL of 50 mM phosphate buffer (PBS 7.0), along with 0.26 μL of ultrapure water. The mixture was incubated at 37°C for 2 hours.

[0359] Antibody and adapter-loador coupling: The above mixture was incubated at 4°C for 10 minutes. The adapter-loador 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) was added to the mixture. The reaction mixture was carried out at 22°C for 30 minutes.

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

[0361] Characterization of antibody-drug conjugates: using operation E(ε) from Example 6 D,280 =6384 and ε D,370 =16180), operation F, operation G and operation H were used to characterize the obtained antibody-drug conjugate.

[0362] The concentration of the antibody-drug conjugate, measured and calculated by operation E, was 4.93 mg / mL, and the average payload per antibody-drug conjugate, measured and calculated by operation E, was 7.67. Figure 3 A shows the detection map of aggregates, and the content of aggregates in the antibody-drug conjugate Hu01-L3H1-LP1-DAR8, measured by operation G, is 1.33%. Figure 3 B shows the detection chromatogram of the hydrophobic interaction 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: Performed according to operation B in Example 6 (the extinction coefficient of the antibody at 280 nm was 1.658 mL mg). - 1 cm -1 Following procedure C, the culture medium for the Hu01-L3H1 antibody was exchanged with PBS 7.0 / EDTA, resulting in an antibody concentration of 22.51 mg / mL. 40 μL of 5 mM TCEP solution (equivalent to 2.5 times the antibody concentration) was added to 533.10 μL of the Hu01-L3H1 antibody aqueous solution, along with 320 μL of 50 mM phosphate-buffered saline (PBS 7.0) and 706.90 μL of ultrapure water. The mixture was incubated at 37°C for 2 hours.

[0365] Antibody and adapter-loador coupling: The above mixture was incubated at 4°C for 10 minutes. The adapter-loador 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 mixture. The reaction mixture was carried out at 22°C for 30 minutes.

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

[0367] Characterization of antibody-drug conjugates: using operation E(ε) from Example 6 D,280 =6384 and ε D,370 =16180), operation F, operation G and operation H were used 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 payload per antibody-drug conjugate, measured and calculated by operation E, was 4.66. Figure 4 A shows the detection map of aggregates, and the content of aggregates in the antibody-drug conjugate Hu01-L3H1-LP1-DAR4, measured by operation G, is 1.16%. Figure 4 B shows a hydrophobic interaction chromatogram of the retention time distribution of antibody-drug conjugates measured by operation H.

[0369] Example 9: Preparation of antibody-drug conjugate Hu02-L1H2-LP1-DAR8

[0370] Antibody reduction: Performed according to operation B in Example 6 (the extinction coefficient of the antibody at 280 nm was 1.382 mL mg). - 1 cm -1 Following procedure C, the culture medium for the Hu02-L1H2 antibody was exchanged with PBS 7.0 / EDTA, resulting in an antibody concentration of 11.98 mg / mL. 106.67 μL of 5 mM TCEP solution (equivalent to 10 times the antibody concentration) was added to 667.78 μL of the Hu02-L1H2 antibody aqueous solution, along with 320 μL of 50 mM phosphate-buffered saline (PBS 7.0) and 505.50 μL of ultrapure water. The mixture was incubated at 37°C for 2 hours.

[0371] Antibody and adapter-loador coupling: The above mixture was incubated at 4°C for 10 minutes. The adapter-loador LP-1 prepared in Example 1 was dissolved in DMA, and then 80 μL (equivalent to 15 times the antibody content) was added to the mixture. The reaction mixture was carried out at 22°C for 30 minutes.

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

[0373] Characterization of antibody-drug conjugates: using operation E(ε) from Example 6 D,280 =6384 and ε D,370 =16180), operation F, operation G and operation H were used to characterize the obtained antibody-drug conjugate.

