An antibody drug conjugate

Abstract

This invention relates to the application of a novel anti-CDCP-1 antibody or its antigen-binding fragment, linker compound, deuterated loading, and linker-loading compound in antibody-drug conjugates. Specifically, it involves an anti-CDCP-1 antibody with good cell-binding activity and affinity, a linker that exhibits strong toxicity and significant inhibitory activity against tumor cells, and a loading compound. Antibody-drug conjugates prepared using the anti-CDCP-1 antibody, linker, and loading compound can produce good inhibitory activity against tumors in both in vivo and in vitro, and have good safety. These advantages are of great value for the development of antibody-drug conjugates in clinical treatment.

Description

An antibody drug conjugate

[0001] Priority

[0002] This application claims priority to the Chinese Patent Application No. CN202410732731.9, filed on June 6, 2024, entitled "A Deuterated Camptothecin Derivative and Ligand Drug Conjugate", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the technical field of biological medicine, in particular to an antibody and antigen-binding fragment thereof, a camptothecin compound, a linker and an antibody drug conjugate thereof. BACKGROUND

[0004] Antibody drug conjugates (ADCs) are a new type of biological drugs coupled by monoclonal antibodies and highly potent cytotoxic payloads through bioactive linkers. The drug action mechanism of ADC is that the ADC realizes specific binding between the target antigen after internalization into the target cancer cells, and the cytotoxic payload exerts cytotoxicity to kill cancer cells after the ADC is internalized into the cancer cells (Fu Z, Li S, Han S, et al. Antibody drug conjugate: the "biological missile" for targeted cancer therapy [J]. Signal Transduction and Targeted Therapy, (2022) 7:93.). ADC can selectively deliver highly potent cytotoxic agents to target cancer cells by specifically targeting cell surface markers that are highly differentially expressed between tumor and healthy cells, and thus is hailed as "biological missile" in the field of anti-tumor therapy. Since 2000, the first ADC drug Mylotarg (gemtuzumab ozogamicin) has been approved for marketing, and as of January 2025, only 16 ADCs (see Table 1 for basic information) have been approved for marketing worldwide. Due to the requirement of patient target point positivity for ADC application and the resistance of ADC after use, there is still a large unmet treatment demand in the clinic. In addition, ADC drugs also have the characteristics of high development difficulty and high technical barriers. Table 1. Basic information of ADC drugs approved for marketing worldwide

[0005] CDCP1 (CUB domain-containing protein 1) is a transmembrane glycoprotein, which is widely expressed in various tumor cells, including colorectal cancer, lung cancer, breast cancer, etc. Studies have shown that CDCP1 plays an important role in the occurrence, invasion and metastasis of tumors, and has become one of the potential targets for tumor treatment (Kaur, H., et al. (2016). "CDCP1: A Novel Target for Cancer Immunotherapy." Journal of Cancer Research and Therapeutics, 12(3), 821-832. DOI: 10.4103 / 0973-1482.188207. Siegel, R. L., et al. (2021). "Cancer Statistics, 2021." CA: A Cancer Journal for Clinicians, 71(1), 7-33. DOI: 10.3322 / caac.21654.). With the gradual recognition of the importance of CDCP1 in tumors, the development of antibody drug conjugates based on CDCP1 is becoming a research hotspot in the field of tumor treatment. The application of new CDCP1 antibody drug conjugates can achieve precise targeted therapy, so that the anti-cancer drugs can act specifically on CDCP1 positive tumor cells, maximizing the therapeutic effect. There are currently antibody drug conjugates targeting CDCP1 in preclinical stage research, and no exploration molecules have entered the clinical stage. The development of CDCP1 antibody drug conjugates still faces many challenges, including antibody specificity, drug load design, conjugation method optimization, drug stability in vivo and release mechanism, etc. How to overcome these technical difficulties and improve the clinical efficacy of CDCP1 antibody drug conjugates has become the core problem of current research.

[0006] From the perspective of drug load design, the types of toxins currently available for constructing effective ADCs are very limited. The 16 marketed ADCs involve only 10 types of toxins, including Calicheamicin, PE38 (a fragment of Pseudomonas aeruginosa exotoxin a), PBD (pyrrolobenzodiazepine), auristatin compounds (MMAE, MMAF), maytansinoid compounds (such as DM1, DM4), camptothecin compounds (such as DXD, SN38, KL610023), etc.

[0007] Among them, the camptothecin compound is a general term for a class of compounds obtained by modifying the group at one or more sites of camptothecin (CPT). Camptothecin (CPT) is a topoisomerase I inhibitor that can be isolated from the bark of Camptotheca acuminata (a traditional Chinese medicine for treating cancer), and its specific molecular structure was first disclosed by Wall et al. in 1966 (Wall M. Plant antitumor agents I. The isolation and structure of camptothecin-A novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata [J]. J Am Chem Soc, 1966, 88.), as shown below,

[0008] Camptothecin (CPT) can bind to topoisomerase and DNA through hydrogen bonds, prevent DNA recombination and cause cell apoptosis by damaging DNA. However, due to the poor water solubility, low targeting, high toxicity to normal tissues, and instability in plasma, camptothecin has many limitations in application (Dancey J, Eisenhauer E A. Current perspectives on camptothecins in cancer treatment [J]. Br J Cancer, 1996; Proulx M E, Desormeaux A, Marquis J F, et al. Treatment of visceral leishmaniasis with sterically stabilized liposomes containing camptothecin [J]. Antimicrobial Agent and Chemotherapy, 2019.). Since the structure of camptothecin was first disclosed more than 50 years ago, although a large number of camptothecin compounds have been disclosed later, and some camptothecin compounds have been applied as toxins to construct antibody drug conjugates. However, the number of antibody drug conjugates containing camptothecin compounds and having significant drug properties is still very small. As can be seen from Table 1, among the 16 currently marketed antibody drug conjugates, only three use camptothecin compounds, namely DXD in trastuzumab deruxtecan, SN38 in Sacituzumab Govitecan and KL6100023 (structures are shown below) in Sacituzumab Tirumotecan. Although antibody drug conjugates developed based on these three loads have achieved remarkable results, due to the different killing effects and safety characteristics of each load on different types of tumor diseases, there is a high degree of difference, and the combination with different linkers will also bring differences in treatment effect. Therefore, there is still a great demand and need for improvement for the development of new camptothecin loads.

[0009] From the perspective of drug composition and technical characteristics, ADC has undergone four iterations in the development process, namely: first-generation ADC drugs, second-generation ADC drugs, third-generation ADC drugs, and fourth-generation ADC drugs. Among them, ① the first-generation ADC drug (Mylotarg, Besponsa) is mainly composed of conventional chemotherapy drugs connected with mouse antibodies through non-cleavable linkers. The efficacy of such ADC drugs is not superior to that of free cytotoxic drugs, and there are often problems such as immunogenicity, uncontrollable release of toxic load, antibody aggregation, metabolic instability, narrow safety window, and high challenges in drug development; ② the second-generation ADC drug (Adcetris, Kadcyla) optimizes monoclonal antibodies, cytotoxic loads, and linkers. Compared with IgG4, this type of antibody is more suitable for biological coupling with small-molecule loads and has higher cancer cell targeting ability. Another breakthrough is the use of more effective cytotoxic drugs, which improves their water solubility and coupling level. In addition, the second-generation ADC improves the linker to achieve better plasma stability and drug uniformity. Through the improvement of the three components of antibodies, linkers, and toxins, the second-generation ADC has good clinical effects and safety, but still has the shortcomings of insufficient therapeutic window due to off-target toxicity, aggregation or rapid clearance of high DAR value ADC; ③ the third-generation ADC drug (such as: Polivy, Padcev) introduces site-specific coupling technology to produce ADC with good characterization DAR value (2 or 4) and expected cytotoxicity. Such ADCs exhibit lower off-target toxicity, lower immunogenicity, and higher pharmacokinetic efficiency, but still have problems such as insufficient effective load release and high drug resistance; ④ the fourth-generation ADC drug (Enhertu, Trodelvy) adopts a high DAR value (4-8) and a low-to-moderate activity cytotoxic load strategy. The load has a high bystander effect and good efficacy for tumors with low and high expression of tumor surface antigens. The two fourth-generation ADC drugs currently on the market, Enhertu and Trodelvy, both use camptothecin compounds as cytotoxic loads, and the linker-toxin of Enhertu will fall off in the body circulation, causing cytotoxicity. Tropelvy uses a payload with weak activity and poor stability in the body circulation.

[0010] Deuterium is a stable form of non-radioactive isotope of hydrogen in nature, which was first discovered and named in 1932. Deuterium and hydrogen have very similar physical and chemical properties. Because deuterium has a larger atomic mass than hydrogen, the vibration frequency of C-D bond is lower than that of C-H bond, the ground state energy is lower, and the dissociation activation energy is increased, so the C-D bond is more stable than the C-H bond (6-9 times). After replacing hydrogen in the drug molecule with deuterium, the metabolic site may be blocked, and the generation of toxic metabolites may be reduced. In addition, deuterated drugs can remain stable under various metabolic enzymes, slow down the clearance rate of the system and prolong the half-life of the drug in the body. Therefore, deuterated drug therapy can reduce the single dose by reducing the side effects of the drug while not affecting the pharmacological activity of the drug. In 2017, the FDA approved the first deuterated drug in history - Austedo of Teva, which was approved for marketing in China in May 2020. Austedo is a deuterated version of tetrabenazine, and the original drug tetrabenazine has been the mainstream drug for treating Huntington's disease, but it has defects such as short half-life and low patient dependence. By replacing the hydrogen atoms in the two methoxy groups on the benzene ring of tetrabenazine with deuterium atoms, the deuterated tetrabenazine significantly reduces the speed of drug metabolism, increases the half-life of the drug, and reduces the amount of drug administration, while also inhibiting the withdrawal reaction caused by the decrease in drug blood concentration. Although deuterium has many advantages in drug discovery, there are still unpredictability of deuterium effect, large difference in the effect of deuterium at different sites, and synthesis challenges in controlling the position and degree of deuterium enrichment. Currently, there is no deuterated antibody-drug conjugate on the market.

[0011] In summary, after decades of development, the number of approved drugs and targets is still limited, and only 16 ADC drugs have been approved, targeting 13 targets. Considering the complexity and diversity of cancer, the existing drugs cannot meet the needs of all patients. Although antibody-drug conjugates containing camptothecin compounds have significantly improved treatment effects, there are still differences in drug efficacy, safety and drug resistance. Therefore, it is crucial to further develop and screen high-affinity antibodies targeting highly expressed tumor-specific targets and suitable payloads and linker structures, which determine the effectiveness and safety of antibody-drug conjugates.

[0012] Therefore, in view of the above problems and challenges, there is still a need to improve the payload, antibody, and linker to develop antibody-drug conjugates with improved safety and efficacy or to impart differential therapeutic effects. SUMMARY

[0013] The present application provides an antibody or antigen-binding fragment targeting CDCP1, a novel linker, a payload, and the use thereof in antibody-drug conjugates.

[0014] The present application also provides an antibody drug conjugate (ADC) comprising the above-mentioned antibody, a nucleotide encoding the above-mentioned CDCP1 antibody, a polynucleotide combination, an expression vector and a host cell, a pharmaceutical composition comprising the above-mentioned CDCP1 antibody, the antibody drug conjugate, and their use in the preparation of a medicament for treating or preventing cancer.

[0015] In one aspect, an antibody drug conjugate targeting CDCP1 is provided, the antibody drug conjugate comprising a targeting moiety targeting CDCP1, the targeting moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0016] (1) the heavy chain variable region (VH) comprises the following 3 CDRs: CDR-H1 of the sequence set forth in SEQ ID NO: 19, CDR-H2 of the sequence set forth in SEQ ID NO: 20, and CDR-H3 of the sequence set forth in SEQ ID NO: 21; and,

[0017] the light chain variable region (VL) comprises the following 3 CDRs: CDR-L1 of the sequence set forth in SEQ ID NO: 22, CDR-L2 of the sequence set forth in SEQ ID NO: 23, and CDR-L3 of the sequence set forth in SEQ ID NO: 24;

[0018] or

[0019] (2) the heavy chain variable region (VH) comprises the following 3 CDRs: CDR-H1 of the sequence set forth in SEQ ID NO: 29, CDR-H2 of the sequence set forth in SEQ ID NO: 30, and CDR-H3 of the sequence set forth in SEQ ID NO: 31; and,

[0020] the light chain variable region (VL) comprises the following 3 CDRs: CDR-L1 of the sequence set forth in SEQ ID NO: 32, CDR-L2 of the sequence set forth in SEQ ID NO: 33, and CDR-L3 of the sequence set forth in SEQ ID NO: 34;

[0021] the CDRs are defined according to the IMGT numbering system.

[0022] In some embodiments, the antibody drug conjugate, wherein,

[0023] the heavy chain variable region (VH) comprises a sequence as set forth in SEQ ID NO: 25 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25, and the light chain variable region (VL) comprises a sequence as set forth in SEQ ID NO: 26 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26; or

[0024] the heavy chain variable region (VH) comprises a sequence as set forth in SEQ ID NO: 35 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35, and the light chain variable region (VL) comprises a sequence as set forth in SEQ ID NO: 36 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36.

[0025] In some embodiments, the targeting moiety of the antibody drug conjugate is selected from an antibody or an antigen binding fragment.

[0026] In some embodiments of the antibody drug conjugate, the antibody or antigen binding fragment is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody, or a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, a Fv fragment, a dAb, a Fd, a single chain antibody (scFv).

[0027] In some embodiments, the targeting moiety of the antibody drug conjugate further comprises a constant region of an immunoglobulin, further preferred wherein the immunoglobulin is selected from IgGl, IgG2, IgG3, or IgG4.

[0028] In some embodiments, the antibody moiety of the antibody drug conjugate comprises a heavy chain and a light chain, wherein,

[0029] the heavy chain comprises a sequence as set forth in SEQ ID NO: 27 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27, and the light chain comprises a sequence as set forth in SEQ ID NO: 28 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28; or

[0030] the heavy chain comprises a sequence as set forth in SEQ ID NO: 37 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37, and the light chain comprises a sequence as set forth in SEQ ID NO: 38 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 38.

[0031] In some embodiments, the antibody drug conjugate has a general structure of Ab-U n , wherein Ab represents a targeting moiety, U represents a linker and a payload moiety; n is an integer selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of U attached to Ab is 1, 2, 3, 4, 5, 6, 7, or 8, respectively; Ab is covalently attached to the payload via a linker.

[0032] In some embodiments, the antibody drug conjugate has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the payload moieties attached to Ab via one or more of the linkers.

[0033] In some embodiments, the antibody drug conjugate has a payload moiety selected from

[0034] is the site where the payload and linker are attached.

[0035] In some embodiments, the antibody drug conjugate has a linker and a payload moiety represented by the following formula (I):

[0036] wherein,

[0037] L is a linker to attach the antibody, selected from the following structures:

[0038] Q1, Q2, Q3, Q4 are each independently a single bond or selected from the group consisting of -(CH2CH20)p-, -NH-(CH20CH2)p-C(O)-, -(NHCH2C(O))-0-, -NH(CH2CH20)p-CH2CH2C(O)-, -(CH2CH20)p-CH2CH2C(O)-, -CH2-Y-(CH2CH20)p-CH2CH2C(O)-, -NHCH2-Y-(CH2CH20)p-CH2CH2C(O)-, -(CH2)-Y-, -NHCH2C(O)CH2C(O)-; said Y is selected from C3-8 cycloalkyl or C3-8 cycloheteroalkyl or C1-8 cyclo- unsaturated heteroalkyl, said heteroalkyl comprising 1-3 atoms selected from N, O or S, p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0039] A1 is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0040] A2 is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0041] A3 is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0042] S1, S3 are independently selected from

[0043] S2, S4 are independently selected from -NH-CH2- or

[0044] D1 is a first drug unit, D2 is a second drug unit;

[0045] W is selected from

[0046] m1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0047] m2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0048] m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0049] m4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0050] m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0051] m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0052] m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0053] m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0054] m9 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0055] m10 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0056] the subscript n is 0 or 1.

[0057] In some embodiments, the payload is independently selected from the following structures:

[0058] is the site of attachment of the payload and linker, wherein: R1, R2, R3, R4, R5are independently selected from H, D, T, at least one of R1, R2, R3, R4, R5is D, R7is independently selected from -NH- or -NH-C(O)-CH2-O-, the drug unit is attached to the linker structure through either of the hydroxyl or amine groups present.

[0059] In some embodiments, the payload is independently selected from the following structures:

[0060]

[0061] is the site of attachment of the payload and linker, wherein: R1, R2, R3, R4, R5are independently selected from H, D, T, at least one of R1, R2, R3, R4, R5is D, R7is independently selected from -NH- or -NH-C(O)-CH2-O-, the drug unit is attached to the linker structure through either of the hydroxyl or amine groups present.

[0062] In some embodiments, the L is selected from the following structures:

[0063] In some embodiments, the A1, A2, A3are independently selected from a single bond or a peptide comprising amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit).

[0064] Preferably, said A1, A2, A3are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

[0065] Preferably, said A1, A2, A3are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

[0066] -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; preferably, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Gly-, -Phe-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Val-Ala-, -Ala-Ala-Ala-; more preferably, said linker comprises the following structure: -Val-Ala-.

[0067] In some embodiments, said Q1, Q2, Q3, Q4linker groups are independently selected from a single bond or the following structures:

[0068] wherein,

[0069] m11is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0070] m12 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0071] m13 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0072] m14 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0073] m15 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0074] m16 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0075] m17 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0076] m18 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0077] Preferably, the Q1, Q2, Q3, Q4linking groups are independently selected from a single bond or the following structures:

[0078] In some embodiments, the S1, S3are independently selected from the following structures: wherein m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0079] Preferably, the S1, S3are independently selected from the following structures:

[0080] In some embodiments, the S2, S4are selected from -NH-CH2- or the following structures: wherein m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0081] Preferably, the S2, S4are selected from -NH-CH2- or the following structures:

[0082] In some embodiments, the W is selected from the following structures:

[0083] In some embodiments, the antibody drug conjugate is selected from the following structures:

[0084] said n8 is selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of Linker- Payload moieties attached to said Ab is 1, 2, 3, 4, 5, 6, 7, or 8; said antibody is attached to the Linker-Payload moieties via a disulfide bond on a cysteine in the antibody.

[0085] In some embodiments, the antibody drug conjugate is selected from the following structures:

[0086] said n2 is selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of Linker- Payload moieties attached to said Ab is 1, 2, 3, 4, 5, 6, 7, or 8; said antibody is attached to the Linker-Payload moieties via a disulfide bond on a cysteine in the antibody.

[0087] In one aspect, a method of treating a tumor is provided, the method comprising administering to a patient an effective amount of any of the above-described antibody drug conjugates.

[0088] In some embodiments, the tumor is CDCP-1 positive.

[0089] In some embodiments, the tumor is lung cancer or pancreatic cancer; further preferred, the tumor is lung cancer squamous carcinoma, lung adenocarcinoma, non-small cell lung cancer, or pancreatic adenocarcinoma.

[0090] In one aspect, an antibody or antigen-binding fragment thereof targeting CDCP1 is provided, the antibody or antigen-binding fragment thereof comprising the following heavy chain variable region (VH) and light chain variable region (VL), wherein:

[0091] (1) a VH comprising the following 3 CDRs: CDR-H1 comprising the sequence set forth in SEQ ID NO: 19, CDR-H2 comprising the sequence set forth in SEQ ID NO: 20, CDR-H3 comprising the sequence set forth in SEQ ID NO: 21; and, a VL comprising the following 3 CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 22, CDR-L2 comprising the sequence set forth in SEQ ID NO: 23, CDR-L3 comprising the sequence set forth in SEQ ID NO: 24; or

[0092] (2) a VH comprising the following 3 CDRs: CDR-H1 comprising the sequence set forth in SEQ ID NO: 29, CDR-H2 comprising the sequence set forth in SEQ ID NO: 30, CDR-H3 comprising the sequence set forth in SEQ ID NO: 31; and,

[0093] a VL comprising a CDR-L1 comprising a sequence as set forth in SEQ ID NO: 32, a CDR-L2 comprising a sequence as set forth in SEQ ID NO: 33, and a CDR-L3 comprising a sequence as set forth in SEQ ID NO: 34;

[0094] The CDRs are defined according to the IMGT numbering system.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant protein or Fab fragment comprising the antigen-binding fragment, a F(ab') fragment, a F(ab')2 fragment, a Fv fragment, a dAb, a Fd, a single chain antibody (scFv).