[0374] The concentration of the antibody-drug conjugate, measured and calculated by operation E, was 5.03 mg / mL, and the average payload per antibody-drug conjugate, measured and calculated by operation E, was 7.83. Figure 5 A shows the detection map of aggregates, and the content of aggregates in the antibody-drug conjugate Hu02-L1H2-LP1-DAR8, measured by operation G, is 1.31%. Figure 5 B shows the detection chromatogram of the measured hydrophobic interaction 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: Performed according to operation B in Example 6 (the extinction coefficient of the antibody at 280 nm was 1.35 mL mg). - 1 cm -1 Following procedure C, the culture medium for human IgG antibodies was exchanged with PBS 7.0 / EDTA, and the antibody concentration after the exchange was 10 mg / mL. 0.267 mL of 5 mM TCEP solution (equivalent to 10 times the antibody concentration) was added to 1.33 mL of human IgG protein aqueous solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number SP001), along with 0.4 mL of 50 mM PBS 7.0. After confirming the pH of the solution was 7.0 ± 0.1, the mixture was incubated at 37°C for 2 hours.

[0377] Antibody and adapter-loador conjugation: Incubate the above mixture at 4°C for 10 minutes. Dissolve the adapter-loador LP-1 in DMA, then add 0.2 mL (equivalent to 15 times the antibody concentration) to the mixture. Incubate the mixture at 22°C for 30 minutes.

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

[0379] Characterization of antibody-drug conjugates: using operation E(ε) from Example 6 D,280 =6384 and ε D,370 =16180), operation G and operation H were used to characterize the obtained antibody-drug conjugate.

[0380] The concentration of the antibody-drug conjugates measured and calculated by operation E was 11.08 mg / mL, and the average payload per antibody-drug conjugate measured and calculated by operation E was 9.15. Figure 6 A shows the detection map of aggregates, and the content of aggregates in the antibody-drug conjugate HuIgG-LP1-DAR8, measured by operation G, is 4.38%. Figure 6 B shows the detection chromatogram of the hydrophobic interaction 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 the antibody-drug conjugate tisoxumab-MMAE (referred to as: reference ADC)

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

[0383] Antibody reduction: Performed according to operation B in Example 6 (the extinction coefficient of the antibody at 280 nm was 1.508 mL mg). - 1 cm -1 Following procedure C, the antibody culture medium was exchanged with PBS 7.0 / EDTA, resulting in an antibody concentration of 7.318 mg / mL. 23.21 μL of 5 mM TCEP solution (equivalent to 2.04 times the antibody concentration) was added to 1127.36 μL of reference antibody aqueous solution, along with 300 μL of 50 mM PBS 7.0 and 49.43 μL of ultrapure water. The mixture was incubated at 37°C for 2 hours.

[0384] Antibody and adapter-loador coupling: The above mixture was incubated at 4°C for 10 minutes. The adapter-loador VC-MMAE (DC Chemicals, DC50025) was dissolved in DMA, and then 79.66 μL (equivalent to 7 times the antibody content) was added to the mixture. The reaction mixture was incubated at 22°C for 30 minutes.

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

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

[0387] The concentration of the antibody-drug conjugate calculated by operation B was 5.01 mg / mL. The average payload per antibody-drug conjugate in the reference ADC, measured and calculated by operations F and G, was 3.73. Figure 7 A shows the detection map of aggregates, and the aggregate content in the reference ADC measured by operation G is 0.92%. Figure 7 B shows the detection chromatogram of the hydrophobic interaction of the reference ADC as measured by operation H. The HNSTD (maximum dose without serious toxicity) of the cynomolgus monkey reference ADC is 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 (Gibco, product number 25200072) in culture dishes. The digested cells were centrifuged at 1000 rpm for 5 minutes and blocked with 10% goat serum (Nanjing Senbeijia Biotechnology Co., Ltd., product number SRJ-SE-GO012) for 30 minutes. Then, Mu01 and Mu02 antibodies prepared in Examples 4 and 5, respectively, were added to 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 (Jackson Immuno Research, product number 115-545-003) was added at a dilution of 1:500 and incubated for 1 hour. After washing twice with PBS, the products were analyzed by flow cytometry.

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

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

[0392] MDA-MB-231 (human breast cancer, from Zhejiang Meisen Cell Technology Co., Ltd.) cells were cultured to a cell density of 80%. Cells were collected and added to 96-well plates, and the cell density was adjusted to 2-5 × 10⁻⁵. 4 / mL. Add 100μl to each well. Serially dilute 3-fold the Mu01 prepared in Example 4, the Mu02 prepared in Example 5, or the negative control IgG (mouse anti-IgG, purchased from Abimate Pharmaceuticals (Shanghai) Co., Ltd., product number M070890) at an initial concentration of 30nM, and then add them to the cell culture medium. Simultaneously add 2μg / mL of goat anti-mouse antibody conjugated with MMAE (goat anti-mouse antibody purchased from Jackson, catalog number 115-545-003). Culture the cells for 3 days. Observe cell viability periodically during this period. After 3 days, add 15μL of CCK-8 kit stock solution (purchased from Icoin Biotechnology Co., Ltd., catalog number ATUOC1303) directly to a 96-well plate. Incubate at 37°C for 0.5 to 2 hours, then measure the absorbance at 450nm using a microplate reader, and plot cell viability curves based on absorbance values ​​and antibody dilution gradients.

[0393] The method for conjugating the goat anti-mouse antibody with MMAE is similar to that in Comparative Example 2.

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

[0395] Experimental Example 3: Affinity determination of Hu01 candidate molecule to BXPC3 cells

[0396] BxPC3 cells (human pancreatic cancer cells, obtained from the Cell Bank of the Chinese Academy of Sciences) were digested with trypsin (Gibco, product number 25200072) in culture dishes. The digested cells were centrifuged at 1000 rpm for 5 minutes and blocked with 10% goat serum (Nanjing Senbeijia Biotechnology Co., Ltd., product number SRJ-SE-GO012) for 30 minutes. Then, the Hu01 antibodies L5H2, L5H1, L3H1, and L3H2 prepared in Example 4 were serially diluted 3-fold at an initial concentration of 500 nM. The Hu01 antibodies were then added to the cells and incubated for 1 hour. After washing twice with PBS, FITC-labeled goat anti-human antibody (Sigma, product number FS9512-2ML) was added at a 1:500 ratio 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 the same as that of Mu01 prepared in Example 4, and whose constant region is the same as that of human IgG1 described in Example 4.

[0398] like Figure 10 As shown, all four candidate molecules of the Hu01 antibody showed good affinity for BXPC3 cells, with L3H1 exhibiting the highest affinity.

[0399] Experiment Example 4: Affinity determination of Hu02 candidate molecules to BXPC3 cells

[0400] BxPC3 cells (human pancreatic cancer cells, obtained from the Cell Bank of the Chinese Academy of Sciences) were digested with trypsin (Gibco, product number 25200072) in culture dishes. The digested cells were centrifuged at 1000 rpm for 5 minutes and blocked with 10% goat serum (Nanjing Senbeijia Biotechnology Co., Ltd., product number SRJ-SE-GO012) for 30 minutes. Then, the Hu02 antibodies L1H1, L1H2, L1H3, L1H4, L1H5, L1H6, L2H1, L2H2, L2H3, L2H5, and L2H6 prepared in Example 5 were serially diluted 3-fold at an initial concentration of 500 nM. The Hu02 antibodies were then added to the cells and incubated for 1 hour. After washing twice with PBS, FITC-labeled goat anti-human antibody (Sigma, product number FS9512-2ML) was added at a dilution of 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 the same as that of Mu02 prepared in Example 5, and whose constant region is the same as that of human IgG1 described in Example 4.