[0096] In some embodiments, the antibody or antigen-binding fragment further comprises a constant region of an immunoglobulin, wherein the immunoglobulin is selected from IgGl, IgG2, IgG3, or IgG4.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups:

[0098] (1) a sequence of the heavy chain variable region is as set forth in SEQ ID NO: 25, or a sequence having the same CDRs 1-3 as SEQ ID NO: 25 and having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25; and a sequence of the light chain variable region is as set forth in SEQ ID NO: 26, or a sequence having the same CDRs 1-3 as SEQ ID NO: 26 and having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26; or

[0099] (2) a sequence of the heavy chain variable region is as set forth in SEQ ID NO: 35, or a sequence having the same CDRs 1-3 as SEQ ID NO: 35 and having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35; and a sequence of the light chain variable region is as set forth in SEQ ID NO: 36, or a sequence having the same CDRs 1-3 as SEQ ID NO: 36 and having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36.

[0100] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups:

[0101] (1) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 25; and the sequence of the light chain variable region is set forth in SEQ ID NO: 26;

[0102] or

[0103] (2) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 35; and

[0104] the sequence of the light chain variable region is set forth in SEQ ID NO: 36.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain selected from any one of the following groups:

[0106] (1) the heavy chain comprises a sequence set forth in SEQ ID NO: 27 or a sequence that is greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27; and

[0107] the light chain comprises a sequence set forth in SEQ ID NO: 28 or a sequence that is greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28;

[0108] or

[0109] (2) the heavy chain comprises a sequence set forth in SEQ ID NO: 37 or a sequence that is greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37; and

[0110] the light chain comprises a sequence set forth in SEQ ID NO: 38 or a sequence that is greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain selected from any one of the following groups:

[0112] (1) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 27; and the amino acid sequence of the light chain is set forth in SEQ ID NO: 28; or

[0113] (2) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 37; and the amino acid sequence of the light chain is set forth in SEQ ID NO: 38.

[0114] In one aspect, there is provided a fusion protein comprising any of the above-mentioned antibodies or antigen-binding fragments thereof.

[0115] In one aspect, there is provided an isolated polynucleotide encoding any of the above-mentioned antibodies or antigen-binding fragments thereof or fusion proteins.

[0116] In one aspect, there is provided a nucleic acid construct comprising the above-mentioned polynucleotide.

[0117] In some embodiments, the nucleic acid construct is an expression vector, wherein the polynucleotide is operably linked to regulatory sequences permitting its expression of the encoded polypeptide in a host cell or a cell-free expression system.

[0118] In one aspect, there is provided a host cell comprising the above-mentioned polynucleotide or the above-mentioned nucleic acid construct; preferably, the host cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, an E. coli cell; more preferably, the host cell is selected from the group consisting of a CHO cell, a NS0 cell, a Sp2 / 0 cell or a BHK cell.

[0119] In one aspect, there is provided a method for producing the above-mentioned antibodies or antigen-binding fragments thereof or fusion proteins, comprising culturing the above-mentioned host cell under conditions permitting expression of the above-mentioned nucleic acid construct, and recovering the expressed protein produced from the culture.

[0120] In one aspect, there is provided the use of the above-mentioned antibodies or antigen-binding fragments thereof or fusion proteins or polynucleotides in the manufacture of a medicament.

[0121] Preferably, the medicament comprises a mono-specific antibody, a bi-specific antibody, a multi-specific antibody, an antibody-drug conjugate or an antibody-nuclide conjugate.

[0122] In one aspect, there is provided a linker as shown in formula (II),

[0123] wherein L is a linker to attach an antibody, selected from the group consisting of:

[0124] wherein Q1, Q2, Q3, Q4are each independently a single bond or selected from the group consisting of -(CH2CH2O)p-, -NH-(CH2OCH2)p-C(O)-, -(NHCH2C(O))p-O-, -NH(CH2CH2O)p-CH2CH2C(O)-, -(CH2CH2O)p-CH2CH2C(O)-, -CH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -NHCH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -(CH2)-Y-, -NHCH2C(O)CH2C(O)-; said Y is selected from C3-8cycloalkyl or C3-8cycloheteroalkyl or C3-8cycloheteroalkenyl, said heteroalkyl comprising 1-3 atoms selected from N, O or S, p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0125] A1is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0126] A2is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0127] A3is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0128] S1, S3are independently selected from

[0129] S2, S4are independently selected from -NH-CH2- or

[0130] W is selected from

[0131] m1is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0132] m2is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0133] m3is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0134] m4is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0135] m5is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0136] m6is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0137] m7is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0138] m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0139] m9 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0140] m10 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0141] the subscript n3 is 0 or 1.

[0142] In some embodiments, the L is selected from the following structures:

[0143] In some embodiments, the A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

[0144] In some embodiments, the A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

[0145] In some embodiments, the A1, A2, A3 are independently selected from a single bond or a peptide consisting of the following structure:

[0146] -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; preferably, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Gly-, -Phe-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Val-Ala-, -Ala-Ala-Ala-; more preferably, the linker comprises the following structure: -Val-Ala-.

[0147] In some embodiments, the Q1, Q2, Q3, Q4linker groups are independently selected from a single bond or the following structures: wherein,

[0148] m11is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0149] m12is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0150] m13is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0151] m14is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0152] m15is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0153] m16is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0154] m17is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0155] m18 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0156] In some embodiments, the Q1, Q2, Q3, Q4linker is independently selected from a single bond or the following structures:

[0157] In some embodiments, the S1, S3is independently selected from the following structures: wherein,

[0158] m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0159] In some embodiments, the S1, S3is independently selected from the following structures:

[0160] In some embodiments, the S2, S4is selected from -NH-CH2- or the following structures: wherein,

[0161] m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0162] In some embodiments, the S2, S4is selected from -NH-CH2- or the following structures:

[0163] In some embodiments, the W is selected from the following structures:

[0164] In some embodiments, the W is selected from the following structures:

[0165] In some embodiments, the linker is selected from the following structures:

[0166] wherein, the is attached to an antibody; the is attached to a drug unit.

[0167] In some embodiments, the linker is selected from the following structures:

[0168] wherein, the is attached to an antibody; the attached to the payload compound.

[0169] In one aspect, a linker-payload compound is provided as shown in formula (III):

[0170] wherein L is an antibody-attaching linker selected from the group consisting of:

[0171] wherein Q1, Q2, Q3, Q4 are each independently a single bond or selected from the group consisting of -(CH2CH2O)p-, -NH-(CH2OCH2)p-C(O)-, -(NHCH2C(O))p-O-, -NH(CH2CH2O)p-CH2CH2C(O)-, -(CH2CH2O)p-CH2CH2C(O)-, -CH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -NHCH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -(CH2)-Y-, -NHCH2C(O)CH2C(O)-; said Y is selected from C3-8 cycloalkyl or C3-8 cycloheteroalkyl or C3-8 cyclo-unsaturated heteroalkyl, said heteroalkyl comprising 1-3 atoms selected from N, O or S, p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0172] A1 is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0173] A2 is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0174] A3 is a single bond or a peptide consisting of 1, 2, 3, 4 amino acid residues;

[0175] S1, S3 are independently selected from

[0176] S2, S4 are independently selected from -NH-CH2- or

[0177] D1 is a first payload compound, D2 is a second payload compound, D1, D2 are independently selected from a compound as shown in formula (IV):

[0178] wherein R1, R2, R3, R4, R5 are independently selected from hydrogen (H), deuterium (D), at least one, two, three or four of R1, R2, R3, R4, R5 are D; R6 is selected from -NH2 or -NH-C(O)-CH2-OH; said D1, D2 are linked to S1, S2 or S4 through either of the present hydroxyl or amine groups;

[0179] W is selected from

[0180] m1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0181] m2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0182] m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0183] m4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0184] m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0185] m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0186] m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0187] m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0188] m9 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0189] m10 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0190] the subscript n5 is 0 or 1.

[0191] In linker-payload compounds in some embodiments, the D1, D2 are independently selected from the following structures:

[0192] In some preferred embodiments, the D1, D2 are independently selected from the following structures:

[0193]

[0194] In some more preferred embodiments, the structure of D1, D2 is:

[0195] In linker-payload compounds in some embodiments, the -L is selected from the following structures:

[0196] In some embodiments, said A1, A2, A3are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

[0197] In some embodiments, said A1, A2, A3are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val).

[0198] In some embodiments, said A1, A2, A3are independently selected from a single bond or a peptide comprising the following structure: -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; preferably, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Gly-, -Phe-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Val-Ala-, -Ala-Ala-Ala-; more preferably, said linker comprises the following structure: -Val-Ala-.

[0199] In some embodiments, the Q1, Q2, Q3, Q4linker groups of the linker-payload compound are independently selected from a single bond or the following structures: wherein,

[0200] m11is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0201] m12is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0202] m13is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0203] m14is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0204] m15is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0205] m16is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0206] m17is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0207] m18is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0208] In some embodiments, the Q1, Q2, Q3, Q4linker groups of the linker-payload compound are independently selected from a single bond or the following structures:

[0209] In some embodiments, the S1, S3of the linker-payload compound are independently selected from the following structures:

[0210] wherein, m5is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m6is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m7is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0211] In some embodiments, the S1, S3of the linker-payload compound are independently selected from the following structures:

[0212] In some embodiments, the S2, S4of the linker-payload compound are selected from -NH-CH2- or the following structures:

[0213] wherein, m8is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0214] In some embodiments, S2, S4 of the linker-payload compound is selected from -NH-CH2- or the following structures:

[0215] In some embodiments, W of the linker-payload compound is selected from the following structures:

[0216] In some embodiments, W of the linker-payload compound is selected from the following structures:

[0217] In some embodiments, the linker-payload compound is selected from the following structures:

[0218] In some preferred embodiments, the linker-payload compound is selected from the following structures:

[0219] In one aspect, a linker-payload compound is provided, which is a stereoisomer, a pharmaceutically acceptable salt, or a solvate of the above linker-payload.

[0220] In one aspect, a deuterated camptothecin compound of Formula (V) is provided:

[0221] wherein: R1, R2, R3, R4, R5are independently selected from hydrogen (H), deuterium (D), at least one, two, three, or four of R1, R2, R3, R4, R5are D; R6is selected from -NH2or -NH-C(O)-CH2-OH.

[0222] In some embodiments, the structure of Formula (V) is selected from the group consisting of the following compounds:

[0223] In some preferred embodiments, the structure of Formula (V) is: In some more preferred embodiments, the structure of the compound is:

[0224] In some embodiments, the structure of the deuterated camptothecin compound of Formula (V) is selected from the group consisting of the following compounds:

[0225] In some preferred embodiments, the structure shown in Formula (V) is:

[0226] In some more preferred embodiments, the structure of the compound is:

[0227] In one aspect, a compound is provided, the compound being a stereoisomer, a pharmaceutically acceptable salt, or a solvate of a compound of I-1 to I-8, I-1a to I-8a.

[0228] In one aspect, a compound is provided, the compound being a stereoisomer, a pharmaceutically acceptable salt, or a solvate of a compound of I-1 to I-8, I-1a to I-8a.

[0229]

[0230] L2 is a linker that attaches an antibody;

[0231] W2 is an optional group;

[0232] A5 is a peptide consisting of 1, 2, 3, or 4 amino acid residues;

[0233] A6 is a peptide consisting of 1, 2, 3, or 4 amino acid residues;

[0234] S2, S4 are independently selected from -NH-CH2- or

[0235] D3 is a first payload compound;

[0236] D4 is a second payload compound;

[0237] m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0238] X3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0239] X4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0240] X5 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0241] In some embodiments, the L2 comprises the following structure:

[0242] wherein R8 is selected from N, O, or S; X1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and X2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0243] In some preferred embodiments, the L2 comprises the following structure:

[0244] wherein R8 is selected from N, O, or S; X1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0245] In some preferred embodiments, the L2 comprises the following structure:

[0246] wherein X2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0247] In some preferred embodiments, the L2 comprises the following structure:

[0248] wherein R8 is selected from N, O, or S.

[0249] In some preferred embodiments, the L2 comprises the following structure:

[0250]

[0251]

[0252] In some preferred embodiments, the L2 structure is as follows:

[0253]

[0254] In some embodiments, the W comprises the following structure: wherein M9 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; M10 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0255] In some preferred embodiments, the W is selected from the following structure:

[0256]

[0257] In some preferred embodiments, the W is selected from the following structure:

[0258]

[0259] In some embodiments, said A1, A2, A3are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val).

[0260] In some embodiments, said A1, A2, A3are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val).

[0261] In some embodiments, said A1, A2, A3are independently selected from a single bond or a peptide comprising the following structure:

[0262] -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; preferably, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Gly-, -Phe-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Val-Ala-, or -Ala-Ala-Ala-; more preferably, said linker comprises the following structure: -Val-Ala-.

[0263] In some embodiments, the first and second payload compounds are independently selected from the group consisting of cytotoxic molecules, immunopotentiators, and radioisotopes, the cytotoxic molecules including but not limited to microtubulin inhibitors or DNA damaging agents; further preferably, the microtubulin inhibitors include but are not limited to dolastatin and auristatin cytotoxic molecules, maytansine cytotoxic molecules; the DNA damaging agents include but are not limited to calicheamicin, duocarmycin, antrmycin derivative PBD, camptothecins and camptothecin derivatives, SN-38, Dxd; further preferably, the auristatin cytotoxic molecules include but are not limited to MMAE or MMAF or their derivatives, the maytansine cytotoxic molecules include but are not limited to DM1, DM4 or their derivatives; further preferably, the camptothecin derivatives are selected from the group consisting of the compounds of claim 65 or 66, which are attached to S2 or S4 by loss of a hydrogen from either of the hydroxyl or amine groups present.

[0264] In some embodiments, S2, S4 are selected from -NH-CH2- or the following structures:

[0265]

[0266] In some embodiments, the compound is selected from the group consisting of: wherein D3 is a first payload compound; D4 is a second payload compound.

[0267] In some preferred embodiments, the compound is selected from the group consisting of: wherein D3 is a first payload compound; D4 is a second payload compound.

[0268] In one aspect, a compound is provided, which is a stereoisomer, a pharmaceutically acceptable salt, or a solvate of the above compound.

[0269] In one aspect, the use of the above linker in the preparation of an antibody drug conjugate is provided.

[0270] In one aspect, the use of the above linker-payload compound in the preparation of an antibody drug conjugate is provided.

[0271] In one aspect, the use of any of the above compounds in the manufacture of an antibody drug conjugate is provided.

[0272] In one aspect, an antibody drug conjugate comprising a targeting unit, a linker, a payload is provided, wherein the linker is any of the above linker structures, and / or the payload is selected from any of the above compounds.

[0273] In some embodiments, the targeting unit of the antibody drug conjugate is selected from any of the above antibodies or antigen binding fragments thereof.

[0274] In some embodiments, the linker-payload of the antibody drug conjugate is selected from any of the above compounds.

[0275] In some embodiments, the antibody drug conjugate is selected from the following structures:

[0276] wherein, Ab is a targeting unit, and n9 is selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0277] In some embodiments, the antibody drug conjugate is selected from the following structures:

[0278] wherein, Ab is a targeting unit; n4 is selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of linker-payload moieties attached to the Ab is 1, 2, 3, 4, 5, 6, 7, or 8.

[0279] In some preferred embodiments, the antibody drug conjugate has the structure of ADC-1300; the targeting unit Ab is an antibody or antigen binding fragment thereof targeting CDCP1; in some more preferred embodiments, the targeting unit Ab is any of the above antibodies or antigen binding fragments thereof.

[0280] In one aspect, a pharmaceutical composition comprising any of the above antibody drug conjugates and / or any of the above antibodies or antigen binding fragments thereof, and a pharmaceutically acceptable carrier is provided.

[0281] In one aspect, the use of any of the above antibodies or antigen binding fragments thereof or fusion proteins, any of the above polynucleotides, any of the above nucleic acid constructs, any of the above antibody drug conjugates, or any of the above pharmaceutical compositions in the manufacture of a medicament for treating or preventing cancer is provided.

[0282] In some implementations, the cancer is CDCP1-positive cancer.

[0283] In some embodiments, the CDCP1-positive cancer is lung cancer or pancreatic cancer; in some preferred embodiments, the CDCP1-positive cancer is squamous cell carcinoma of lung cancer, adenocarcinoma of lung cancer, non-small cell lung cancer, or adenocarcinoma of pancreas.

[0284] On the one hand, it relates to a recombinant protein comprising any of the aforementioned antibody or antigen-binding fragments.

[0285] In some embodiments, the recombinant protein is a bispecific antibody or a multispecific antibody.

[0286] On the one hand, it involves the application of any of the aforementioned antibodies or antigen-binding fragments in the preparation of recombinant proteins.

[0287] All references cited in this article, including patents, patent applications, and various publications, are incorporated in their entirety by reference, as if each reference were individually and specifically cited and presented in its entirety in this article.

[0288] The references and inclusions of patent documents in this article are for convenience only and do not reflect any opinion on the validity, patentability, and / or enforceability of such patent documents.

[0289] All headings and subheadings used herein are for convenience only and should not be construed as limiting the invention in any way.

[0290] The use of any and all examples or exemplary wording provided herein (e.g., such as) is intended only to better illustrate the invention and not to limit the scope of the invention, unless otherwise stated. Nothing in the specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0291] This invention includes all modifications and equivalents to the subject matter set forth in the appended claims, where permitted by applicable law. Attached Figure Description

[0292] Figure 1 is a comparison of the internalization efficiency of ADCs with Dxd and dDxd payloads (both antibodies are Ab1 and the linker is DL01) in EBC-1 cells (human lung adenocarcinoma cells).

[0293] Figure 2 is a comparison of the internalization efficiency of ADCs with Dxd and dDxd payloads (both antibodies are Ab2 and both linkers are DL01) in BXPC-3 cells (human in situ pancreatic cancer cells).

[0294] Figure 3 is a graph of xenograft tumor growth inhibition data for anti-CDCPl antibody drug conjugate (MMAE payload) on EBC-1 cells (human lung adenocarcinoma cells);

[0295] Figure 4 is a graph of xenograft tumor growth inhibition data for anti-CDCPl antibody drug conjugate (MMAE payload) on BxPC3 cells (human in situ pancreatic carcinoma cells);

[0296] Figure 5 is a graph of xenograft tumor growth inhibition data for anti-CDCPl antibody drug conjugate (camptothecin class payload) on EBC-1 cells (human lung adenocarcinoma cells) for a 5 mg / kg dose experiment;

[0297] Figure 6 is a graph of xenograft tumor growth inhibition data for anti-CDCPl antibody drug conjugate (camptothecin class payload) on EBC-1 cells (human lung adenocarcinoma cells) for a 2.5 mg / kg dose experiment;

[0298] Figure 7 is a graph of toxicity tolerance test data for anti-CDCPl antibody drug conjugate (payloads dDxd, Dxd, respectively); DETAILED DESCRIPTION DEFINITIONS

[0299] The practice of the present application will employ, unless otherwise indicated, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, and many of these techniques are fully explained in the literature. Such techniques are described in detail in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001); and Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Techniques, 4th Ed., John Wiley & Sons, Inc., Hoboken, N.J. (1999).

[0300] Unless otherwise indicated, the term "alkyl" alone or in part, in the present invention refers to a straight or branched, saturated or unsaturated hydrocarbon group of the indicated number of carbon atoms, which is substituted or unsubstituted (e.g., C1-8alkyl refers to an alkyl group having from 1 to 8 carbon atoms), representative straight chain "C1-8alkyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl; while branched -C3-C8alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated -C2-C8alkyl groups include, but are not limited to, -ethenyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-iso-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl, which alkyl groups can be unsubstituted or can be substituted by one or more groups.

[0301] Unless otherwise indicated, the term "C1-8heteroalkyl" alone or in part, in the present invention refers to a stable straight or branched chain hydrocarbon or combinations thereof having from 1 to 8, preferably 1 to 3, heteroatoms selected from O, N, and S, and which is completely saturated or which contains one or two units of unsaturation, and the nitrogen and sulfur atoms can be optionally oxidized and the nitrogen heteroatom can be optionally quaternized. Heteroatom O, N, S can be placed at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Representative heteroalkyl groups include, but are not limited to, -O-CH3, -CH2-O-CH3, -CH2-CH2-O-CH3, -NH-CH3, -CH2-NH-CH3, -CH2-N(CH3)-CH3, -S-CH3, -CH2-S-CH3, -CH2-CH2-S-CH3. Typically, a C1 to C4heteroalkyl group has 1 to 4 carbon atoms and 1 or 2 heteroatoms.

[0302] Unless otherwise indicated, the term "C3-8cycloalkyl" alone or in part, in the present invention refers to a 3-, 4-, 5-, 6-, 7-, or 8-membered monocyclic or bicyclic, monovalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic carbocyclic ring obtained by removing a hydrogen atom from a ring atom of a parent ring system, representative C3-C8cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.

[0303] Unless otherwise indicated, the term "C3-8heterocycloalkyl" by itself or as part of another term means a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having from 3 to 8 carbon atoms (also referred to as ring members) and from one to four heteroatom ring members independently selected from N, O, P, or S and obtained by removing one hydrogen atom from a ring atom of a parent ring system. Heterocycles in which all ring atoms are involved in the aromatic compound structure are referred to as heteroaryls, otherwise heterocarbocycles.

[0304] Unless otherwise indicated, the term "any functional group" in the term "any functional group substituted group" in the present application includes, but is not limited to, the following groups: -halogen, -Ci-C8alkyl, -C2-C8alkenyl, -C2-C8alkynyl, -O-(Ci-C8alkyl), -O-(C2-C8alkenyl), -O-(C2-C8alkynyl), -aryl, -C(O)R ’ , -OC(O)R ’ , -C(O)OR ’ , -C(O)NH2, -C(O)NHR ’ , -C(O)N(R ’ )2, -NHC(O)R ’ , -SR ’ , -SO3R ’ , -S(O)2R ’ , -S(O)R ’ , -OH, -NO2, -N3, -NH2, -NH(R ’ ), -N(R ’ )2, and -CN, wherein each R ’ is independently selected from -H, -Ci-C8alkyl, -C2-C8alkenyl, -C2-C8alkynyl, or -aryl, wherein said -Ci-C8alkyl, -C2-C8alkenyl, -C2-C8alkynyl, O-(Ci-C8alkyl), -O-(C2-C8alkenyl), -O-(C2-C8alkynyl), and -aryl can be further optionally substituted with one or more groups including, but not limited to: -Ci-C8alkyl, -C2-C8alkenyl, -C2-C8alkynyl, -halogen, -O-(Ci-C8alkyl), -O-(C2-C8alkenyl), -O-(C2-C8alkynyl), -aryl, -C(O)R ” , -OC(O)R ” , -C(O)OR ” , -C(O)NH2, -C(O)NHR ” , -C(O)N(R ” )2, -NHC(O)R ” , -SR ” , -SO3R” -S(O)2R ” -S(O)R ” -OH, -N3, -NH2, -NH(R ” ), -N(R ” )2, and -CN, wherein each R ” is independently selected from -H, -Ci-C8alkyl, -C2-C8alkenyl, -C2-C8alkynyl, or -aryl.

[0305] The term "amino acid" in the present invention includes natural amino acids, molecules containing N and carboxylic acid capable of forming amide bond, molecules of general formula NH2-CHR-COOH or residues within a peptide bearing the parent amino acid, wherein "R" is one of many different side chains. "R" can be a substituent found in natural amino acids. "R" can also refer to a substituent that is not a natural amino acid.

[0306] The term "amino acid residue" in the present invention refers to the portion of an amino acid that remains after the loss of a water molecule when linked to another amino acid.

[0307] The term "stereoisomer" in the present invention refers to a compound having the same chemical composition and connectivity but different positioning of atoms in space that cannot be interconverted by rotation about single bonds.

[0308] The term "pharmaceutically acceptable salt" in the present invention refers to a pharmaceutically acceptable organic or inorganic salt of a compound (linker-drug or its ligand drug conjugate). Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, tartrate, oleate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate salts. Pharmaceutically acceptable salts can involve the incorporation of another molecule such as an acetate ion, a succinate ion, or other counter ion, which can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Additionally, pharmaceutically acceptable salts can have more than one charged atom in its structure. Where multiple charged atoms are part of a pharmaceutically acceptable salt, multiple counter ions can be present, thus a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ions.

[0309] The term "antibody drug conjugate" in the present invention refers to a substance in which a bioactive molecule (a drug molecule) is linked to a targeting unit. In some embodiments of the present invention, the bioactive molecule is linked to the targeting unit via a linker, which is capable of being cleaved under certain conditions (e.g., hydrolytic enzymes and / or pH conditions within a tumor) and / or under certain actions (e.g., lysosomal protease action), thereby separating the bioactive molecule from the targeting unit. In some embodiments of the present invention, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present invention, the bioactive molecule is directly linked to the targeting unit via a covalent bond, which is capable of being cleaved under certain conditions or actions, thereby separating the bioactive molecule from the targeting unit.

[0310] The term "targeting unit" in the present invention refers to a structure that binds or associates with a biological moiety or fragment thereof, including but not limited to an antibody fragment or functional fragment, a surrogate, a variant, a protein ligand, a protein scaffold, an RNA, a DNA, an RNA or DNA fragment, a small molecule ligand, or an antigen binding fragment.

[0311] The term "monoclonal antibody" in the present invention includes, but is not limited to, a human monoclonal antibody, a humanized monoclonal antibody, or a chimeric human-mouse (or other species) monoclonal antibody. Antibodies include full-length antibodies and antigen-binding fragments thereof. Human monoclonal antibodies can be prepared by any of a variety of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982, Meth. Enzymol. 92:3-16).

[0312] The term "antibody" in the present invention can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to a target cell (e.g., a cancer cell antigen, a viral antigen or a microbial antigen), or other antibody that binds to a tumor cell or stroma, "functionally active" referring to the ability of the fragment, derivative or analog to immunospecifically bind to a target cell. Other useful antibodies include, but are not limited to, fragments of antibodies such as, without limitation, F(ab')2 fragments, Fab fragments, Fvs, single-chain antibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, bispecific antibodies, multispecific antibodies, or any other molecule with the same specificity as an antibody; antibodies include modified analogs and derivatives, i.e., analogs and derivatives modified by covalent attachment of any type of molecule, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, and without limitation, derivatives and analogs of antibodies include those that have been further modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be introduced into an antibody by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Antibodies immunospecific for a cancer cell antigen are commercially available or produced by any method known to one skilled in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank database or similar database, from a literature publication, or by routine cloning and sequencing.

[0313] The term "antigen-binding fragment" in the present invention refers primarily to a functional fragment of an antibody that is capable of specifically binding to an antigen, such as a scFv (single-chain antibody), Fv, Fab, F(ab')2, Fab', scFv-Fc, a single-domain antibody, an antibody-like binding protein, a bispecific antibody, a multispecific antibody, or any antibody fragment modified by chemical modification.

[0314] The term "linker" in the present invention has two reactive ends, one end for binding or associating with a biological moiety or fragment thereof, such as a targeting unit, and the other end for conjugating, e.g., a drug. Unless otherwise indicated, the -MC-VC-PAB- linker structure in the present invention is as follows: Unless otherwise indicated, the -MC-GGFG- linker structure in the present invention is as follows:

[0315] "Cancer" in the present invention refers to or describes the physiological condition or disorder that is typically characterized by uncontrolled growth of cells in a mammal, and "tumor" comprises one or more cancerous cells.

[0316] The term "therapeutic agent" in the present application refers to a cytotoxic agent, an immunopotentiator, a radioisotope, a cytostatic agent, a tubulin inhibitor, or a DNA damaging agent, and specifically includes, but is not limited to, cytotoxic molecules of the dolastatin and auristatin classes, cytotoxic molecules of the maytansine class, calicheamicin class, duocarmycin class, the antitumor drug PBD, a camptothecin and camptothecin derivatives, SN-38, Dxd, etc.

[0317] The present application also includes compounds with various radioactive or non-radioactive isotopes of atoms. The atoms comprising the compounds of the present application can also contain unnatural proportions of atomic isotopes at one or more of the atoms, or can be substituted with an atom having an atomic mass different from the atomic mass usually found in nature for these atoms. As examples of isotopes that can be found in nature, there can be mentioned for example hydrogen ( 2 H), tritium ( 3 H), or carbon-14 ( 14 C).

[0318] Unless otherwise indicated, the term "substituted" in the present application means that any group denotes the point of attachment of a structural fragment to the rest of the molecule.

[0319] Unless otherwise indicated, the terms "may be substituted," "may contain," "contains," and other variations thereof in the present application are inclusive or open-ended and do not exclude additional, unrecited elements or method steps, unless otherwise specified.

[0320] Unless otherwise defined, the term "camptothecin derivative" in the present application refers to a compound comprising a camptothecin parent structure substituted with any functional group, wherein the camptothecin parent structure is as shown below:

[0321] Unless otherwise indicated, the term "independently selected from a group" in the present application means that each substituent is selected independently of the other. Thus, each substituent can be the same as or different from the other substituent(s).

[0322] Unless otherwise indicated, the term "linker" in the present application has two reactive ends, one end for binding or associating with a biological moiety or fragment thereof, such as a targeting unit, and the other end for conjugating, for example, a payload.

[0323] Unless otherwise indicated, "treatment" in the present application refers to the reduction or elimination of the disease or condition being addressed. A subject is successfully "treated" if at least one indicator and symptom of the subject shows observable and / or detectable alleviation and / or improvement after the subject has received a therapeutic amount of a compound of the present application or a pharmaceutically acceptable form thereof or a pharmaceutical composition of the present application. It is understood that treatment includes not only complete treatment, but also treatment that does not achieve complete treatment, but that achieves some biologically or medically relevant result.

[0324] Embodiments of the present application will now be described in detail in the following examples, but a person skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Examples

[0325] 1. Preparation of deuterated camptothecin compounds

[0326] Synthesis of compound QSC-200 of Example 1

[0327] Synthesis of QSC-5-01: N-(3-fluoro-5-iodo-4-methylphenyl)acetamide (60.0 g, 0.20 mol) and 3-butyn-1-ol (28.7 g, 0.41 mol) were weighed into 300 mL of DMF (N,N-Dimethylformamide) and stirred to dissolve. TEA (Triethylamine) (82.7 g, 0.82 mol) was added, and after stirring for 10 min, PdCl2(PPh3)2(3.6 g, 0.005 mol) and CuI (3.9 g, 0.02 mol) were added. The reaction was stirred for 4 h. The reaction solution was added dropwise to 3 L of water, and after the dropwise addition was completed, stirring was continued for 1 h. The filter cake was transferred to a 2 L single-neck flask, dissolved in 1 L of EA, and then 12 g of silica gel was added and stirred for 30 min. The color was removed by passing through a silica gel column, and the filtrate was concentrated by rotary evaporation to about 500 mL. 1 L of Heptane was added, and the mixture was slurried for 16 h. The mixture was filtered, and the filter cake was dried to obtain 33.8 g of QSC-5-01, with a yield of 70.3%. LC-MS (ESI+) 235.10 [M+H] + .

[0328] Synthesis of QSC-5-02: QSC-5-01 (30.0 g, 0.13 mol), Pt02(3.0 g, 10% w / w) were weighed, added into 1200 mL THF (tetrahydrofuran), 300 mL D20, and dissolved under deuterium atmosphere, and reacted at room temperature for 16 h. 2 L EA (Ethyl acrylate), 1 L H20, 5.0 g activated carbon were added to the system, stirred for 0.5 h, and filtered with diatomite. The filtrate was separated, the organic phase was concentrated to obtain a white solid, which was slurried with MTBE (Methyl tert-Butyl Ether): Heptane = 1:1 500 mL for 2 h, filtered, and the filter cake was dried to obtain 29.3 g of QSC-5-02 with a yield of 94.5%. LC-MS (ESI+) 243.16 [M+H] + .

[0329] Synthesis of QSC-5-03: QSC-5-02 (28.9 g, 0.12 mol) was weighed, added into 600 mL acetone, and stirred and dispersed. 58 mL Cr03·H2SO4 was slowly added dropwise at 0°C, and the reaction was continued for 10-30 min. 50 mL isopropyl alcohol was added to the reaction system, stirred for 20 min, filtered, and the filter cake was eluted with 200 mL EA. After the acetone was removed from the filtrate by rotary evaporation, 1 L EA and 500 mL H20 were added to stir and separate. The organic phase was washed with purified water twice, 500 mL each time, and the organic phase was retained. NaOH solution (NaOH solution was prepared by adding 7.2 g NaOH to 300 mL purified water and stirring and dissolving) was added to the organic phase, stirred for 5 min, separated, and the aqueous phase was retained. The aqueous phase was washed with EA twice, 300 mL each time. The aqueous phase was adjusted to pH 2-3 with 6N hydrochloric acid, and a large amount of solid was precipitated. The aqueous phase was extracted with EA three times, 300 mL each time, the organic phases were combined, concentrated, and the obtained solid was stirred with Heptane for 2 h, filtered, and dried to obtain 18.0 g of QSC-5-03 with a yield of 58.9%. LC-MS (ESI+) 257.14 [M+H] + .

[0330] Synthesis of QSC-5-04: QSC-5-03 (18.0 g, 0.07 mol) was weighed, 33 mL TFA (Trifluoroacetic acid) was added, stirred, and cooled to 0 °C. 29 mL TFAA (Trifluoroacetic anhydride) was added dropwise, and the reaction was carried out for 30 min. The reaction solution was added dropwise to 500 mL H2O, and after the dropwise addition was completed, stirring was continued for 30 min. Filtration was performed, and the filter cake was washed with purified water. The filter cake was transferred to a 2 L bottle, dissolved with 500 mL EA, 250 mL purified water was added, stirred for 10 min, and the liquid was separated. The organic phase was washed with 250 mL saturated Na2CO3, dried over anhydrous sodium sulfate, and rotary evaporation was performed to obtain a solid. The solid was slurried with 250 mL Heptane for 2 h, filtered and dried to obtain 14.2 g of QSC-5-04, with a yield of 85.0%. LC-MS (ESI+) 239.13 [M+H] + .

[0331] Synthesis of QSC-5-05: 400 mL THF and 140 mL tert-butyl alcohol were added to a 500 mL bottle, and the temperature was cooled to 0 °C. 19.7 g of potassium tert-butoxide (19.7 g, 0.17 mol) was added and stirred for 10 min. QSC-5-04 (14.0 g, 0.06 mol) was dissolved in 130 mL THF and slowly added dropwise to the reaction system. After the dropwise addition was completed, stirring was continued for 10 min. Butyl nitrite (9.6 g, 0.09 mol) was dissolved in 30 mL THF and slowly added dropwise to the reaction system. After the dropwise addition was completed, the reaction was continued for 30 min. 200 mL saturated NH4Cl was added to the reaction system, stirred for 10 min, and the liquid was separated. 1 L EA, 400 mL purified water was added to the organic phase, stirred for 10 min, and the liquid was separated. The organic phase was washed once more with 400 mL H2O, and rotary evaporation was performed to obtain a solid. The obtained solid was slurried with 200 mL Heptane for 2 h, filtered and dried to obtain 13.7 g of QSC-5-05, with a yield of 87%. LC-MS (ESI+) 268.12 [M+H] + .

[0332] Synthesis of QSC-5: QSC-5-05 (13.5 g, 0.05 mol) was weighed into a flask, THF (810 mL) was added, Pt / C (1.4 g, 10% w / w) was added, acetic anhydride (13.5 g, 0.10 mol) was added, and the reaction was stirred under a hydrogen atmosphere for 16 h. The system was filtered using celite to aid filtration, and the organic phase was concentrated by rotary evaporation to give a solid. The solid was slurried in 200 mL EA for 2 h and filtered. The filter cake was dissolved in 400 mL DCM (dichloromethane), 5 g of activated carbon was added, and the mixture was stirred for 1 h. The mixture was filtered using celite to aid filtration, and the organic phase was retained. The organic phase was concentrated by rotary evaporation to give a solid. The solid was slurried in 200 mL heptane for 1 h, filtered, and dried to give 9.0 g of QSC-5 in 60.4% yield. LC-MS (ESI+) 296.15 [M+H] + .

[0333] Synthesis of QSC-6: QSC-5 (8.01 g, 27.0 mmol) was dissolved in 320 mL of 2N HCl and the reaction was stirred at 35 °C for 2.0 h. After the reaction was complete, the pH was adjusted to 7-8 using 0.5N NaCO3 solution, and the reaction was extracted three times with dichloromethane. The organic phase was dried and concentrated by rotary evaporation, and then dried under vacuum to give QSC-6 6.76 g in 98.5% yield. LC-MS (ESI + ) 255.1 [M+H] + .

[0334] Synthesis of QSC-7: QSC-6 (6.76 g, 26.6 mmol), (S)-4-ethyl-4-hydroxy-7,8- dihydro-lH-pyrano[3,4-F]indolizine-3,6,10(4H)-one (8.57 g, 32.6 mmol), and p-toluenesulfonic acid monohydrate (2.53 g, 13.3 mmol) were dissolved in 272 mL of toluene and 27 mL of DMF, and the reaction was refluxed for 10 h. After the reaction was complete, the reaction was allowed to cool to room temperature, MTBE and n-heptane were added, and the reaction was filtered to give a crude product, which was dried under vacuum to give QSC-7 15.36 g in 120.4% yield. LC-MS (ESI + ) 482.2 [M+H] + .

[0335] Synthesis of QSC-200: QSC-7 crude (15.36 g) was weighed into a reaction bottle, 150 mL purified water and 75 mL methanesulfonic acid were added for dissolution, and the reaction was stirred at 110°C for 5 h. After the reaction was completed, the mother liquor was filtered while hot, 750 mL anhydrous ethanol was added to the mother liquor, and the mixture was filtered and dried under vacuum to obtain 5.4 g of crude product; the crude product was dissolved in 81 mL purified water and 81 mL methanesulfonic acid, 324 mL anhydrous ethanol was slowly added dropwise for crystallization, and the mixture was filtered and dried under vacuum to obtain 4.46 g of QSC-200, with a yield of 30.9%. LC-MS (ESI+): 439.2 [M+H] + .

[0336] Synthesis of compounds dDxd, d1Dxd of Example 2

[0337] 1. Synthesis of compound dDxd

[0338]

[0339] QSC-200 (43.9 mg, 0.1 mmol), hydroxyacetic acid (15.1 mg, 0.2 mmol), and HATU (76.1 mg, 0.2 mmol) were dissolved in 2 mL of DMF, DIPEA (49.7 μL, 0.3 mmol) was added, and the mixture was stirred at room temperature for 3 h. The mixture was separated and purified by preparative liquid chromatography, and then freeze-dried to obtain 25.9 mg of dDxd, with a yield of 52.1%. LC-MS (ESI+): 498.5 [M+H] + .

[0340] 2. Synthesis of compound d1Dxd

[0341] 1) Synthesis of compound d1DX8951

[0342]

[0343] Compound DX8951 (5 g, 9.4 mmol) was stirred in 50 mL of ethanol and 40 mL of water, and then triethylamine (1.9 g, 18.8 mmol), sodium tungstate dihydrate (3.4 g, 10.3 mmol), and 25 mL of hydrogen peroxide were added dropwise. The mixture was stirred at 50°C for 8 h. Then 100 mL of purified water was added to the system, and the mixture was stirred for 1 h and then filtered. The filter cake was slurried in 250 mL of N,N-dimethylformamide for 4 h, and then filtered to obtain 3.1 g of target compound d1DX8951-01, with a yield of 73%. LC-MS (ESI+) 450.15 [M+H] + .