[0402] like Figure 11 As shown in Table 5, all 11 candidate molecules of the Hu02 antibody showed good affinity for BXPC3 cells, with L2H6 showing the highest affinity and L1H2 showing good affinity.

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

[0404]

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

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

[0407] 30 μL of PPP reagent (a mixture of 5 pM tissue factor and 4 μM phospholipids, purchased from Stago, product number TS30.00) was added to human plasma containing 50 μg / mL CTI (corn trypsin inhibitor, purchased from Enzyme Research) and diluted antibody Hu01-L3H1 prepared in Example 4, Hu02-L1H2 prepared in Example 5, or negative control phosphate-buffered saline (PBS), and incubated at 37°C for 10 minutes. 120 μL of the mixture was added to a reaction vessel, and then a tissue factor-dependent thrombin formation reaction was initiated by adding 30 μL of FluCa reagent (containing calcium and thrombin substrate, purchased from Stago, product number TS50.00). Parameters were automatically exported using a TC TechnoClone instrument.

[0408] like Figure 12 As shown, the inhibition rates of thrombin peak value by Hu01-L3H1 and Hu02-L1H2 were lower than those of the reference antibody. These results indicate that Hu01-L3H1 and Hu02-L1H2 have less impact on coagulation function and lower coagulation toxicity compared to the in vitro reference antibody.

[0409] Experiment 6: In vitro killing effect of ADCs Hu01-L3H1-LP1-DAR8, Hu01-L3H1-LP1-DAR4, and Hu02-L1H2-LP1-DAR8 on 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 achieve a cell density of 80%. Cells were collected and seeded in 96-well plates, and the cell density was adjusted to 2-5 × 10⁻⁵ cells / well. 4 / ml. 100 μL of cells were seeded in each well. ADC molecules were serially diluted 3-fold at an initial concentration of 300 nM and then added to the cell culture medium and incubated for 5 days. Cell apoptosis was observed periodically during this period. After 5 days, 15 μL of CCK-8 kit stock solution (purchased from Acinetobacter Biotechnology Co., Ltd., product number ATUOC1303) was added directly to each 96-well plate and incubated at 37°C for 0.5-2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell survival curves were plotted based on the absorbance values ​​and antibody dilution gradients.

[0411] The Hu01-L3H1-LP1-DAR8 prepared in Example 7, the Hu01-L3H1-LP1-DAR4 prepared in Example 8, the Hu02-L1H2-LP1-DAR8 prepared in Example 9, and the isotype control HuIgG-LP1-DAR8 prepared in Comparative Example 1 were selected as ADC molecules.

[0412] Figure 13 , Figure 14 and Figure 15 Cell survival curves of Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 in KYSE150, 5637, and SW780 cells are shown. The results indicate that Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 exhibit significant cytotoxic effects on these cell lines in vitro, with Hu01-L3H1-LP1-DAR8 showing better cytotoxicity.

[0413] Figure 16 Cell survival curves of Hu01-L3H1-LP1-DAR8, Hu01-L3H1-LP1-DAR4, and Hu02-L1H2-LP-DAR8 against Detroit 562 cells in vitro are shown. The results indicate that the cytotoxicity of the ADCs is significant in vitro, and the cytotoxicity from highest to lowest is Hu01-L3H1-LP1-DAR8 > Hu01-L3H1-LP1-DAR4 > Hu02-L1H2-LP1-DAR8.