[0344] Compound d1DX8951-01 (2.0 g, 4.4 mmol), methanesulfonic acid (856 mg, 8.8 mmol), 100 mg 5% palladium on carbon (1 mg, 0.005 mmol) were dispersed with 60 mL tetrahydrofuran and 30 mL deuterium oxide and stirred under deuterium gas for 4 h. The tetrahydrofuran was removed by rotary evaporation and 30 mL methanesulfonic acid was added to dissolve the residue. The target compound d1DX8951 was obtained by crystallization from 300 mL ethanol and filtration, 1.1 g, yield 46%. LC-MS (ESI+) 437.18 [M+H] + .

[0345] 2) Synthesis of compound d1Dxd

[0346]

[0347] d1DX8951 (53.2 mg, 0.1 mmol), hydroxyacetic acid (15.0 mg, 0.2 mmol), HATU (76.0 mg, 0.2 mmol) were dissolved in 2 mL DMF, DIPEA (N,N- Diisopropylethylamine) (50.1 μL, 0.3 mmol) was added, stirred at room temperature for 3 h, prepared liquid phase separation and purification, freeze-drying, obtained the target compound d1Dxd 27.3 mg, yield 57.2%. LC-MS (ESI+) 495.5 [M+H] + .

[0348] Example 3 Elimination half-life and AUC analysis

[0349] 1. In vitro liver microsomal half-life evaluation

[0350] Different species incubation mixture: PBS (pH = 7.4), different species liver microsomal protein (final concentration 0.5 mg / mL), compound (final concentration 0.5 μg / mL). 180 μL of incubation solution was taken respectively, pre-incubated in a 37 °C constant temperature water bath for 5 min, then 20 μL of NADPH (nicotinamide adenine dinucleotide phosphate) (final concentration 1 mM) was added to start the reaction, and 0.8 mL of ice-cold acetonitrile was added to terminate the reaction at 0, 5, 10, 20, 30, 60 min respectively. The content of organic solvent in the incubation system should be controlled within 1%.

[0351] The in vitro incubation of Dxd and dDxd was carried out using liver microsomes of cynomolgus monkey and human. The remaining amount of Dxd and dDxd was determined by high performance liquid chromatography. The concentration of the test substance at 0 min was taken as 100%, and the remaining percentage of the test substance at each time point was obtained by comparing the concentration at each time point with the concentration at 0 min. The natural logarithm of the remaining percentage of the test substance at each time point was linearly regressed with the incubation time to obtain the slope (k), and the elimination half-life T 1 / 2 in various liver microsomes was obtained by the formula

[0352] The data of the metabolic stability evaluation of dDxd and Dxd in liver microsomes are shown in Table 2. The half-life of dDxd was shorter than that of Dxd in the liver microsomes of cynomolgus monkey and human in vitro, indicating that dDxd is metabolized faster in the liver and has better safety.

[0353] 2. In vivo elimination half-life and AUC (Area Under Curve) analysis

[0354] The quarantine qualified SD rats were taken, 6 rats in each group, half male and half female. The compounds were prepared on the day of administration in the examples, and the solvent was 5% dimethyl sulfoxide (DMSO) + 10% polyoxyethylene castor oil (solutol) + 85% saline. The rats were weighed before administration, and the administration amount was calculated according to the body weight. The administration dose was 1 mg / kg, and the administration volume was 5 mL / kg. The rats were administered by intravenous injection. Before administration, at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after administration, blood was collected from the jugular vein, about 0.2 mL of each sample was collected, anticoagulated with sodium heparin, placed on ice after collection, and centrifuged to separate the plasma (centrifugation conditions: 6800g, 6 minutes, 2-8°C) within 1 hour. 40 μL of the plasma sample was added with 160 μL of acetonitrile for precipitation, mixed, and centrifuged at 3500 x g for 5-20 minutes. 100 μL of the supernatant after treatment was subjected to LC / MS / MS analysis to determine the concentration of the test compound. The pharmacokinetic parameters were calculated by using Phoenix WinNonlin 7.0 based on the blood drug concentration data at different time points, and AUC 0-t , and T 1 / 2 and their mean and standard deviation were provided.

[0355] The pharmacokinetic test data of dDxd and Dxd in rats are shown in Table 3. dDxd has a shorter half-life than Dxd; the AUC 0-24h of Dxd is 174.99 ± 87.65 h*ng / mL, and the AUC 0-24h90.84 ± 12.23 h*ng / mL, dDxd has lower in vivo exposure, and has better safety. Table 3. Elimination half-life and AUC data of dDxd and dDxd in rats in vivo

[0356] Through the performance test of d1Dxd compound, the test results show that the d1Dxd compound has surprisingly high metabolic stability, good pharmacokinetic characteristics, high cytotoxicity and good safety.

[0357] Preparation of linker-load compound

[0358] Synthesis of compound INT-1 of Example 4

[0359] N-benzyloxycarbonyl-L-phenylalanine (150 g, 0.50 mol), N-hydroxysuccinimide (69.21 g, 0.60 mol) were dissolved with 750 mL of tetrahydrofuran, and the reaction solution was cooled to 0°C. DCC (N,N'-Dicyclohexylcarbodiimide) (124.08 g, 0.60 mol) was weighed and dissolved with 310 mL of tetrahydrofuran under ultrasonic, and the DCC THF solution was added dropwise at a temperature of 0-5°C. The reaction was carried out at room temperature for 4 h. The system was cooled to 5°C, filtered, and the filter cake was washed with a small amount of tetrahydrofuran. The filtrate was replaced with EtOH twice and stirred overnight. Filtration gave 185.42 g of INT-1-1 with a yield of 93%. LC-MS (ESI+) 397.27 [M+H] + .

[0360] Compound INT-1-1 (185.42 g, 0.47 mol) was dissolved with 1390 mL of tetrahydrofuran, and bisglycine (92.70 g, 0.70 mol), sodium bicarbonate (47.16 g, 0.56 mol) were weighed and dissolved with 705 mL of water under ultrasonic. The bisglycine sodium bicarbonate aqueous solution was added dropwise at room temperature. The reaction was carried out at room temperature for 2 h. The reaction solution was adjusted to PH≤2, extracted with DCM three times, washed with 1N HCl once, and dried. Filtration was carried out, and the filtrate was washed with DCM and dried. The filter cake was dried by adding a system of EA:Hep.(1-pyridin-2-yl-ethanol)=2:1 and stirring overnight. Filtration was carried out, and the filter cake was washed with EA and Hep. three times, respectively. The filter cake was dried to obtain the target compound INT-1-2 162.02 g with a yield of 93.78%. LC-MS (ESI+) 414.34 [M+H] + .

[0361] Compound INT-1-2 (20.00 g, 48.38 mmol) was dissolved in 240 mL of tetrahydrofuran, and lead tetraacetate (27.05 g, 128.68 mmol) was added at room temperature. The reaction was allowed to proceed overnight at room temperature. Filtration was performed, and the filter cake was washed with a small amount of tetrahydrofuran. The mother liquor was concentrated, and column chromatography was performed to obtain the target compound INT-1-3 14.55 g, yield 70.36%. LC-MS (ESI+) 450.34 [M+Na] + .

[0362] Compound INT-1-3 (14.00 g, 32.7 mmol) was dissolved in 280 mL of DCM, and pyridine p-toluenesulfonic acid salt (PPTS) (1.62 g, 0.033 mmol) was added at room temperature. Hydroxyacetic acid (3.73 g, 49 mmol) was added, and the reaction was allowed to proceed overnight at reflux. Filtration was performed, and the filter cake was washed with a small amount of dichloromethane. Column chromatography was performed to obtain the target compound INT-1 8.75 g, yield 60.25%. LC-MS (ESI+) 444.34 [M+H] + .

[0363] Synthesis of compound INT-2

[0364] Benzyl oxycarbonyl-L-valyl-L-alanine (160 g, 500 mmol) and N-hydroxysuccinimide (85.71 g, 750 mol) were dissolved in 2000 mL of dichloromethane, and the temperature was lowered to 0°C. The temperature was controlled at 0-10°C, and 2,4,6-collidine (180.64 g, 1490 mmol) was added. The mixture was stirred for 1 h while maintaining the temperature at 0-10°C. A solution of TFAA (156.54 g, 750 mmol) in 300 mL of dichloromethane was added, and the mixture was stirred for 3 h. Filtration was performed to obtain INT-2-1 199 g, yield 95.0%. LC-MS (ESI+) 420.4 [M+H] + .

[0365] Compound INT-2-1 (199 g, 470 mmol) was dissolved in 1600 mL of tetrahydrofuran, and a 5% aqueous solution of sodium bicarbonate (1000 mL) was added. Glycine (42.74 g, 570 mmol) was added, and DME (1,2-Dimethoxyethane) 400 mL was added. The reaction was allowed to proceed at 40°C for 5 h. The reaction solution was added to 2N hydrochloric acid 350 mL, and dichloromethane 2 L was added for extraction. The organic phase was collected and concentrated to obtain the target compound INT-2-2 150.0 g, yield 84.0%. LC-MS (ESI+) 380.3 [M+H] + .

[0366] Compound INT-2-2 (20.0 g, 52.71 mmol) was dissolved in 800 mL of tetrahydrofuran, 5 mL of glacial acetic acid was added, and 35.0 g of lead tetraacetate (118.13 mmol) was added with rapid stirring. The reaction was stirred at room temperature for 3 h. The reaction was centrifuged, and the supernatant was concentrated. 600 mL of dichloromethane was added, and the mixture was washed twice with 100 mL of 1 N hydrochloric acid each time. The mixture was washed once with 100 mL of saturated brine. The mixture was dried over anhydrous sodium sulfate for 1 h, filtered, and the filtrate was concentrated to give compound INT-2-3 (14.3 g, 68.9% yield) as a white solid. LC-MS (ESI+) 395.3 [M+H] + .

[0367] Compound INT-2-3 (7.0 g, 17.8 mmol) was dissolved in 200 mL of dichloromethane, and 29.57 g of benzyl glycolate (178 mmol) and 0.45 g of PPTS (0.18 mmol) were added. The mixture was stirred at reflux overnight. The reaction was concentrated to give INT-2-4 (7.1 g, 80.2% yield) as a light yellow oil. LC-MS (ESI+) 500.3 [M+H] + .

[0368] Compound INT-2-4 (7.1 g, 14.2 mmol) was dissolved in 150 mL of methanol, and 2.5 g of 10% palladium-carbon (0.35 w) was added with a hydrogen balloon. The mixture was stirred at room temperature for 3 h. The reaction was diluted with 70 mL of water, filtered, and the filtrate was concentrated to give compound INT-2 (3.7 g, 95.3% yield) as a white solid. LC-MS (ESI+) 276.1 [M+H] + .

[0369] Synthesis of compound INT-3

[0370] Maleimide hexanoic acid (2112.1 mg, 10 mmol) and N-hydroxysuccinimide (1.38 g, 12 mmol) were dissolved in 50 mL of tetrahydrofuran, and the reaction was cooled to 0 °C. DCC (2.48 g, 12 mmol) was weighed, and 40 mL of tetrahydrofuran was added thereto and sonicated to dissolve. The DCC THF solution was added dropwise at a temperature of 0-5 °C. The reaction was stirred at room temperature for 4 h. The system was cooled to 5 °C, filtered, and the filter cake was washed with a small amount of tetrahydrofuran. The filtrate was replaced with EtOH twice and was agitated overnight. The mixture was filtered to give 2.86 g of INT-3 (93% yield). LC-MS (ESI+) 331.3 [M+Na] + .

[0371] Synthesis of compound INT-4

[0372] INT-3 (616.3 mg, 2.00 mmol), bisglycine (264.2 mg, 2.00 mmol) were dissolved in 10 mL of dichloromethane, TEA (415.9 μΐ, 3.00 mmol) was added, and the mixture was stirred at room temperature for 3 h. The reaction solution was concentrated under reduced pressure, and the solid crude product was purified by preparative liquid chromatography to give 572.2 mg of INT-4-01 with a yield of 88%. LC-MS (ESI+) 326.1 [M+H] + .

[0373] INT-4-01 (572.2 mg, 1.75 mmol) was dissolved in 10 mL of tetrahydrofuran, and N-hydroxysuccinimide (402.2 mg, 3.50 mmol) and DCC (722.1 mg, 3.50 mmol) were added at room temperature. The reaction was allowed to proceed overnight at room temperature. The system was cooled to 5 °C, filtered, the filter cake was washed with a small amount of tetrahydrofuran, and the mother liquor was concentrated under reduced pressure. The target compound INT-4 was obtained by slurrying in ethanol. LC-MS (ESI+) 423.2 [M+H] + .

[0374] Synthesis of compound INT-5

[0375] N3-PEG2-NHS (10 g, 33 mmol) was dissolved in 100 mL of tetrahydrofuran, and a 5% sodium bicarbonate (100 mL) aqueous solution of glycylglycine (5.28 g, 40 mmol) was added, followed by the addition of 40 mL of DME. The reaction was allowed to proceed at 40 °C for 5 h, 2N hydrochloric acid (35 mL) was added to the reaction solution, dichloromethane (200 mL) was added for extraction, and the organic phase was collected and concentrated under reduced pressure to give the target compound INT-5-1 (9.3 g) with a yield of 88.0%. LC-MS (ESI+) 318.3 [M+H] + .

[0376] INT-5-1 (9 g, 28.4 mmol), N-hydroxysuccinimide (3.59 g, 31.2 mmol) were dissolved in 80 mL of tetrahydrofuran, the reaction solution was cooled to 0 °C, DCC (6.43 g, 31.2 mmol) was weighed, and a 20 mL tetrahydrofuran solution of DCC was prepared by ultrasonic dissolution, and the DCC THF solution was added dropwise at a temperature of 0-5 °C. The reaction was allowed to proceed at room temperature for 4 h. The system was cooled to 5 °C, filtered, the filter cake was washed with a small amount of tetrahydrofuran, the filtrate was replaced with EtOH twice, and the slurry was allowed to stand overnight. The target compound INT-5 (10.58 g) was obtained by filtration with a yield of 90%. LC-MS (ESI+) 415.2 [M+H] + .

[0377] Synthesis of compound INT-6

[0378] INT-3 (380.3 mg, 1.0 mmol) was dissolved in 5 mL of dichloromethane, and propargylamine (55.1 mg, 1.0 mmol) and N,N-diisopropylethylamine (66.1 mg, 1.2 mmol) were added at room temperature. It was stirred at room temperature overnight. The system was rotary evaporated under reduced pressure, and purified by preparative liquid phase to obtain the target compound INT-6 181.8 mg of crude product. Yield 73.2%. LC-MS (ESI+) 249.3 [M+H] + .

[0379] Synthesis of compound INT-7 in Example 10

[0380] 1,3,5-triacryloylhexahydro-1,3,5-triazine (25 g, 100 mmol) was dissolved in 500 mL of dichloromethane, and TEA (4159 μl, 30 mmol) and mercaptoacetic acid (2.8 g, 30 mmol) were added, and stirred at room temperature for 3 h. The reaction solution was concentrated under reduced pressure, and the solid crude product was purified by flash preparation to obtain 3.1 g of INT-7-1, with a yield of 30%. LC-MS (ESI+) 342.1 [M+H] + .

[0381] Compound INT-7-1 (1000 mg, 2.86 mmol) was dissolved in 10 mL of DMF, and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1100 mg, 2.89 mmol), amino-PEG4 tert-butyl ester (919 mg, 2.86 mmol), and N,N-diisopropylethylamine (DIPEA) (370 mg, 2.86 mmol) were added dropwise at room temperature with stirring. The reaction was stirred at room temperature for 2 h. The reaction solution was rotary evaporated, and column chromatography was performed with DCM / MeOH=30:1 as the eluent to obtain the target compound INT-7-2 1.04 g, with a yield of 56.6%. LC-MS (ESI+) 645 [M+H] + .

[0382] Compound INT-7-2 (1000 mg, 1.55 mmol) was weighed into 20 mL of DCM, and trifluoroacetic acid 4 mL was added with stirring. It was stirred at room temperature overnight, and the reaction solution was rotary evaporated, and then dissolved in 20 mL of DCM. It was washed with water three times, 5 mL each time, and then washed once with saturated brine 5 mL. The organic phase was rotary evaporated to obtain the target compound 0.91 g of INT-7 as a light yellow oil, with a yield of 100%, LC-MS (ESI+) 589 [M+H] + .

[0383] Synthesis of compound INT-8 in Example 11

[0384] 1,3,5-Triacrylylhexahydro-1,3,5-triazine (16 g, 64 mmol) was dissolved in 320 mL of dichloromethane, and TEA (2662 μl, 19 mmol) and mercaptoacetic acid (1.8 g, 19 mmol) were added. The mixture was stirred at room temperature for 3 h. The reaction solution was concentrated under reduced pressure, and the crude solid was purified by rapid preparative analysis to give 2.1 g of INT-8-1, with a yield of 30%. LC-MS (ESI+) 342.1 [M+H] + .

[0385] Compound INT-8-1 (2 g, 5.86 mmol) was dissolved in 40 mL of DCM and added at room temperature to prepare 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (2.67 g, 7.03 mmol), amino-PEG-4 tert-butyl ester (3.21 g, 6.44 mmol), and then DIPEA (N,N-Diisopropylethylamine) (908.6 mg, 7.03 mmol) was added dropwise with stirring at room temperature. The reaction was allowed to proceed for 2 h at room temperature. The reaction solution was evaporated to dryness and subjected to column chromatography with DCM / MeOH eluent of 80:1 to give 900 mg of the target compound INT-7-2, in a yield of 49.5%. LC-MS (ESI+) 822 [M+H] + .

[0386] Weigh 900 mg (1.10 mmol) of compound INT-8-2 and add 8 mL of DCM. Add 2 mL of trifluoroacetic acid while stirring. Stir overnight at room temperature. Reduce the reaction solution to dryness by rotary evaporation, then add 20 mL of DCM to dissolve. Wash three times with 5 mL of water each time. Wash once with 5 mL of saturated brine. Reduce the organic phase to dryness to give 750 mg of the target compound INT-7, a pale yellow oil, in 100% yield. LC-MS (ESI+) 764 [M+H] + .

[0387] Example 12 Synthesis of compound INT-9

[0388] INT-5 (7.7 g, 18.6 mmol) was dissolved in 70 mL of DMF, and INT-13 (6.9 g, 22.3 mmol) and DIPEA (4.8 g, 37.2 mmol) were added. The reaction was carried out at room temperature for 5 h, and the mixture was concentrated and purified using flash chromatography. The purified product was lyophilized to obtain 9.3 g of INT-9, with a yield of 84.5%. LC-MS (ESI+) 609.6 [M+H] + .

[0389] Example 13 Synthesis of compound INT-10

[0390] INT-2 (1 g, 3.6 mmol), N3-PEG2-NHS (1.2 g, 4.0 mmol), with 10 mL DMF, DIPEA (0.56 g, 4.4 mmol) was added, and the mixture was stirred at room temperature for 24 h. Concentration, flash purification, and lyophilization gave INT-10 1.28 g, yield 80%. LC-MS (ESI+) 461.2 [M+H] + .

[0391] Synthesis of compound INT-11

[0392] INT-10 (594 mg, 1.29 mmol), QSC-200 (515 mg, 1.17 mmol), and HATU (535 mg, 1.41 mmol) were weighed into a reaction bottle, DMF was added at room temperature, stirred uniformly, and DIPEA (454 mg, 3.51 mmol) was added dropwise. The reaction was stopped after 2 h, and the product was purified by reverse phase preparative purification. INT-11 was obtained in a yield of 73%. LC-MS (ESI+): 882.3 [M+H] + .

[0393] Synthesis of compound INT-12

[0394] Benzyl oxycarbonyl-L-alanine (30 g, 110 mmol) was dissolved in 900 mL of tetrahydrofuran, 9 mL of glacial acetic acid was added, and lead tetraacetate (104.0 g, 240 mmol) was added with rapid stirring. The mixture was stirred at 40 °C for 4 h. Filtration, concentration of the filtrate, and obtained compound INT-12-1 27.72 g in the form of a white solid with a yield of 88.1%. LC-MS (ESI+) 295.3 [M+H] + .

[0395] Compound INT-12-1 (12 g, 40.8 mmol) was dissolved in 120 mL of dichloromethane, benzyl glycolate (67.7 g, 408 mmol), and PPTS (Pyridinium p-Toluenesulfonate) (1.02 g, 4.08 mmol) were added, and the mixture was stirred at reflux overnight. Concentration of the reaction mixture gave INT-12-2 13.8 g in the form of a yellowish oil with a yield of 85.2%. LC-MS (ESI+) 401.5 [M+H] + .

[0396] Compound INT-12-2 (10 g, 25 mmol) was dissolved in 300 mL of methanol, 10% palladium on carbon (3.5 g, 0.35 w) was added, and hydrogen gas was bubbled through the solution at room temperature for 3 h. The reaction was filtered, and the filtrate was concentrated to give compound INT-12-3 as a white solid (3.5 g, 80.1% yield). LC-MS (ESI+) 177.2 [M+H] + .

[0397] Compound INT-12-2 (10 g, 25 mmol) was dissolved in 300 mL of methanol, 10% palladium on carbon (3.5 g, 0.35 w) was added, and hydrogen gas was bubbled through the solution at room temperature for 3 h. The reaction was filtered, and the filtrate was concentrated to give compound INT-12-3 as a white solid (3.5 g, 80.1% yield). LC-MS (ESI+) 177.2 [M+H] + .

[0398] Compound INT-12-2 (10 g, 25 mmol) was dissolved in 300 mL of methanol, 10% palladium on carbon (3.5 g, 0.35 w) was added, and hydrogen gas was bubbled through the solution at room temperature for 3 h. The reaction was filtered, and the filtrate was concentrated to give compound INT-12-3 as a white solid (3.5 g, 80.1% yield). LC-MS (ESI+) 177.2 [M+H] + .

[0399] Example 16 Synthesis of compound INT-13

[0400] Compound INT-12-2 (10 g, 25 mmol) was dissolved in 300 mL of methanol, 10% palladium on carbon (3.5 g, 0.35 w) was added, and hydrogen gas was bubbled through the solution at room temperature for 3 h. The reaction was filtered, and the filtrate was concentrated to give compound INT-12-3 as a white solid (3.5 g, 80.1% yield). LC-MS (ESI+) 177.2 [M+H] + .

[0401] Compound INT-12-2 (10 g, 25 mmol) was dissolved in 300 mL of methanol, 10% palladium on carbon (3.5 g, 0.35 w) was added, and hydrogen gas was bubbled through the solution at room temperature for 3 h. The reaction was filtered, and the filtrate was concentrated to give compound INT-12-3 as a white solid (3.5 g, 80.1% yield). LC-MS (ESI+) 177.2 [M+H]+ .

[0402] Synthesis of compound INT-14

[0403] Fmoc-L-propargylglycine (503 mg, 1.5 mmol) was dissolved with 10 mL tetrahydrofuran, and then added with NHS (N-Hydroxy succinimide) (190 mg, 1.65 mmol) and DCC (340 mg, 1.65 mmol) at room temperature. The system was stirred at room temperature overnight. The system was filtered, and the filter cake was washed with 1 mL tetrahydrofuran for three times. The mother liquor was dried under reduced pressure to obtain the target compound INT-14-1 940 mg of crude product. LC-MS (ESI+) 455.5 [M+Na] + .

[0404] The crude compound INT-14-1 (470 mg, 0.75 mmol) was dissolved with 8 mL dichloromethane, and then added with mPEG8-NH2 (288 mg, 0.75 mmol) and N,N- diisopropylethylamine (96 mg, 0.75 mmol) at room temperature. The system was stirred at room temperature overnight. The system was dried under reduced pressure, and then purified by preparative liquid chromatography to obtain the target compound INT-14-2 368 mg of crude product. Yield 70%. LC-MS (ESI+) 702.1 [M+H] + .

[0405] The crude compound INT-14-1 (470 mg, 0.75 mmol) was dissolved with 8 mL dichloromethane, and then added with NH2-PEG4-COOH (200 mg, 0.75 mmol) and N,N- diisopropylethylamine (96 mg, 0.75 mmol) at room temperature. The system was stirred at room temperature overnight. The system was dried under reduced pressure, and then purified by preparative liquid chromatography to obtain the target compound INT-14-3 247 mg of crude product. Yield 56%. LC-MS (ESI+) 584.3 [M+H] + .

[0406] The compound INT-14-2 (200 mg, 0.29 mmol) was dissolved with 5 mL dichloromethane, and then added with diethylamine (1 mL) at room temperature. The system was stirred at room temperature overnight. The system was dried under reduced pressure, and then dissolved with 5 mL dichloromethane again, and then dried under reduced pressure again. The above operation was repeated for five times. The target compound INT-14-4 was obtained as a crude product. LC-MS (ESI+) 479.9 [M+H] + .

[0407] Compound INT-14-4 crude was dissolved with 5 mL dichloromethane, compound INT-14-3 (183 mg, 0.31 mmol), HOBT (1-Hydroxybenzotriazole) (58 mg, 0.43 mmol) and EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (82 mg, 0.43 mmol) were added at room temperature. Stirring at room temperature overnight. The system was rotary evaporated under reduced pressure, purified by preparative liquid phase to obtain the target compound INT-14 167 mg. Yield 56%. LC-MS (ESI+) 1044.5 [M+H] + .

[0408] Example 18 Synthesis of compound INT-15

[0409] Compound INT-14 (146 mg, 0.14 mmol) was dissolved with 4 mL dichloromethane, diethylamine (0.4 mL) was added at room temperature. Stirring at room temperature for 2 h. The system was rotary evaporated under reduced pressure. Dissolved with 5 mL dichloromethane again, rotary evaporated under reduced pressure again, repeated five times. The target compound INT-15-1 crude was obtained. LC-MS (ESI+) 820.5 [M+H] + .

[0410] Compound INT-15-1 crude was dissolved with 2 mL N,N-dimethylformamide, 6-(maleimido)hexanoic acid succinimidyl ester (65 mg, 0.21 mmol) and N,N-diisopropylethylamine (36 mg, 0.28 mmol) were added at room temperature. Stirring at room temperature overnight. The system was purified by preparative liquid phase to obtain the target compound INT-15 mg. Two-step yield 62%. LC-MS (ESI+) 1014.5 [M+H] + .

[0411] Example 19 Synthesis of compound INT-16

[0412] Fmoc-Pra-5Sar-OMe (705 mg, 1 mmol) was dissolved with 10 mL dichloromethane, diethylamine (2 mL) was added at room temperature. Stirring at room temperature for 3 h. The reaction system was rotary evaporated to obtain the target compound INT-16-1 crude, which was directly used in the next step without purification. LC-MS (ESI+) 483.26 [M+H] + .

[0413] Compound INT-14-3 (583 mg, 1 mmol) was dissolved in 15 mL of N,N- dimethylformamide, N,N-diisopropylethylamine (194 mg, 1.5 mmol) and HATU (456 mg, 1.2 mmol) were added at room temperature, stirred at room temperature for 1 h. Compound INT-16-1 crude was added at room temperature, stirred at room temperature overnight. The system was purified by preparative liquid phase to obtain the target compound INT-16-2 456 mg, yield 44%. LC-MS (ESI+) 1048.41 [M+H] + .

[0414] Compound INT-16-2 (456 mg, 0.44 mmol) was dissolved in 6 mL of dichloromethane, diethylamine (1.2 mL) was added at room temperature, stirred at room temperature for 3 h. The reaction system was spin-dried to obtain the target compound INT-16 crude, which was directly used in the next step without purification. LC-MS (ESI+) 825.64 [M+H] + .

[0415] Synthesis of compound INT-17

[0416] Compound INT-7 (306 mg, 0.52 mmol) was dissolved in 10 mL of N,N- dimethylformamide, N,N-diisopropylethylamine (84 mg, 0.65 mmol) and HATU (456 mg, 0.52 mmol) were added at room temperature, stirred at room temperature for 1 h. Compound INT-16 crude was added at room temperature, stirred at room temperature overnight. The system was purified by preparative liquid phase to obtain the target compound INT-17 200 mg, yield 33%. LC-MS (ESI+) 1048.41 [M+H] + .

[0417] Synthesis of compound INT-18

[0418] INT-14-1 (300 mg, 694 μmol) was weighed into a 50 mL single-mouth bottle, 10 mL of dichloromethane was added to dissolve, stirred at room temperature. NH2-PEG4-OMe (143.79 mg, 694 μmol) was added to the system. DIPEA 145 μL was added dropwise to the reaction system, and the reaction was carried out at room temperature overnight. After the reaction was completed by LC-MS monitoring, the solvent was removed by rotary evaporation, and purified by reverse flash preparation system. The target product INT-18-1 267 mg, purity 99.79, yield 71.71%. ESI-MS (m / z): 526.24 [M+H] + .

[0419] INT-18-1 (267 mg, 509 μmol) was weighed into a 10 mL single neck flask, dissolved in 3 mL of dichloromethane, and stirred at room temperature. 600 μL of diethylamine was added dropwise to the system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was spin-dried, redissolved in 5 mL of dichloromethane, spin-dried, and repeated 3 times. The obtained INT-18-2 crude product was directly used in the next step.

[0420] INT-14-3 (294.82 mg, 506 μmol) was weighed into a 25 mL single neck flask, dissolved in 6 mL of dichloromethane, and stirred at room temperature. HATU (288.60 mg, 759 μmol) was added to the reaction system. 177 μL of DIPEA was added dropwise to the reaction system, and stirred at room temperature for 30 min. INT-18-2 (153 mg, 506 μmol) was added to the reaction system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the solvent was removed by spin evaporation, and purified by a reverse flash preparation system. The target product INT-18-3 263.0 mg, purity 99%, yield 59.95% was obtained. LC-MS (ESI+): 868.67 [M+H] + .

[0421] INT-18-3 (511 mg, 589.38 μmol) was weighed into a 50 mL single neck flask, dissolved in 20 mL of dichloromethane, and stirred at room temperature. 3 mL of diethylamine was added dropwise to the system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was spin-dried. 20 mL of dichloromethane was used to dissolve and spin-dry, washed with n-hexane, repeated 3 times, and the obtained INT-18 crude product 158 mg. LC-MS (ESI+) 588.7 [M+H] + .

[0422] Synthesis of compound INT-19

[0423] INT-14-1 (432.4 mg, 1.0 mmol), NH2PEG2-COOH (177.3 mg, 1.0 mmol) were weighed into a 25 mL single neck flask, dissolved in 10 mL of dichloromethane, and 50 μL of DIPEA was added dropwise and stirred at room temperature overnight. After the reaction was completed, the solvent was removed by spin evaporation, and purified by a reverse flash preparation system. The target product INT-19-1 441.5 mg, yield 89.3% was obtained. LC-MS (ESI+): 495.5 [M+H] + .

[0424] INT-14-1 (432.4 mg, 1.0 mmol), 10-Sar-COOHMe (742.8 mg, 1.0 mmol) were weighed into a 50 mL single-neck flask, dissolved in 15 mL of dichloromethane, and stirred at room temperature overnight. After the reaction was completed by LC-MS monitoring, the solvent was removed by rotary evaporation, and the product was purified by reverse flash preparation. The target product INT-19-2 was obtained in a yield of 983.7 mg, 92.7%. LC-MS (ESI+): 1061.2 [M+H] + .

[0425] INT-19-2 (503.2 mg, 0.5 mmol) was weighed into a 25 mL single-neck flask, dissolved in 5 mL of dichloromethane, and stirred at room temperature. 1 mL of diethylamine was added dropwise to the system, and the reaction was carried out at room temperature overnight. After the reaction was completed by LC-MS monitoring, the reaction solution was rotary evaporated. The residue was dissolved in 5 mL of dichloromethane, rotary evaporated, washed with n-hexane, and repeated 3 times to obtain INT-19-3 crude product in a yield of 707.1 mg, 89.6%. LC-MS (ESI+): 852.9 [M+H] + .

[0426] INT-19-1 (247.2 mg, 0.5 mmol), INT-19-3 (425.7 mg, 0.5 mmol), HATU (288.60 mg, 759 μmol) were weighed into a 25 mL single-neck flask, dissolved in 6 mL of DMF, and stirred at room temperature. After the reaction was completed by LC-MS monitoring, the product was purified by reverse flash preparation. The target product INT-19-4 was obtained in a yield of 497.9 mg, 76.6%. LC-MS (ESI+): 1301.4 [M+Na] + .

[0427] INT-19-4 (497.9 mg, 0.38 mmol) was weighed into a 25 mL single-neck flask, dissolved in 8 mL of dichloromethane, and stirred at room temperature. 1.5 mL of diethylamine was added dropwise to the system, and the reaction was carried out at room temperature for 3 h. After the reaction was completed by LC-MS monitoring, the reaction solution was rotary evaporated. The residue was dissolved in 5 mL of dichloromethane, rotary evaporated, washed with n-hexane, and repeated 3 times to obtain INT-19-5 crude product in a yield of 378.9 mg, 91.3%. LC-MS (ESI+): 1093.2 [M+H] + .

[0428] INT-3 (95.1 mg, 0.25 mmol), INT-19-5 (189.3 mg, 0.175 mmol) were taken in a 25 mL vial, 10 mL dichloromethane was added, stirred at room temperature overnight. After monitoring the reaction completion by LC-MS, purified by reverse phase flash. Obtained the target product INT-19165.2 mg, yield 82.3%. LC-MS (ESI+) 1286.4 [M+H] + .

[0429] Synthesis of compound INT-20

[0430] INT-9 (304 mg, 0.5 mmol), QSC-200 (219.2 mg, 0.5 mmol), HATU (184.9 mg, 0.6 mmol) were taken in a 25 mL vial, 10 mL DMF, 125 μL DIPEA was added, stirred at room temperature overnight. After monitoring the reaction completion by LC-MS, purified by preparative liquid chromatography. Obtained the target product INT-20 427.9 mg, yield 83.1%. LC-MS (ESI+) 1031.1 [M+H] + .

[0431] Synthesis of compound INT-21

[0432] INT-3 (2.0 g, 6.49 mmol), 3-[2-(2-aminoethoxy)ethoxy]-propanoic acid (1.1 g, 6.49 mmol), were dissolved in 40 mL N,N-dimethylformamide, N,N-diisopropylethylamine (1.3 g, 9.7 mmol) was added, stirred at room temperature for 2.0 h. After the reaction was completed, purified by preparative liquid chromatography to obtain the target compound INT-21-1 2.1 g, yield 88%. LC-MS (ESI+) 371.2 [M+H] + .

[0433] INT-21-1 (400 mg, 1.08 mmol), N-hydroxysuccinimide (150 mg, 1.32 mmol), DCC (268 mg, 1.3 mmol) were dissolved in 10 mL tetrahydrofuran, stirred at room temperature for 4.0 h. After the reaction was completed, insoluble solid was removed by filtration to obtain the target compound INT-21 452 mg, yield 90%. LC-MS (ESI+) 468.2 [M+H] + .

[0434] Synthesis of compound INT-22

[0435]

[0436] INT-17 (500.00 mg, 0.36 mmol) was dissolved in 10 mL acetonitrile and 10 mL purified water, DIPEA was added, the pH was adjusted to about 10, and stirring was performed at room temperature for 2 h. After the reaction was completed as monitored by LC-MS, the reaction solution was concentrated under reduced pressure to obtain the target compound INT-22481.36 mg, with a yield of 96.93%. LC-MS (ESI+) 1379.6 [M-H] - .

[0437] Synthesis of compound INT-23 in Example 26

[0438]

[0439] INT-22 (481.36 mg, 0.35 mmol), methylamine hydrochloride (28.23 mg, 0.42 mmol), and HATU (198.72 mg, 0.52 mmol) were placed in a reaction bottle, 5 mL of DMF was added at room temperature, stirred uniformly, and DIPEA (112.58 mg, 0.87 mmol) was added dropwise. The reaction was allowed to proceed for 1 h, and then the reaction was stopped and purified by reverse phase preparative purification. INT-23 was obtained in an amount of 276.37 mg, with a yield of 56.88%. LC-MS (ESI+) 1394.7 [M+H] + .

[0440] Synthesis of compound INT-24 in Example 27

[0441]

[0442] Fmoc-O-tert-butyl-L-glutamic acid (2 g, 4.7 mmol) was dissolved in 40 mL of tetrahydrofuran, and N-hydroxysuccinimide (594 mg, 4.17 mmol) and DCC (1067 mg, 5.17 mmol) were added at room temperature. The reaction was allowed to proceed overnight at room temperature. The system was filtered, the filter cake was washed with a small amount of tetrahydrofuran, and the mother liquor was rotary evaporated to obtain the target compound INT-24-1 in crude form, which was directly used in the next step without purification. LC-MS (ESI+) 523.2 [M+H] + .

[0443] Compound INT-24-1 (110 mg, 0.21 mmol) was dissolved in 3 mL of dichloromethane, TEA (21 mg, 0.21 mmol) and mPEG4-NH2 (40 mg, 0.21 mmol) were added at room temperature. Stirring at room temperature overnight. The system was spin-dried solvent, purified by preparative liquid phase to obtain the target compound INT-24-2 132 mg, yield 80%. LC-MS (ESI+) 615.4 [M+H] + .

[0444] Compound INT-24-2 (110 mg, 0.139 mmol) was dissolved in 1 mL of dichloromethane, diethylamine (200 μL) was added at room temperature, stirring at room temperature for 3 h. The reaction system was spin-dried solvent to obtain the target compound INT-24-03 crude, which was directly used in the next step without purification. LC-MS (ESI+) 393.4 [M+H] + .

[0445] Compound INT-24-1 (220 mg, 0.42 mmol) was dissolved in 5 mL of dichloromethane, TEA (42 mg, 0.42 mmol) and NH2-PEG4-COOH (111 mg, 0.42 mmol) were added at room temperature. Stirring at room temperature overnight. The system was spin-dried solvent, purified by preparative liquid phase to obtain the target compound INT-24-4 221 mg, yield 78%. LC-MS (ESI+) 673.4 [M+H] + .

[0446] Compound INT-24-4 (85 mg, 0.13 mmol) was dissolved in 1.5 mL of DMF, DIPEA (25 mg, 0.19 mmol) and TSTU (46 mg, 0.15 mmol) were added at room temperature. Stirring at room temperature for 1 h. Compound INT-24-03 crude was added at room temperature, stirring at room temperature overnight. The system was purified by preparative liquid phase to obtain the target compound INT-24-5 72 mg, yield 53%. LC-MS (ESI+) 1047.7 [M+H] + .

[0447] Compound INT-24-5 (72 mg, 0.06 mmol) was dissolved in 4 mL of dichloromethane, diethylamine (800 μL) was added at room temperature. Stirring at room temperature for 1 h. The system was spin-dried under reduced pressure to obtain the target compound INT-24-6 crude. LC-MS (ESI+) 825.7 [M+H] + .

[0448] Compound INT-24-6 crude was dissolved with 4 mL dichloromethane, TEA (18 mg, 0.18 mmol) and INT-7 (28 mg, 0.09 mmol), HATU (68.35 mg, 0.18 mmol) were added at room temperature. Stirring at room temperature overnight. The system was rotary evaporated under reduced pressure, the system was purified by preparative liquid phase to obtain the target compound INT-24-7 27 mg, two-step yield 38%. LC-MS (ESI+) 1394.8 [M+H] + .

[0449] Compound INT-24-7 (10 mg, 0.08 mmol) was dissolved with 1 mL dichloromethane, TFA (200 μL) was added at room temperature. Stirring at room temperature for 2 h. The system was rotary evaporated under reduced pressure to obtain the target compound INT-24 crude 10 mg. LC-MS (ESI+) 1283.6 [M+H] + .

[0450] Synthesis of compound INT-25

[0451] Compound INT-2-3 (14.00 g, 32.7 mmol) was dissolved with 280 mL DCM, PPTS (1.62 g, 0.033 mmol) was added at room temperature, glycolic acid (3.73 g, 49 mmol) was reacted overnight under reflux. Filtration, the filter cake was washed with a small amount of dichloromethane, and column chromatography to obtain the target compound INT-25-1 10.53 g, yield 78.31%. LC-MS (ESI+) 410.2 [M+H] + .