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

[0415] Six-week-old Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. They were treated with 1.0 × 10⁻⁶ ppm. 7 NCI-H292 (human lung cancer cells, obtained from the Chinese Academy of Sciences Cell Bank) cells were subcutaneously injected into each mouse to construct a CDX (cell-derived xenograft) mouse model. When the average tumor volume was approximately 100 mm², the cells were used. 3 At the same time, Hu01-L3H1-LP1-DAR8 prepared in Example 7 at 2.5 mpk (mg / kg), 5 mpk, and 10 mpk, Hu02-L1H2-LP1-DAR8 prepared in Example 9 at 10 mpk, the reference ADC prepared in Comparative Example 2 at 3 mpk, and HuIgG-LP1-DAR8 prepared in Comparative Example 1 at 10 mpk were administered intravenously. After administration, tumor volume was measured twice weekly using calipers, and tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0416] Figure 17 The in vivo tumor-suppressive effects of Hu01-L3H1-LP1-DAR8, Hu02-L1H2-LP1-DAR8, and the reference ADC in the NCI-H292 CDX mouse model were demonstrated. It showed that Hu01-L3H1-LP1-DAR8 and Hu02-L1H2-LP1-DAR8 exhibited similar tumor-suppressive efficacy to the reference ADC. Furthermore, the tumor-suppressive effect of Hu01-L3H1-LP1-DAR8 showed a dose-dependent effect based on the tumor inhibition curves at different doses.

[0417] Figure 18 The effects of Hu01-L3H1-LP1-DAR8, Hu02-L1H2-LP1-DAR8, and the reference ADC on mouse body weight are shown. The results indicate that the above ADC molecules have minimal effect on mouse body weight, suggesting that they do not cause significant gastrointestinal toxicity at this dose.

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

[0419] Six-week-old Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. They were treated with 5.0 × 10⁻⁶ mice. 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 a CDX mouse model. When the average tumor volume was approximately 130 mm², the cells were used. 3 At the same time, Hu01-L3H1-LP1-DAR8 prepared in Example 7 at 5 mg / kg and 10 mg / kg, the reference ADC prepared in Comparative Example 2 at 3 mg / kg, and HuIgG-LP1-DAR8 prepared in Comparative Example 1 at 10 mg / kg were injected intravenously. Tumor volume was measured and calculated according to the method in Experimental Example 7.

[0420] Figure 19 The in vivo tumor-suppressive effects of Hu01-L3H1-LP1-DAR8 and the reference ADC on the NCI-H226 CDX mouse model were demonstrated. Results showed that the tumor-suppressive effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg was superior to that of the reference ADC at a dose of 3 mg / kg. The tumor-suppressive effect of Hu01-L3H1-LP1-DAR8 at a dose of 5 mg / kg was similar to that of the reference ADC at a dose of 3 mg / kg.

[0421] Experimental Example 9: Tumor inhibitory effects of Hu01-L3H1-LP1-DAR8 and reference ADC on a mouse model constructed using IGROWV1 cells.

[0422] Six-week-old Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. They were treated with 1.0 × 10⁻⁶ ppm. 7 IGROV1 cells (human ovarian cancer, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) were subcutaneously injected into each mouse to construct a CDX mouse model. When the average tumor volume was approximately 100 mm², the CDX mouse model was established. 3 At the same time, 10 mpk of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 3 mpk of the reference ADC prepared in Comparative Example 2, and 10 mpk of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were injected intravenously. Tumor volume was measured and calculated according to the method in Experimental Example 7.

[0423] Figure 20 The in vivo tumor-suppressive effects of Hu01-L3H1-LP1-DAR8 and the reference ADC on a CDX mouse model constructed using IGROV1 cells were demonstrated. The results showed that the tumor-suppressive effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg was superior to 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 using SW780 cells.

[0425] Six-week-old Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. 4.0 × 10⁻⁶ mice were used. 6 SW780 cells (human bladder cancer, obtained from Zhejiang Meisen Cell Technology Co., Ltd.) were subcutaneously injected into each mouse to construct a CDX mouse model. When the average tumor volume was approximately 100 mm², the CDX mouse model was established. 3 At the same time, 10 mg / kg of Hu01-L3H1-LP1-DAR8 prepared in Example 7, 3 mg / kg of the reference ADC prepared in Comparative Example 2, and 10 mg / kg of HuIgG-LP1-DAR8 prepared in Comparative Example 1 were injected intravenously. Tumor volume was measured and calculated according to the method in Experimental Example 7.