[0452] INT-25-1 (410.3 mg, 1 mmol), QSC-200 (440.5 mg, 1 mmol), HATU (571.3 mg, 1.5 mmol) were placed in a 25 mL single-necked flask, 5 mL DMF, 125 μL DIPEA were added, and stirring at room temperature overnight. After the reaction was completed, it was purified by preparative liquid chromatography. The target product INT-25-2 was obtained 665.3 mg, yield 80.1%. LC-MS (ESI+) 831.4 [M+H] + .

[0453] INT-25-2 (415.0, 0.5 mmol), palladium on carbon (83.2 mg, 20%), were dissolved with 10 mL tetrahydrofuran, stirring at room temperature under hydrogen for 2.0 h. After the reaction was completed, the palladium on carbon was removed by filtration, the filter cake was washed with methanol, and the filtrate was rotary evaporated to obtain the target compound INT-25 335 mg, yield 95%. LC-MS (ESI+) 697.3 [M+H] + .

[0454] Synthesis of compound INT-26

[0455] Compound INT-16-1 (337 mg, 0.7 mmol), Fmoc-Pra-Sar5-COOH (483 mg, 0.7 mmol) and HATU (418 mg, 1.1 mmol) were dissolved in 15 mL of N,N-dimethylformamide, and N,N- diisopropylethylamine (108 mg, 0.84 mmol) was added. The system was stirred at room temperature for 3 h. The target compound INT-26-1 was obtained by preparative liquid phase purification, with a yield of 380 mg and a yield of 58.2%. LC-MS (ESI+) 1155.57 [M+H] + .

[0456] Compound INT-26-1 (462 mg, 0.4 mmol) was dissolved in 6 mL of dichloromethane, and diethylamine (1.2 mL) was added at room temperature. The system was stirred at room temperature for 3 h. The solvent was rotary evaporated, and the target compound INT-26-2 was obtained by freeze-drying, with a yield of 370 mg. LC-MS (ESI+) 933.47 [M+H] + .

[0457] Compound INT-26-2 (205 mg, 0.22 mmol), INT-7 (118 mg, 0.2 mmol) and HATU (114 mg, 0.3 mmol) were dissolved in 5 mL of N,N-dimethylformamide, and N,N- diisopropylethylamine (31 mg, 0.24 mmol) was added. The system was stirred at room temperature for 3 h. The target compound INT-26 was obtained by preparative liquid phase purification, with a yield of 83 mg and a yield of 70.5%. LC-MS (ESI+) 1503.7 [M+H] + .

[0458] Synthesis of compound INT-27

[0459] Compound INT-26-1 (160 mg, 0.17 mmol), Fmoc-Sar2-COOH (61 mg, 0.16 mmol) and HATU (73 mg, 0.19 mmol) were weighed into a 25 mL round-bottom reaction bottle, dissolved in 6 mL of N,N-dimethylformamide, and N,N- diisopropylethylamine (62 mg, 0.48 mmol) was added. The system was stirred at room temperature for 3 h. The target compound INT-27-1 was obtained by preparative liquid phase purification, with a yield of 157 mg and a yield of 71.2%. LC-MS (ESI+) 1298.61 [M+H] + .

[0460] Compound INT-27-1 (156 mg, 0.12 mmol) was dissolved in 6 mL of dichloromethane, diethylamine (1.2 mL) was added at room temperature, and stirring was performed at room temperature for 3 h. The reaction system was spin-dried to remove the solvent, and freeze-drying was performed to obtain 129 mg of the crude product of the target compound INT-27-2, which was directly used in the next step without purification. LC-MS (ESI+) 1075.55 [M+H] + .

[0461] Compound INT-27-2 (117 mg, 0.11 mmol), INT-7-1 (53 mg, 0.11 mmol), and HATU (49 mg, 0.13 mmol) were weighed into a 25 mL round-bottom reaction bottle, dissolved in 6 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (22 mg, 0.17 mmol) was added, and stirring was performed at room temperature for 3 h. The system was purified by preparative liquid chromatography to obtain 65 mg of the target compound INT-27 with a yield of 40.9%. LC-MS (ESI+) 1398.64 [M+H] + .

[0462] Synthesis of compound DL01-dDxd of Example 31

[0463] Intermediate INT-13 (105 mg, 0.34 mmol) and INT-4 (215 mg, 0.51 mmol) were dissolved in 6.5 mL of N,N-dimethylformamide, and stirring was performed at room temperature for 2 h. The system was purified by preparative liquid chromatography to obtain 186.4 mg of the target compound DL01-01 with a yield of 89.3%. LC-MS (ESI+) 615.34 [M+H] + .

[0464] Compound DL01-01 (52 mg, 0.085 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and QSC-200 (25 mg, 0.057 mmol), EDCI (32 mg, 0.17 mmol), and HOBT (23 mg, 0.17 mmol) were added at room temperature. Stirring was performed at room temperature for 2 h. The system was purified by preparative liquid chromatography to obtain 19.4 mg of the target compound DL01-dDxd with a yield of 22%. LC-MS (ESI+) 1038.58 [M+H] + .

[0465] Synthesis of compound DL02-dDxd of Example 32

[0466] Diazido-ethanediol-acetic acid (238 mg, 1.17 mmol), HATU (326 mg, 0.86 mmol) were dissolved in 15 mL of N,N-dimethylformamide, N,N-diisopropyl ethylamine (201 mg, 1.56 mmol) was added, and the mixture was stirred at room temperature for 1.0 h. INT-13 (240 mg, 0.78 mmol) was added to the reaction solution, and the mixture was stirred for 2.0 h. After the reaction was completed, the target compound DL02-dDxd-019 was obtained by preparative liquid phase purification. 3.9 mg, yield 24%. LC-MS (ESI+) 495.4 [M+H] + .

[0467] DL02-dDxd-01 (25 mg, 0.051 mmol), HATU (19 mg, 0.051 mmol), QSC-200 (15 mg, 0.034 mmol) were dissolved in 15 mL of N,N-dimethylformamide, N,N-diisopropyl ethylamine (8.8 mg, 0.068 mmol) was added, and the mixture was stirred at room temperature for 2.0 h. After the reaction was completed, the target compound DL02-dDxd-02 was obtained by preparative liquid phase purification. 6.3 mg, yield 14%. LC-MS (ESI+) 916.8 [M+H] + .

[0468] Compound DL02-dDxd-02 (6.3 mg, 0.0069 mmol) was dissolved in 1 mL of acetone, INT-6 (2.5 mg, 0.01 mmol), copper sulfate pentahydrate (2.5 mg, 0.01 mmol) were added at room temperature, and the mixture was stirred in a low-temperature bath at 10 °C under nitrogen protection. Vitamin C sodium (2.0 mg, 0.01 mmol) was weighed and dissolved in 1 mL of water, and then added dropwise to the above reaction solution, and the mixture was stirred at 10 °C for 1 h. The reaction was quenched with 1 mL of acetonitrile. The target compound DL02-dDxd was obtained by preparative liquid phase purification. 5.2 mg, yield 65%. LC-MS (ESI+) 1164.8 [M+H] + .

[0469] Synthesis of compound DL05-dDxd

[0470] Intermediate INT-2 (60 mg, 0.218 mmol) and INT-4 (138 mg, 0.327 mmol) were dissolved in 5 mL of N,N-dimethylformamide, and the mixture was stirred at room temperature for 2 h. The system was purified by preparative liquid phase purification to obtain the target compound DL05-01 104 mg, yield 81.9%. LC-MS (ESI+) 583.29 [M+H] + .

[0471] Compound DL05-01 (50 mg, 0.086 mmol) was dissolved in 5 mL of N,N- dimethylformamide, QSC-200 (38 mg, 0.072 mmol), EDCI (41 mg, 0.21 mmol) and HOBT (29 mg, 0.21 mmol) were added at room temperature. Stirring at room temperature for 2 h. The system was purified by preparative liquid phase to obtain the target compound DL05-dDxd 64.6 mg, yield 74.9%. LC-MS (ESI+) 1004.28 [M+H] + .

[0472] Example 34 Synthesis of compound DL06-dDxd

[0473] Compound INT-11 (100 mg, 0.113 mmol) was dissolved in 4 mL of acetone, INT-6 (28 mg, 0.113 mmol), copper sulfate pentahydrate (42.5 mg, 0.17 mmol) were added at room temperature, and the temperature was transferred to a low-temperature tank at 10 °C. Vitamin C sodium (33.7 mg, 0.17 mmol) was weighed and dissolved in 2 mL of water, and added dropwise to the above reaction solution, and stirred at 10 °C for 1 h. The reaction was quenched with acetonitrile (2 mL). Purified by preparative liquid phase to obtain the target compound DL06-dDxd 76.8 mg, yield 60.2%. LC-MS (ESI+) 1130.93 [M+H] + .

[0474] Example 35 Synthesis of compound DL07-dDxd

[0475] INT-12 (500 mg, 1.1 mmol), HATU (501 mg, 1.32 mmol), QSC-200 (531 mg, 1.21 mmol) were dissolved in 30 mL of N,N-dimethylformamide, N,N- diisopropylethylamine (213 mg, 1.65 mmol) was added, and stirred at room temperature for 2.0 h. After the reaction was completed, the target compound DL07-01-dDxd 672 mg was obtained by preparative liquid phase purification, yield 70%. LC-MS (ESI+) 874.5 [M+H] + .

[0476] Compound DL07-01-dDxd (672 mg, 0.77 mmol) was dissolved in 20 mL of methanol. Palladium on carbon (200 mg, 30%) was added at room temperature. Hydrogen was replaced three times and stirred at room temperature for 2 h. The system was filtered, and the palladium on carbon was washed with 5 mL of methanol three times, and the filtrate was rotary evaporated to obtain the crude DL07-02-dDxd which was directly used in the next step. LC-MS (ESI+) 740.3 [M+H] + .

[0477] Compound DL07-02-dDxd (150 mg, 0.20 mmol), INT-21 (112 mg, 0.24 mmol) were dissolved in 10 mL N,N-dimethylformamide, N,N-diisopropyl ethylamine (39 mg, 0.3 mmol) was added, stirred at room temperature for 2.0 h. After the reaction was completed, it was purified by preparative liquid phase to obtain the target compound DL07-dDxd 75 mg, yield 34%. LC-MS (ESI+) 1092.8 [M+H] + .

[0478] Synthesis of compound DL14-dDxd

[0479] Maleimide-octaglycol-succinimidyl acrylate (200 mg, 0.32 mmol), INT-2 (89 mg, 0.32 mmol) were added to a 25 mL single-neck flask, 5 mL DMF, 60 μL TEA, stirred at room temperature overnight, purified by preparative liquid phase to obtain DL14-01 127.8 mg, yield 50.9%. LC-MS (ESI+) 779.4 [M+H] + .

[0480] DL14-01 (127.2 mg, 0.16 mmol), EDCI (34.6 mg, 0.18 mol), HOBT (11.1 mg, 0.082 mmol), QSC-200 (96.4 mg, 0.18 mmol) were added to 5 mL DMF, 41 μL DIPEA, stirred at room temperature for 6 h, purified by preparative liquid phase to obtain DL14-dDxd 95.5 mg, yield 48.5%. LC-MS (ESI+) 1201.3 [M+H] + .

[0481] Synthesis of compound DL03-dDxd

[0482] INT-9 (218 mg, 0.36 mmol) was dissolved in 5 mL N,N-dimethylformamide, QSC-200 (192 mg, 0.36 mmol), HATU (150 mg, 0.39 mmol) and N,N-diisopropyl ethylamine (95 mg, 0.72 mmol) were added at room temperature. Stirred at room temperature for 1 h. The system was purified by flash to obtain the target compound DL03-01-dDxd 274 mg. Yield 74%. LC-MS (ESI+) 1029.9 [M+H] + .

[0483] Compound INT-15 (178 mg, 173 μmol) was dissolved with 8 mL acetone and 4 mL water, compound DL03-01-dDxd (360 mg, 344 μmol) and copper sulfate pentahydrate (134 mg, 536 μmol) were added at room temperature. The system was placed in a 10 °C low temperature bath and stirred. An aqueous solution (4 mL) containing sodium ascorbate (106.7 mg, 536 μmol) was slowly added dropwise to the reaction. Stirred at 10 °C for 1 h. The system was quenched with acetonitrile (2 mL). The system was purified by preparative liquid phase to obtain the target compound DL03-dDxd 361 mg, yield 68%. LC-MS (ESI+) 1537.1 [M / 2+H] + .

[0484] Example 38 Synthesis of compound DL04-dDxd

[0485] INT-15 (404.5 mg, 0.40 mmol), INT-11 (723.0 mg, 0.82 mmol), copper sulfate pentahydrate (237.5 mg, 0.95 mmol) were dissolved in 6 mL acetone, 1 mL glacial acetic acid was added, stirred at 10 °C under argon. Sodium L-ascorbate (239.3 mg, 1.20 mmol) was dissolved with 2 mL water and added dropwise to the reaction, stirred at 10 °C for 1.0 h. After the reaction was completed, 2 mL acetonitrile was added to quench, purified by preparative liquid phase to obtain DL04-dDxd 242.5 mg, yield 22%. LC-MS (ESI + ) 2778.8 [M+H] + .

[0486] Example 39 Synthesis of compound DL08-dDxd

[0487] INT-19 (40 mg, 31.12 pmol) was weighed into a 25 mL three-necked flask, 2 mL of acetone and 2 mL of purified water were added to completely dissolve it, and it was stirred at 0°C. INT-20 (70.52 mg, 68.46 pmol) was weighed into the reaction system in turn, copper sulfate (14.9 mg, 93.35 pmol) was dissolved in 1 mL of water and slowly added to the reaction system, and sodium ascorbate (19.73 mg, 99.58 pmol) was dissolved in 1 mL of water and slowly added to the reaction system. After stirring at 0°C for 30 min, the reaction was monitored by LC-MS. After the reaction was completed, 100 pL of ACN was added to the reaction system to quench the reaction, and reverse phase liquid chromatography purification was performed. The target product DL08-dDxd was obtained in an amount of 51 mg, with a purity of 97.41% and a yield of 48.99%. ESI-MS (m / z): 1672.78 [M / 2+H] + .

[0488] Example 40 Synthesis of compound DL09-dDxd

[0489] INT-19 (40 mg, 31.12 pmol) was weighed into a 25 mL three-necked flask, 2 mL of acetone and 2 mL of purified water were added to completely dissolve it, and it was stirred at 0°C. INT-11 (60.38 mg, 68.46 pmol) was weighed into the reaction system in turn, copper sulfate pentahydrate (23.31 mg, 93.35 pmol) was dissolved in 1 mL of water and slowly added to the reaction system, and sodium ascorbate (19.73 mg, 99.58 pmol) was dissolved in 1 mL of water and slowly added to the reaction system. After stirring at 0°C for 30 min, the reaction was monitored by LC-MS. After the reaction was completed, 100 pL of ACN was added to the reaction system to quench the reaction, and reverse phase liquid chromatography purification was performed. The target product DL09-dDxd was obtained in an amount of 49 mg, with a purity of 98.11% and a yield of 51.64%. ESI-MS (m / z): 1524.56 [M / 2+H] + .

[0490] Example 41 Synthesis of compound DL10-dDxd

[0491] Compound INT-15-1 (830 mg, 1 mmol), INT-7 (590 mg, 1 mmol) were dissolved in 10 mL DMF, HATU (380 mg, 10 mmol) was added. DIPEA (130 mg, 10 mmol) was added under stirring at room temperature. The reaction was stirred at room temperature for 2 h. The reaction solution was diluted with acetonitrile and water, and then filtered through a 0.45 pm filter membrane. The filtrate was purified by high-pressure preparative liquid chromatography to obtain DL10-01-dDxd 97 mg, yield 69.8%, LC-MS (ESI+) 1392 [M+H] +

[0492] Compound DL10-01-dDxd (97 mg, 0.07 mmol), INT-11 (129.3 mg, 015 mmol), copper sulfate (18.5 mg, 0.21 mmol) were dissolved in 5 mL acetone solution. Stirring at 10 °C for 10 min. Vitamin C sodium (13.8 mg, 0.21 mmol) was weighed and dissolved in 5 mL water, and the vitamin C sodium aqueous solution was added to the reaction solution. Continue stirring at 10 °C for 1 h. The acetone was removed by rotary evaporation, and the reaction solution was diluted with acetonitrile and water and then purified by high-pressure preparative liquid chromatography to obtain 65 mg of DL10-dDxd, yield 30%, LC-MS (ESI+) 1758.3 [M / 2] + .

[0493] Synthesis of compound DL11-dDxd in Example 42

[0494] INT-7 (403.3 mg, 0.68 mmol), HATU (195.2 mg, 0.51 mmol) were dissolved in 10 mL N,N-dimethylformamide, and N,N-diisopropylethylamine 90.3 pL was added and stirred at room temperature for 1.0 h. INT-18 (221.4 mg, 0.34 mmol) was added to the reaction solution, and the reaction was carried out for 2.0 h. After the reaction was completed, the target compound DL11-01-dDxd 147.6 mg was obtained by preparative liquid chromatography, with a yield of 36%. LC-MS (ESI + )1216.3[M+H] + .

[0495] DL11-01-dDxd (147.6 mg, 0.12 mmol), INT-11 (236.8 mg, 0.27 mmol), copper sulfate pentahydrate (99.8 mg, 0.40 mmol) were dissolved in 6 mL of acetone and stirred at 10 °C under argon. Sodium L-ascorbate (84.7 mg, 0.43 mmol) was dissolved in 2 mL of water and added dropwise to the reaction. The reaction was stirred at 10 °C for 1.0 h. After the reaction was completed, 2 mL of acetonitrile was added to quench the reaction, and purified by preparative liquid chromatography to give DL11-dDxd 57.3 mg, yield 16%. LC-MS (ESI + ) 2978.8 [M+H] + .

[0496] Synthesis of compound DL12-dDxd

[0497] INT-9 (188 mg, 245.79 pmol) was weighed into a 50 mL single-neck flask, dissolved in 10 mL of DMF and stirred at room temperature. HATU (102.8 mg, 270 pmol) was added to the reaction system. 52 pL of DIPEA was measured and added dropwise to the reaction system, and stirred at room temperature for 50 min. INT-18 (158 mg, 245.8 pmol) was weighed into the reaction system and reacted at room temperature overnight. After the reaction was completed, the product was purified by high-pressure reverse-phase preparative system. The target product DL12-01-dDxd was obtained in 185 mg, purity 98%, yield 59.95%. LC-MS (ESI + ): 1392.59 [M+H] + .

[0498] DL12-01-dDxd (121 mg, 86.95 pmol) was weighed into a 50 mL three-neck flask, dissolved in 5 mL of acetone and 1 mL of purified water, stirred at 0 °C, and INT-11 (153.37 mg, 173.9 pmol) was added to the reaction system. Copper sulfate pentahydrate (54.27 mg, 217.37 pmol) was dissolved in 2 mL of water and slowly added to the reaction system. Sodium ascorbate (51.67 mg, 260.85 pmol) was dissolved in 2 mL of water and slowly added to the reaction system. The reaction was stirred at 0 °C for 30 min. After the reaction was completed, 100 pL of ACN was added to quench the reaction, and purified by reverse-phase preparative liquid chromatography. The target product DL12-dDxd was obtained in 89 mg, yield 32.44%. ESI-MS (m / z): 1578.76 [M+2H] 2+ .

[0499] Synthesis of compound DL13-dDxd of Example 44

[0500] Compound INT-17 (200 mg, 0.14 mmol) was dissolved with 10 mL of acetone, INT-11 (256 mg, 0.29 mmol), copper sulfate pentahydrate (105 mg, 0.42 mmol) and sodium ascorbate (83 mg, 0.42 μmol) were added at room temperature, and stirred at room temperature for 1 h. The reaction was quenched with acetonitrile (5.0 mL). The system was purified by preparative liquid phase to obtain the target compound DL13-dDxd 181 mg, with a yield of 41%. LC-MS (ESI+) 1579.76 [M / 2+H] + .