[0426] Figure 21 The in vivo tumor-suppressive effects of Hu01-L3H1-LP1-DAR8 and the reference ADC on a CDX mouse model constructed using SW780 cells were demonstrated. The results showed that the tumor-suppressive effect of Hu01-L3H1-LP1-DAR8 at a dose of 10 mg / kg was superior to 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 a PDX1 mouse model constructed from human lung cancer tissue

[0428] Six-week-old female nude mice (NU / NU) weighing 18g-21g were purchased from Beijing Vital River Laboratory 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 (PDX) model. The average tumor volume was approximately 160 mmHg. 3 At that time, 10 mg / kg of Hu01-L3H1-LP1-DAR8 prepared in Example 7 and PBS were injected intravenously, respectively. Tumor volume was measured and calculated according to the method in Experimental Example 7.

[0429] Figure 22 The in vivo tumor-suppressive effects of Hu01-L3H1-LP1-DAR8 and PBS on a PDX1 mouse model were demonstrated. Results showed that a 10 mg / kg dose of Hu01-L3H1-LP1-DAR8 significantly inhibited tumor growth compared to PBS. Furthermore, on day 22, tumors were completely eliminated in all five mice, indicating that Hu01-L3H1-LP1-DAR8 has a significant therapeutic effect on tumor suppression.

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

[0431] Six-week-old female nude mice (NU / NU) weighing 18g-21g were purchased from Beijing Vital River Laboratory 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. The tumor volume was approximately 160 mm². 3 At the same time, 10 mg / kg of Hu01-L3H1-LP1-DAR4 prepared in Example 8, 3 mg / kg of the reference ADC prepared in Comparative Example 2, and PBS were injected intravenously, respectively. Tumor volume was measured and calculated according to the method in Experimental Example 7.

[0432] Figure 23 The in vivo tumor-suppressive effects of Hu01-L3H1-LP1-DAR4 and the reference ADC on the PDX2 mouse model were demonstrated. The results showed that the tumor-suppressive effect of Hu01-L3H1-LP1-DAR4 at a dose of 10 mg / kg was significantly greater than that of the reference ADC, indicating that Hu01-L3H1-LP1-DAR4 has significant therapeutic efficacy in tumor suppression.

[0433] Experimental Example 13: Toxicological Experiment of Hu01-L3H1-LP1-DAR8 on Cynomolgus Monkeys

[0434] Toxicological experiments were conducted on cynomolgus monkeys to test the efficacy of Hu01-L3H1-LP1-DAR8 through repeated administration.

[0435] Six cynomolgus macaques (purchased from Guangxi Frontier Biotechnology Co., Ltd.) were randomly divided into three groups, with one male and one female in each group. The solvent, 10 mg / kg Hu01-L3H1-LP1-DAR8, and 30 mg / kg Hu01-L3H1-LP1-DAR8 were administered intravenously on days 1, 22, and 43, respectively. During the experiment, any abnormalities in the animals were observed, and blood samples were collected for complete blood count and blood biochemical analysis. On day 50, the animals were euthanized, and samples were taken for pathological analysis. As shown in Table 6, none of the test substances caused death in this experiment. The target organs associated with the test substances were bone marrow, intestine, thymus, and spleen. The HNSTD (maximum dose without serious toxicity) of Hu01-L3H1-LP1-DAR8 was 30 mg / kg, indicating that Hu01-L3H1-LP1-DAR8 is safe.

[0436] Table 6. Results of toxicological experiments on cynomolgus monkeys.

[0437]

[0438]

[0439] ADCs with the novel linker presented in this paper exhibit better safety profiles than ADCs with a payload MMAE analog, a microtubule inhibitor toxin. Specifically, ADCs linked to VC-MMAE (e.g., vetisolumumab) have an HNSTD of 3 mpk (data from the U.S. Food and Drug Administration) and are linked to ZymeLink. TM Auristatin-linked ADCs (e.g., XB002 / ICON-2) have HNSTDs ranging from 10 mpk to 18 mpk (World ADC Digital, September 15–18, 2020). The ADC presented in this article has a significantly higher HNSTD than existing technologies and a longer therapeutic window. Furthermore, the ADC presented in this article demonstrates superior in vivo antitumor efficacy.

[0440] While specific embodiments have been described, the applicant or a person skilled in the art may conceive of alternatives, modifications, variations, improvements, and substantial equivalents that are currently unforeseeable or unforeseeable. Therefore, the appended claims and their possible modifications are intended to cover all such alternatives, modifications, variations, 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 HCDR 1, 2, and 3, and the VL comprises LCDR 1, 2, and 3; wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is as shown in IYPGX1GDX2 (SEQ ID NO: 2), the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3; and the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is as shown in LTS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 5; CDRs are defined or numbered by the IMGT system; Where X1 is D or Q, and X2 is S or A.

2. The isolated antibody or its antigen-binding fragment according to claim 1, wherein... X1 is D, and X2 is S; X1 is Q, and X2 is S; or X1 is Q, and X2 is A.

3. The isolated antibody or its antigen-binding fragment according to claim 1, comprising: VH, which has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 6, and VL, which has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 7; VH, which has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8, and VL, which has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 15; VH, which has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8, and VL, which has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 17; VH having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9, and VL having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 15; or VH has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9, and VL has at least 95% identity with the amino acid sequence shown in SEQ ID NO:

17.

4. The isolated antibody or its antigen-binding fragment according to claim 1, wherein... The VH contains the amino acid sequence shown in SEQ ID NO: 6, and the VL contains the amino acid sequence shown in SEQ ID NO: 7; The VH contains the amino acid sequence shown in SEQ ID NO: 8, and the VL contains the amino acid sequence shown in SEQ ID NO: 15; The VH contains the amino acid sequence shown in SEQ ID NO: 8, and the VL contains the amino acid sequence shown in SEQ ID NO: 17; The VH contains the amino acid sequence shown in SEQ ID NO: 9, and the VL contains the amino acid sequence shown in SEQ ID NO: 15; or The VH contains the amino acid sequence shown in SEQ ID NO: 9, and the VL contains the amino acid sequence shown in SEQ ID NO:

17.

5. The isolated antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO: 35 and a light chain constant region containing the amino acid sequence shown in SEQ ID NO:

36.

6. The isolated antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain containing the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 35, and a light chain containing the amino acid sequences shown in SEQ ID NO: 15 and SEQ ID NO:

36.

7. A nucleic acid encoding the antibody of claim 1 or an antigen-binding fragment thereof.

8. The nucleic acid according to claim 7, wherein the nucleic acid encoding the VH comprises the nucleotide sequence shown in SEQ ID NO: 18, and the nucleic acid encoding the VL comprises the nucleotide sequence shown in SEQ ID NO:

19.

9. A vector comprising the nucleic acid of claim 7.

10. A host cell comprising the nucleic acid of claim 7.

11. An antibody-drug conjugate of Formula I, Ab-(LD)n(I) Or its pharmaceutically acceptable salt; wherein Ab is the isolated antibody or its antigen-binding fragment as described in claim 1; L is a connector covalently connected to Ab and D respectively; D represents the payload; n is an integer from 1 to 10.

12. The antibody-drug conjugate of claim 11, wherein the L comprises a cleavable peptide cleaved by cathepsin B.

13. The antibody-drug conjugate of claim 11, wherein the L comprises an amino acid unit, the amino acid unit comprising a dipeptide, tripeptide, tetrapeptide or pentapeptide.

14. The antibody-drug conjugate according to claim 13, wherein the amino acid unit is Val Cit, Val Ala, Glu Val Cit, Ala Ala Asn, Gly-Val-Cit, Gly-Gly-Gly or Gly-Gly-Phe-Gly.