[0501] Synthesis of compound DL25-dDxd of Example 45

[0502] INT-23 (51.00 mg, 0.04 mmol), INT-11 (63.11 mg, 0.07 mmol) were weighed and dissolved in acetone (1.5 mL), purified water (0.5 mL) and stirred and replaced with nitrogen for 3 times; copper sulfate pentahydrate (8.87 mg, 0.04 mmol), sodium ascorbate (7.05 mg, 0.04 mmol) were weighed and dissolved in purified water (0.2 mL) respectively and used as prepared, and the copper sulfate pentahydrate solution and the sodium ascorbate solution were added in turn, and stirred at room temperature for 0.5 h. After the reaction was completed by LC-MS monitoring, the system was purified by preparative liquid phase to obtain DL25-dDxd 60.13 mg with a yield of 54.02%. LC-MS (ESI+) 3157.5 [M+H] + .

[0503] Synthesis of compound DL01-d1Dxd of Example 46

[0504] Compound DL01-01 (850 mg, 1.4 mmol), d1DX85951 (620 mg, 1.2 mmol) were dissolved in 20 mL of N,N-dimethylformamide, HATU (684 mg, 1.8 mmol), N,N-diisopropyl ethylamine (387 mg, 3 mmol) were added, and stirred at room temperature for 2 h. The system was purified by preparative liquid phase to obtain the target compound DL-d1Dxd 868 mg with a yield of 72%. LC-MS (ESI+) 1035.40 [M+H] + .

[0505] Synthesis of compound DL13-d1Dxd of Example 47

[0506] Take INT-10 (594.0 mg, 1.29 mmol), dlDX8951 (507.24 mg, 1.16 mmol) and HATU (535 mg, 1.41 mmol) into a reaction bottle, add 10 mL of DMF at room temperature, stir evenly, and drop DIPEA (454 mg, 3.51 mmol). The reaction is 2 h, the reaction is stopped, and the reverse phase preparation purification is carried out. INT-11-d1 649.25 mg, yield 57.27%. LC-MS (ESI+) 879.3 [M+H] + .

[0507] Take INT-17 (50.00 mg, 0.04 mmol), INT-11-d1 (63.02 mg, 0.07 mmol) into acetone (1.5 mL), purified water (0.5 mL), stir to dissolve and replace with nitrogen for 3 times; take copper sulfate pentahydrate (8.96 mg, 0.04 mmol), sodium ascorbate (7.10 mg, 0.04 mmol) into purified water (0.2 mL) respectively, dissolve for standby, add copper sulfate pentahydrate solution and sodium ascorbate solution in turn, stir at room temperature for 0.5 h, monitor the reaction completion by LC-MS, prepare liquid phase purification, get DL13-d1Dxd 50.86 mg, yield 45.01%. LC-MS (ESI+) 3152.4 [M+H] + .

[0508] Synthesis of compound DL25-d1Dxd in example 48

[0509] Take INT-23 (50.00 mg, 0.04 mmol), INT-11-d1 (63.02 mg, 0.07 mmol) into acetone (1.5 mL), purified water (0.5 mL), stir to dissolve and replace with nitrogen for 3 times; take copper sulfate pentahydrate (8.96 mg, 0.04 mmol), sodium ascorbate (7.10 mg, 0.04 mmol) into purified water (0.2 mL) respectively, dissolve for standby, add copper sulfate pentahydrate solution and sodium ascorbate solution in turn, stir at room temperature for 0.5 h, monitor the reaction completion by LC-MS, prepare liquid phase purification, get DL25-d1Dxd 53.79 mg, yield 47.59%. LC-MS (ESI+) 3151.4 [M+H] + .

[0510] Preparation of antibody drug conjugate

[0511] Preparation of antibody drug conjugate by using general conjugation method in example 49

[0512] Preparation of antibody drug conjugate by using general conjugation method in example 49The preparation of antibody drug conjugate adopts the general conjugation method: the reducing agent and the protective agent are respectively prepared with purified water as follows: 1-20 mM TCEP (Tris-2-carboxyethyl-phosphine), 1-20 mM DTPA (Diethylene triamine pentacetate acid) mother liquor, the amount of reducing agent can be added within a certain concentration range according to the required conjugation rate, mixed with a certain concentration of monoclonal antibody (such as: 5-30 mg / mL), the final molar ratio of TCEP to antibody is 8-14:1, and the reaction is stirred at 25°C for 1 h. The TCEP-reduced antibody can be directly conjugated.

[0513] A certain concentration (5 mM) of linker-active drug unit compound is prepared by dissolving in DMSO (dimethyl sulfoxide), and the drug is slowly added according to the molar ratio of drug to thiol of 1.2-1.5:1, and the reaction is stirred at 25°C for 1-4 h. After the reaction is completed, centrifugal ultrafiltration is carried out with HIS buffer, and free small molecules such as residual unreacted drugs and DMSO are removed by purification, and the conjugation is detected by LC-MS method, and the purity of the conjugated sample is detected by SEC.

[0514] Example 50: Preparation of antibody drug conjugate by using engineered cysteine conjugation method

[0515] The preparation of antibody drug conjugates adopts the coupling method of engineered cysteine: the reducing agent is prepared with purified water as follows: 1-100 mM TCEP (Tris-2-carboxyethyl-phosphine); the oxidizing agent is prepared with DMSO (dimethyl sulfoxide) as follows: 1-100 mM DHAA (Dehydroacetic acid) stock solution, mixed with a certain concentration of monoclonal antibody (such as: 1-20 mg / mL), the final molar ratio of TCEP to antibody is 10-100:1, and the reaction is stirred at 25°C for 2-18h. After reduction, remove excess TCEP by ultrafiltration centrifugation with Tris-HCl buffer, add DHAA to the system, the final molar ratio of DHAA to antibody is 10-100:1, and the reaction is stirred at 25°C for 1-4h. After the reaction is completed, remove excess DHAA by ultrafiltration centrifugation with Tris-HCl buffer, prepare a certain concentration (5mM) of linker-active drug unit compound dissolved in DMSO (dimethyl sulfoxide), add the drug slowly according to the molar ratio of drug to thiol of 1.2-2:1, and stir at 25°C for 1-4h. After the reaction is completed, remove the residual unreacted drug and free small molecules such as DMSO by centrifugal ultrafiltration with Tris-HCl buffer, and detect the coupling condition by liquid chromatography-mass spectrometry (LC-MS) method, and detect the purity of the coupled sample by size exclusion chromatography (SEC).

[0516] Example 51: Quality verification by preparing target Trop2 antibody drug conjugates (random coupling) The conjugation linker-toxin compound DL01-dDxd, DL02-dDxd, DL05-dDxd, DL06-dDxd, DL07-dDxd, DL05-Dxd, DL06-Dxd, DL07-Dxd is coupled with the antibody molecule Ab (Ab: RD219A1 antibody, a self-expressed and produced target Trop2 antibody) by the general coupling method of this embodiment 49. The average drug / antibody ratio (DAR value) of the conjugate product is detected by liquid chromatography-mass spectrometry (LC-MS), and the purity of the conjugate product is detected by size exclusion chromatography (SEC). Table 4. Structure of antibody drug conjugates prepared by general coupling method Table 5. Test results of antibody drug conjugates prepared by general coupling method The ADC number means that three parts are connected by two dashes “-”, in order of the target part used, the linker and the load part, for example, RD219A1-DL01-dDxd, the target part is RD219A1 antibody, the linker is DL01, and the load is dDxd compound.

[0517] By using the verified ADC target (Trop2) antibody, the preparation and quality detection of antibody drug conjugate were carried out, and the results (Table 5) showed that using the linker or linker-load structure of the application, an antibody conjugated drug with high DAR value and high purity can be prepared.

[0518] Example 52: Quality verification by preparing Trop2 antibody drug conjugate (site-directed conjugation)

[0519] The conjugation linker-toxin compound DL03-dDxd, DL04-dDxd, DL08-dDxd, DL09-dDxd, DL03-Dxd, DL04-Dxd was coupled with an engineered antibody molecule Ab (RD219B1 antibody, a Trop2 targeting antibody after specific site cysteine mutation) using the engineered cysteine coupling method of this embodiment 50. The average drug / antibody ratio (DAR value) of the conjugate product was detected by liquid chromatography-mass spectrometry (LC-MS), and the purity of the conjugate product was detected by size exclusion chromatography (SEC). Table 6. Antibody drug conjugate structure prepared by using engineered cysteine coupling method Table 7. Test results of antibody drug conjugate prepared by using engineered cysteine coupling method ADC number meaning: three parts are connected by two dashes "-", in order, the target part used, the linker and the load part, for example, RD219B1-DL03-dDxd, the target part is RD219B1 antibody, the linker is DL03, and the load is dDxd compound.

[0520] By using the verified ADC target (Trop2) cysteine site-directed mutant antibody, the preparation and quality detection of site-directed conjugated antibody drug conjugate were carried out, and the experimental results (Table 7) showed that using the linker or linker-load structure of the application, a cysteine site-directed conjugated antibody conjugated drug with high DAR value and high purity can be prepared.

[0521] In vitro toxicity test of antibody drug conjugate

[0522] Example 53: Cytotoxic activity of antibody drug conjugate

[0523] The cells BXPC-3 (human in situ pancreatic cancer cells) in the exponential growth phase were seeded at 5E4-1E5 / mL (scientific notation (the same below), representing 5x10 4 -1x10 5The density of the cells was adjusted to 1 x 106 / mL, and 100 μL was inoculated into each well of a 96-well plate. The antibody-drug conjugate was prepared in a gradient of 0-333 nM with complete culture medium, and 100 μL was added to each well. Three wells were set for each concentration, and a blank control group was also set. After 168±2 h of drug treatment, CCK-8 reagent was added, and the plate was incubated at 37°C in a 5% CO2 incubator for 1-4 h. The OD value of each well was detected at 450 nm on a microplate reader. The inhibition rate IR% was calculated as (ODblank-ODdrug) x 100 / ODblank. The inhibition rate and the drug concentration were fitted by four parameters to calculate the IC 50 Table 8. Cytotoxic activity of antibody-drug conjugates on BXPC-3 (human in situ pancreatic adenocarcinoma cells) The ADC number means that, except for "RD219A1-MC-GGFG-DXD", the three parts of the used targeting moiety, linker and payload moiety are connected by two dashes "-". For example, for RD219A1-DL01-dDxd, the targeting moiety is the RD219A1 antibody, the linker is DL01, and the payload is the dDxd compound. "RD219A1-MC-GGFG-DXD" means that the antibody is "RD219A1", the linker is "MC-GGFG", and the payload is DXD.

[0524] As can be seen from Table 8, the ADC prepared by using the linker-toxin structure of the application shows high toxicity to the BXPC-3 (human in situ pancreatic adenocarcinoma cell) tumor cell line. Compared with the currently clinically validated linker-toxin structure of MC-GGFG-DXD in the ADC field, DL01-dDxd, DL02-dDxd, DL03-dDxd, DL04-dDxd, DL05-dDxd, DL06-dDxd, DL07-dDxd, DL08-dDxd and DL09-dDxd show better or comparable cytotoxic activity. The cytotoxic activity test results in this embodiment show that the linker-toxin structure of the application has broad application prospects.

[0525] Example 54. Screening and development of anti-CDCP1 antibodies

[0526] 1. Preparation of anti-CDCP-1 murine antibodies

[0527] (1) Animal immunization scheme

[0528] Animal immunization was performed by immunizing Balb / C and CD1 mice with the extracellular full-length CDCP-1 protein from ACRO (Phe30-Thr667, Cat# CD1-H52H6) as immunogen. The mice were injected subcutaneously with 40 μg of the protein every other week for four times, with Freund's complete or incomplete adjuvant. Seven days after the last immunization, the mice were bled from the eye orbit, and the serum was separated and used to determine the antibody titer by flow cytometry (FACS). After the serum titer reached the level required for the preparation of conventional hybridoma antibodies, the mice were subjected to a boost immunization with 40 μg of the protein, and three days later, the mice were used for cell fusion.

[0529] (2) Hybridoma preparation and screening

[0530] The spleen cells of the mice that met the fusion conditions and had been subjected to the boost immunization were removed and fused with mouse myeloma cells SP2 / 0 under the action of PEG to prepare hybridoma cells. Specifically, the removed spleen cells were fused with the mouse myeloma cells SP2 / 0 at a ratio of 2:1 under the action of 50% PEG1500, and then suspended in a culture medium containing HAT (hypoxanthine (H), aminopterin (A), and thymidine (T)), and then inoculated into a 96-well cell plate at 2-3E6 cells per well, and then cultured in a 37°C, 5% CO2 incubator. After 5 days of culture, the culture medium was replaced with HT medium, and the culture was continued for another 5 days. Then, the binding activity of the antibodies secreted by the hybridoma cells to the extracellular full-length hCDCP1 protein was determined by FACS using MDA-MB-231 (human breast cancer cells) as the cells. Specifically, 100 μL of the hybridoma supernatant was added to an E-tube containing 5E5 cells, and then incubated at room temperature for 1 h, and then centrifuged at 3000 rpm for 3 min, and then washed twice with PBS, and then 100 μL of 1:2000 diluted Anti-Mouse IgG-APC antibody was added to each sample, and then incubated at room temperature for 1 h, and then washed twice with PBS, and then resuspended, and then subjected to machine detection (machine: Invitrogen Attune NxT flow cytometer). The FACS detection results showed that the listed hybridoma supernatants all had good affinity to CDCP1.

[0531] The positive hybridomas with good binding were selected for subcloning to obtain monoclonal hybridoma cells that could produce specific mouse-derived antibodies. To determine the specificity of the produced antibodies, the binding activity of the hybridoma supernatants to negative cell lines was further determined, and the selected negative cell line was Caki-1 cells. The cells that were positive for MDA-MB-231 and negative for Caki-1 in the supernatant detection and screening were monoclonal cells that produced specific mouse-derived antibodies. The FACS detection results showed that the hybridoma supernatants all had good specific binding activity.

[0532] 2. Preparation of anti-CDCP-1 chimeric antibodies

[0533] (1) Preparation of chimeric antibodies

[0534] The hybridomas with good specificity obtained above were subjected to sequence fishing to obtain the complete variable region sequence of the murine antibody. The current method for obtaining the variable region sequence of the murine antibody is Race technology. A monoclonal hybridoma cell strain was selected for expansion culture, genomic RNA was extracted, reverse transcription was performed using a SMARTer RACE 5' / 3' Kit (Clontech 634913) kit, cDNA sequence was obtained, and then a universal primer was used to amplify the variable region sequence of the murine antibody light and heavy chain.

[0535] According to the instructions of the SMARTer RACE 5' / 3' Kit (Clontech 634913), the variable region sequences of 136 hybridomas were successfully fished. The heavy chain variable region sequence and the light chain variable region sequence were respectively constructed on the pcDNA3.1 expression vector containing the human IgG1 heavy chain constant region and the human kappa light chain constant region by homologous recombination method, and the chimeric antibody light and heavy chain expression plasmids were obtained accordingly. After expression in a 293T cell small system, FACS binding screening verification was performed. Finally, 19 chimeric antibodies were prepared through expression and verification in a large system: #2, #5, #6, #13, #31, #32, #33, #38, #66, #89, #92, #94, #96, #108, #110, #111, #118, #119, #120. Among them, the information of chimeric antibodies #6 and #31 is as follows:

[0536] SEQ ID NO: 1 (#6 heavy chain variable region sequence)

[0537]

[0538] SEQ ID NO: 2 (#6 light chain variable region sequence)

[0539]

[0540] SEQ ID NO: 3 (#31 heavy chain variable region sequence)

[0541]

[0542] SEQ ID NO: 4 (#31 light chain variable region sequence)

[0543]

[0544] 3. Chimeric antibody property analysis

[0545] (1) Chimeric antibody affinity detection

[0546] (1.1) Affinity kinetics detection - OCTET

[0547] Affinity kinetics detection was performed using Octet RED96e (purchased from GE Healthcare). The specific operation and method were according to the instrument instruction and the detailed method provided by the manufacturer. Specifically, affinity detection was performed using ProA biosensor (purchased from GE Healthcare). The chimeric antibody of CDCP1 was diluted to a final concentration of 400 nM, 200 nM, 100 nM using PBS containing 0.02% (v / v) Tween, pH 7.4, and then reacted with the ProA biosensor for 1 min, and then reacted with the antigen diluted to 10 μg / L using PBS containing 0.02% (v / v) Tween, pH 7.4, for 1.5 min, and the antigen was human extracellular full-length CDCP1 protein (CD1-H52H6, ACRO, Phe30-Thr667), human extracellular distal membrane CDCP1 protein (13262-H08H, Yikai, Met1-Glu343), mouse extracellular full-length CDCP1 protein (51110-M08H, Yikai, Met1-Leu666), and monkey extracellular full-length CDCP1 protein (90296-C08H, Yikai, Met1-Thr667). The binding and dissociation of the antibody and various antigens were detected by detecting the change in the interference wavelength of the Octet instrument, and then the binding constant and dissociation constant were fitted using Octet User Software, and the affinity constant was the ratio of the dissociation constant to the binding constant. The results showed that #6 and #31 had good binding ability.

[0548] (1.2) Chimeric antibody affinity FACS detection

[0549] The CDCP1 chimeric antibody was diluted to a final concentration of 20 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 0.2 μg / mL, 0.04 μg / mL, 0.004 μg / mL, 0.0004 μg / mL, respectively, and incubated with 5E5 MDA-MB-231 (human breast cancer cells), HCT116 (human colon cancer cells), PC-3 cells at room temperature for 1 h, then centrifuged at 3000 rpm for 3 min, washed with PBS twice, added 100 μL of 1:2000 diluted Anti-Mouse IgG-APC antibody to each sample, reacted at room temperature for 1 h, washed with PBS twice, resuspended, and detected by machine (machine: Invitrogen Attune NxT flow cytometer). The FACS detection results showed that #6 and #31 had good affinity.

[0550] Example 55 Humanization and optimization of anti-CDCP1 antibody

[0551] 1. Preparation of anti-CDCP1 humanized antibody

[0552] (1) Humanization of #6 chimeric antibody

[0553] According to the affinity evaluation results of the chimeric antibody, the #6 chimeric antibody was selected for humanization of the antibody. After humanization of the antibody, the #6 chimeric antibody obtained 6 heavy chains (6-H1, 6-H2, 6-H3, 6-H4, 6-H5, 6-H6) and 6 light chains (6-L1, 6-L2, 6-L3, 6-L4, 6-L5, 6-L6) variable region amino acid sequences, as shown below:

[0554] SEQ ID NO: 5: (#6 humanized heavy chain variable region sequence H1)

[0555]

[0556] SEQ ID NO: 6: (#6 humanized heavy chain variable region sequence H2)

[0557]

[0558] SEQ ID NO: 7: (#6 humanized heavy chain variable region sequence H3)

[0559]

[0560] SEQ ID NO: 8: (#6 humanized heavy chain variable region sequence H4)

[0561]

[0562] SEQ ID NO: 9: (#6 humanized heavy chain variable region sequence H5)

[0563]

[0564] SEQ ID NO: 10: (#6 humanized heavy chain variable region sequence H6)

[0565]

[0566] SEQ ID NO: 11: (#6 humanized light chain variable region sequence LI)

[0567]

[0568] SEQ ID NO: 12: (#6 humanized light chain variable region sequence L2)

[0569]

[0570] SEQ ID NO: 13: (#6 humanized light chain variable region sequence L3)

[0571]

[0572] SEQ ID NO: 14: (#6 humanized light chain variable region sequence L4)

[0573]

[0574] SEQ ID NO: 15: (#6 humanized light chain variable region sequence L5)

[0575]

[0576] SEQ ID NO: 16: (#6 humanized light chain variable region sequence L6)

[0577]

[0578] The obtained variable region amino acid sequences of the heavy chain and the light chain were codon-optimized, enzyme digestion sites, signal peptides, and constant regions of antibody light and heavy chains were added, and pCDNA3.1 vectors were constructed. The obtained complete light and heavy chain genes were plasmid-extracted, and the extracted plasmids were transfected into Expi293 cells in a light-heavy chain cross-combination manner, 100 mL system expression and purification were performed, and property analysis was performed.