15. The antibody-drug conjugate of claim 11, wherein the L comprises at least one spacer in the form of at least one self-degrading spacer.

16. The antibody-drug conjugate according to claim 15, wherein the self-degrading spacer is p-aminophenoxycarbonyl (PABC) or p-aminobenzyl (PAB).

17. The antibody-drug conjugate of claim 15, wherein the L comprises a cleavable peptide, and the cleavable peptide is directly spliced ​​to the spacer.

18. The antibody-drug conjugate of claim 15, wherein L comprises -L1-L2-L3-, L1 represents -(succinimide-3-yl-N)-(CH2)m 1 -C(=O)-, -CH2-C(=O)-NH-(CH2)m 2 -C(=O)- or -C(=O)-(CH2)m 3 -C(=O)-, where m 1 m represents an integer from 2 to 8. 2 Represents integers from 2 to 8, and m 3 L1 represents an integer from 2 to 8; L2 represents an amino acid unit; L3 represents a self-degrading spacer.

19. The antibody-drug conjugate according to claim 11, wherein L is: -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O-GGFG-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O-GGFG-PABC-; -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-PABC-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2o-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O-VA-PABC-); -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-PABC-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2CH2CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2-O-CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-O-CH2-C(=O-); -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; or -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-.

20. The antibody-drug conjugate of claim 16, wherein the p-aminophenoxycarbonyl (PABC) or p-aminobenzyl (PAB) group comprises polysarcosine (poly-N-methylglycine) residues.

21. The antibody-drug conjugate according to claim 11, wherein L is... ； ； ； ;or 。 22. The antibody-drug conjugate of claim 11, wherein the payload is at least one cytotoxic agent, wherein the at least one cytotoxic agent includes a tubulin inhibitor or a topoisomerase inhibitor; the tubulin inhibitor includes aurestatin or a derivative thereof, maytansine or a derivative thereof; the topoisomerase inhibitor includes camptothecin or a derivative thereof.

23. The antibody-drug conjugate according to claim 11, wherein the antibody-drug conjugate is ， ， ， ,or n represents the DAR value from 1 to 10 or from 4 to 10.

24. The antibody-drug conjugate according to claim 23, wherein the antibody-drug conjugate is Furthermore, where Ab is An isolated antibody or antigen-binding fragment thereof that binds to CD142, including The amino acid sequences of LCDR1 (as shown in SEQ ID NO: 4), LCDR2 (as shown in LTS), and LCDR3 (as shown in SEQ ID NO: 5) are as follows: The amino acid sequences are as shown in SEQ ID NO: 1 for HCDR1, as shown in IYPGX1GDX2 (SEQ ID NO: 2) for HCDR2, where X1 is D and X2 is S, and as shown in SEQ ID NO: 3 for HCDR3. CDRs are defined or numbered by the IMGT system; or An isolated antibody or antigen-binding fragment thereof that binds to CD142, including VL, which has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 15, and VH having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8, wherein any sequence variation is located outside the complementary variable region; or, An isolated antibody or antigen-binding fragment thereof that binds to CD142, including A light chain containing the variable region of the amino acid sequence shown in SEQ ID NO: 15, and a constant region containing the amino acid sequence shown in SEQ ID NO: 36, and The heavy chain comprising the variable region of the heavy chain shown in SEQ ID NO: 8, and the constant region comprising the amino acid sequence shown in SEQ ID NO:

35.

25. The antibody-drug conjugate according to claim 24, wherein n represents a DAR value of 4 to 8.

26. A pharmaceutical composition comprising the antibody-drug conjugate of claim 11, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

27. A kit comprising the antibody-drug conjugate of claim 11.

28. Use of the antibody-drug conjugate of claim 11 in the preparation of a therapeutic agent for treating ovarian cancer, gastric cancer, esophageal cancer, cervical cancer, prostate cancer, pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer, bladder cancer, melanoma, and renal cancer.

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