[0579] 2. Screening of #6 humanized antibodies

[0580] (1) Affinity kinetic detection of #6 humanized antibodies

[0581] Octet RED96e (purchased from Sartorius) was used for affinity kinetic detection. The specific operation and method were according to the instrument instruction and the detailed method provided by the manufacturer. Specifically, ProA biosensor (purchased from Sartorius) was used for affinity determination. The #6 chimeric antibody of CDCP1 and 36 humanized antibodies were diluted to a final concentration of 400 nM, 200 nM, 100 nM with PBS solution containing 0.02% (v / v) Tween, pH 7.4, and then reacted with ProA biosensor for 1 min, and then reacted with 10 μg / mL of antigen diluted with PBS solution containing 0.02% (v / v) Tween, pH 7.4, for 1.5 min, i.e. human extracellular full-length CDCP1 protein (CD1-H52H6, ACRO, Phe30-Thr667), human extracellular distal membrane CDCP1 protein (13262-H08H, Yiqi God, Met1-Glu343), and monkey extracellular full-length CDCP1 protein (90296-C08H, Yiqi God, Met1-Thr667). The binding and dissociation of the antibody and various antigens were detected by detecting the change of interference wavelength by Octet instrument, and then the binding constant and dissociation constant were fitted by Octet User Software software, and the affinity constant was the ratio of the dissociation constant to the binding constant. The experimental results showed that the 36 humanized antibodies of #6 had good binding affinity to the three antigens.

[0582] (2) Affinity FACS detection of #6 humanized antibodies

[0583] The 36 humanized antibodies of CDCP1 were diluted to a final concentration of 20 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 0.2 μg / mL, 0.04 μg / mL, 0.004 μg / mL, 0.0004 μg / mL, respectively, and 5E5 MDA-MB-231 (human breast cancer cells), PC-3 cells were incubated at room temperature for 1 h, then centrifuged at 3000 rpm for 3 min, washed with PBS twice, and 100 μl of 1:2000 diluted Anti-Mouse IgG-APC antibody was added to each sample, reacted at room temperature for 1 h, washed with PBS twice, resuspended, and detected by machine (machine: Invitrogen Attune NxT flow cytometer). The FACS detection results are shown in Table 9, and the experimental results show that the following humanized antibodies have excellent affinity. Table 9. Affinity FACS detection results of #6 humanized antibodies in PC-3 cells

[0584] Note: "nd" means not detected.

[0585] (3) Endocytosis rate detection of #6 humanized antibodies derived from chimeric antibodies

[0586] The 18 humanized antibodies selected were labeled according to the promega PHAb antibody dye labeling kit instructions, and the concentration before antibody labeling was 2 mg / mL. The labeled antibodies were incubated with PC-3 (human prostate cancer cells) and HCT116 (human colon cancer cells) at 37°C. The time gradient was set as 0 h, 3 h, 6 h, 8 h, 16 h, and 24 h, and the concentration was set as 100 nM. After each sampling point, the fluorescence intensity was detected by a cell flow cytometer. The experimental results show that H1+L6 (humanized antibody Ab1), H5+L2, and H6+L2 have a faster endocytosis rate.

[0587] 3. Humanization of #31 chimeric antibody

[0588] (1) Humanization screening of #31 chimeric antibody

[0589] According to the affinity evaluation results of the chimeric antibody, the #31 chimeric antibody was selected for humanization of the antibody. After humanization of the antibody, the humanized antibody hAb2 sequence was finally selected as follows:

[0590] The variable region sequence of the humanized antibody hAb2 is as follows:

[0591] SEQ ID NO: 17: (#31 humanized heavy chain variable region sequence H1)

[0592]

[0593] SEQ ID NO: 18: (#31 humanized light chain variable region sequence L1)

[0594]

[0595] The screening process of the CDCP1 antibodies of the present application is outlined as follows, Ab1, Ab2 are subjected to heavy chain A114C mutation (Kabat numbering) to obtain C1, C2, respectively.

[0596] 4. Performance characterization of the optimized humanized antibodies

[0597] (1) Yield of anti-CDCP1 antibodies Ab1 and Ab2

[0598] The antibodies were expressed in Expi293 cells and purified from the culture medium by Protein A column, followed by further purification using a size exclusion column. The expression titer of the antibodies was determined using the quantitative protocol on the Gator BLI system. The data showed that the yield of Ab1 was 400 mg / L, and the yield of Ab2 was 400 mg / L, both Ab1 and Ab2 had high yield.

[0599] (2) Melting temperature of anti-CDCP1 antibodies Ab1 and Ab2

[0600] The melting point of the antibodies was measured by differential scanning fluorimetry (DSF) on a Bio-Rad CFX Opus 96 real-time PCR system. The antibodies were mixed with SYPRO Orange dye and incubated at a temperature gradient of 20°C to 95°C. The fluorescence emission was monitored in real time at 570 nm, and the melting point was calculated by Bio-Rad CFX Maestro software. The data showed that the melting temperature Tm of Ab1 and Ab2 was 67.5°C, both had good thermal stability.

[0601] (3) Purity of anti-CDCP1 antibodies Ab1 and Ab2

[0602] The purity of the antibodies was evaluated by ACQUITY UPLC Protein BEH SEC column (1.7 μm, 4.6 mm x 300 mm) on an Agilent 1290 system. The mobile phase was 205 mM NaCl, 4 mM KCl, 15 mM NaH2PO4, 2.7 mM K2HPO4, pH 7.4. Data analysis was completed using Agilent Chemstation software. The data showed that Ab1 and Ab2 had excellent SEC purity, both were 100%. 1.7 μm, 4.6 mm x 300 mm). The mobile phase was 205 mM NaCl, 4 mM KCl, 15 mM NaH2PO4, 2.7 mM K2HPO4, pH 7.4. Data analysis was completed using Agilent Chemstation software. The data showed that Ab1 and Ab2 had excellent SEC purity, both were 100%.

[0603] (4) Protein binding activity of anti-CDCP1 antibody Ab1 and Ab2

[0604] Protein binding assays were performed on a Biacore T200 system. Antibodies were immobilized on CM5 chips prepared using an anti-human Fc capture kit. A series of diluted full-length CDCP-1 was injected into the multi-cycle kinetics assay. The binding rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD) were calculated using Biacore evaluation software. As shown in Table 10, the data indicate that Ab1 and Ab2 exhibit good protein binding activity. Table 10. Protein binding activity of anti-CDCP1 antibodies Ab1 and Ab2.

[0605] (5) Endocytosis of anti-CDCP1 antibodies Ab1 and Ab2

[0606] Cells were seeded at a density of 20,000 cells per well in 96-well black transparent plates and incubated overnight. The FabFluor-pH Red antibody labeling kit is used to label individual antibodies. Fluorescently labeled antibodies are added to cell plates and then... Fluorescence signals were measured using SX5. As shown in Table 11, the data indicate that Ab1 and Ab2 exhibit good endocytosis effects. Table 11. Endocytosis of anti-CDCP1 antibodies Ab1 and Ab2.

[0607] Example 56: Preparation of anti-CDCP1 antibody and determination of its amino acid sequence

[0608] Based on the screening results of Example 55, the preferred anti-CDCP1 antibodies are Ab1 (#6 humanized H1+L6) and Ab2 (#31 humanized H1+L1).

[0609] Ab1 Heavy chain variable region and light chain variable region CDR1-3 amino acid sequence

[0610] Table 12. CDR amino acid sequence of anti-CDCP1 antibody Ab1 Note: The CDR is obtained based on the IMGT definition scheme.

[0611] Ab1 heavy chain variable region amino acid sequence SEQ ID NO:25:

[0612] Ab1 light chain variable region amino acid sequence SEQ ID NO:26

[0613] Ab1 heavy chain amino acid sequence SEQ ID NO:27

[0614] Ab1 light chain amino acid sequence SEQ ID NO: 28

[0615] Ab2 heavy chain variable region and light chain variable region CDR1-3 Table 13. CDR amino acid sequences of anti-CDCPl antibody Ab2 Note: the CDRs are obtained based on IMGT definition scheme

[0616] Ab2 heavy chain variable region amino acid sequence SEQ ID NO: 35

[0617] Ab2 light chain variable region amino acid sequence SEQ ID NO: 36

[0618] Ab2 heavy chain amino acid sequence SEQ ID NO: 37

[0619] Ab2 light chain amino acid sequence SEQ ID NO: 38

[0620] Example 57 Preparation of anti-CDCPl antibody drug conjugate (MMAE load)

[0621] The preparation of antibody drug conjugate adopts a general conjugation method: the reducing agent and the protecting agent are prepared with purified water as follows: 1-20 mM TCEP (Tris-2-carboxyethyl-phosphine), 1-20 mM DTPA (Diethylene triamine pentacetate acid) stock solution, the amount of reducing agent added can be within a certain concentration range according to the desired conjugation rate, mixed with a certain concentration of monoclonal antibody (such as: 5-30 mg / mL), the final molar ratio of TCEP to antibody is 2-2.5: 1, and the reaction is stirred at 25°C for 1-2h. The TCEP-reduced antibody can be directly conjugated.

[0622] The sequence information of anti-CDCPl antibody C1 is as follows:

[0623] C1 heavy chain amino acid sequence SEQ ID NO: 39

[0624] C1 light chain amino acid sequence SEQ ID NO: 40

[0625] The sequence information of anti-CDCPl antibody C2 is as follows:

[0626] C2 heavy chain amino acid sequence SEQ ID NO: 41

[0627] C2 light chain amino acid sequence SEQ ID NO: 42

[0628] The linker-active drug unit compound was prepared in a certain concentration (5 mM) in DMSO (dimethyl sulfoxide), and the drug was slowly added according to the molar ratio of drug to thiol of 1.2-1.5:1, and stirred at 25°C for 1-2 h. After the reaction was completed, centrifugal ultrafiltration was carried out with HIS buffer to remove residual unreacted drugs and free small molecules such as DMSO, and the coupling was detected by hydrophobic high performance liquid chromatography (HIC-HPLC) and liquid chromatography-mass spectrometry (LC-MS) methods, and the sample purity after coupling was detected by size exclusion chromatography (SEC). Table 15. Test results of antibody drug conjugates

[0629] The ADC number means that, except for "Ab1-MC-VC-PAB-MMAE, Ab2-MC-VC-PAB-MMAE", the three parts of the used targeting part, linker and load part are connected by two dashes "-" in sequence. For example, C1-DL02-MMAE, the targeting part is C1 antibody, the linker is DL02, and the load is MMAE compound. "Ab1-MC-VC-PAB-MMAE" means that the antibody is "Ab1", the linker is "MC-VC-PAB", and the load is MMAE. "Ab2-MC-VC-PAB-MMAE" means that the antibody is "Ab2", the linker is "MC-VC-PAB", and the load is MMAE.

[0630] Example 58 Preparation of anti-CDCP1 antibody drug conjugates (camptothecin class load)

[0631] (1) General conjugation method

[0632] The preparation of antibody drug conjugates adopts a general conjugation method (DAR4 and DAR8): the reducing agent and metal chelating agent are prepared with purified water as follows: 1-20 mM TCEP (Tris-2-carboxyethyl-phosphine), 1-20 mM DTPA (Diethylene triamine pentacetate acid) stock solution, the amount of reducing agent can be added within a certain concentration range according to the desired conjugation rate, mixed with a certain concentration of monoclonal antibody (such as: 5-30 mg / mL), according to the different DAR values, such as DAR4 and DAR8, the final molar ratio of TCEP to antibody is 5-14:1, and the reaction is stirred at 25°C for 1h. The reduced antibody can be directly conjugated.

[0633] A certain concentration (5mM) of linker-active drug unit compound is prepared in DMSO (dimethyl sulfoxide), and according to the different DAR values, such as DAR4 and DAR8, the drug is slowly added according to the molar ratio of drug to thiol of 1.5-5:1, and the reaction is stirred at 25°C for 1-4h. After the reaction is completed, the ADC buffer (20mM histidine hydrochloride, 36.5g / L mannose, ph6.0) is centrifuged and ultrafiltrated to remove residual unreacted drugs and free small molecules such as DMSO, and the conjugation is detected by hydrophobic high performance liquid chromatography (HIC-HPLC) and liquid chromatography-mass spectrometry (LC-MS) methods, and the purity of the conjugated sample is detected by size exclusion chromatography (SEC). Table 16. Structure of antibody drug conjugates prepared by general conjugation method Table 17. Test results of antibody drug conjugates prepared by general conjugation method ADC number meaning: except "Ab1-MC-VC-PAB-MMAE", other numbers are connected by two dashes "-" to connect three parts, in turn, the used targeting part, linker and load part, for example, Ab1-DL01-dDxd, the targeting part is Ab1 antibody, the linker is DL01, and the load is dDxd compound. "Ab1-MC-VC-PAB-MMAE" means that the antibody is "Ab1", the linker is "MC-VC-PAB", and the load is MMAE.

[0634] Example 59 Internalization efficiency of anti-CDCP1 antibody drug conjugates in tumor cells

[0635] The cells EBC-1 (human lung adenocarcinoma cells) in the exponential growth phase were evenly spread in 6-well plates one day before the experiment. About 5E5 cells were spread in each well, 2 mL. The antibody was dialyzed into 10 mM sodium bicarbonate buffer (pH 8.5), and the pHAb Amine Reactive Dye dye was added, and ultrafiltration dialysis was performed after 1 h of reaction. The antibody-dye of calculated concentration was diluted to 10 μg / mL with cell culture medium, 100 μL was taken, and 2 mL of cell well was added, and the cells were incubated at 37°C, with time gradients of 0 h, 1 h, 3 h, 5 h, 7 h, and 24 h. After the endocytosis time, the fluorescence intensity was detected by flow cytometry.

[0636] The results of the internalization efficiency test of two ADCs (Ab1-DL01-Dxd, Ab1-DL01-dDxd) constructed using antibody Ab1 and linker DL01 and with comparative loads of Dxd and dDxd in human lung adenocarcinoma cells EBC-1 are shown in FIG. 1, and the results show that in human lung adenocarcinoma cells EBC-1, the internalization efficiency of the ADC with load dDxd is higher than that of the ADC with load Dxd.

[0637] The results of the internalization efficiency test of two ADCs (Ab2-DL01-Dxd, Ab2-DL01-dDxd) constructed using antibody Ab2 and linker DL01 and with comparative loads of Dxd and dDxd in pancreatic ductal carcinoma cell line XPC-3 are shown in FIG. 2, and the results show that in pancreatic ductal carcinoma cells BXPC-3, the internalization efficiency of the ADC with load dDxd is higher than that of the ADC with load Dxd.

[0638] It was surprisingly found that according to the different antibodies selected, the internalization performance of the antibody drug conjugate prepared by using dDxd as the load according to the present application is better than that of the antibody drug conjugate prepared by using Dxd as the load for the same antibody in specific tumor cell types.

[0639] Example 60 Inhibition of tumor cells by anti-CDCP1 antibody drug conjugate

[0640] Human lung adenocarcinoma cell EBC-1 and human non-small cell lung cancer cell HCC827 in exponential growth phase are inoculated in 96-well plates at a density of 5000-10000 / mL, 100 μL per well. Incubate overnight in a 37°C, 5% CO2 incubator. The ADC is gradiently prepared with complete culture medium at a concentration of 0-3333.3 nM, 9 concentration points, 100 μL is added to the well, 3 replicates are set for each concentration, and a blank control group is also set. After 72-144 h of drug action, add CCK-8 reagent, incubate in a 37°C, 5% CO2 incubator for 1-4 h, detect the OD value of each well at 450 nm on an enzyme label instrument. Calculate the inhibition rate IR% = (OD blank-OD drug) * 100 / OD blank. Perform four-parameter fitting on the inhibition rate and drug concentration, use GraphPad Prism to calculate IC 50 . Table 18. Inhibition effect of anti-CDCP1 antibody drug conjugate on human lung adenocarcinoma cell EBC-1 Table 19. Inhibition effect of anti-CDCP1 antibody drug conjugate on human non-small cell lung cancer cell HCC827

[0641] The test results show that the antibody drug conjugate prepared by the plurality of linker-load L-D structures in the application has obvious proliferation inhibition effect on tumor cells.

[0642] Example 61 Comparison of cell inhibition effects of different load antibody drug conjugates

[0643] 1) Human lung adenocarcinoma cell EBC-1 in exponential growth phase is inoculated in 96-well plates at a density of 5000-10000 / mL, 100 μL per well. Incubate overnight in a 37°C, 5% CO2 incubator.

[0644] 2) The ADC is gradiently prepared with complete culture medium at a concentration of 0-3333.3 nM, 9 concentration points, 100 μL is added to the well, 3 replicates are set for each concentration, and a blank control group is also set.

[0645] 3) After 72-144 h of drug action, add CCK-8 reagent, incubate in a 37°C, 5% CO2 incubator for 1-4 h, detect the OD value of each well at 450 nm on an enzyme label instrument. Calculate the inhibition rate IR% = (OD blank-OD drug) * 100 / OD blank. Perform four-parameter fitting on the inhibition rate and drug concentration, use GraphPad Prism to calculate IC 50 .

[0646] The experimental results show that the antibody-drug conjugates of this embodiment have a significant inhibitory effect on tumor cell proliferation, as shown in Table 20. With the same antibody and linker, the ADC with a loading of dDxd exhibits superior inhibitory activity compared to the ADC with a loading of Dxd; the inhibition rates of the two ADCs are comparable. Table 20. Comparison of EBC-1 cell inhibitory effects.

[0647] Example 62: Inhibition of EBC-1 cell (human lung adenocarcinoma cell) xenograft growth by anti-CDCP1 antibody-drug conjugate (MMAE payload).

[0648] 1) EBC-1 human lung adenocarcinoma cells were cultured in MEM medium (Minimum Essential Medium) containing 10% FBS (Fetal Bovine Serum) and maintained in a 37°C saturated humidity incubator containing 5% CO2.

[0649] 2) Collect EBC-1 cells in the logarithmic growth phase, resuspend them in MEM basal medium containing 50% Matrigel, and adjust the cell density to 1E8 / mL.

[0650] 3) Under aseptic conditions, 0.1 mL of cell suspension was injected subcutaneously into the right axilla of a mouse at a density of 1E7 cells / 0.1 mL / mouse.

[0651] 4) Seven days after animal inoculation, tumors that are too large, too small, or irregular were removed, and regular tumors with a volume of 120.61 mm were selected. 3 -207.69mm 3 The animals were divided into groups of 6, and the average tumor volume of each group was 146.34 mm. 3 When the animals were in the same group, the day was recorded as Day 0, and the medication was administered according to the animals' body weight.

[0652] 5) Measure animal weight and tumor volume twice a week, and observe and record animal clinical symptoms daily.

[0653] In the EBC-1 (human lung adenocarcinoma cell) tumor model, as shown in Figure 3, at a dose of 1 mg / kg, Ab1-MC-VC-PAB-MMAE (antibody Ab1, linker MC-VC-PAB, loading MMAE) and C1-DL02-MMAE had inhibitory effects on tumors. The efficacy of Ab1-MC-VC-PAB-MMAE was significantly better than that of C1-DL02-MMAE. C1-DL02-MMAE also showed good efficacy with increasing dosage, exhibiting a clear dose-dependent effect.

[0654] Example 63 Anti-CDCPl antibody drug conjugate (MMAE load) xenograft tumor growth inhibition experiment on BxPC3 cells (human orthotopic pancreatic cancer cells)

[0655] 1) BxPC-3 cells (human orthotopic pancreatic cancer cells) were cultured in RPMI-1640 medium containing 10% FBS, and maintained in a 37°C saturated humidity incubator containing 5% CO2.

[0656] 2) Collect the BxPC-3 cells (human orthotopic pancreatic cancer cells) in the logarithmic growth phase, resuspend in MEM basal medium containing 50% Matrigel, and adjust the cell density to 1E8 / mL.

[0657] 3) Under sterile conditions, inoculate 0.1 mL of cell suspension into the right axillary of mice subcutaneously, with an inoculation density of 1E7 cells / 0.1 mL / mouse.

[0658] 4) 27 days after inoculation of the animals, remove the tumors that are too large, too small, and irregular, and select regular tumors with a volume of 100.5 mm 3 - 207.86 mm 3 The average tumor volume of each group of animals after grouping was 145.33 mm 3 left and right, record the day of grouping as Day 0, and administer the drug according to the body weight of the animals.

[0659] 5) Two administrations were performed on days 0 and 11, respectively, animal body weight and tumor volume were measured twice a week, and animal clinical symptoms were observed and recorded daily.

[0660] The experimental results show (Figure 4) that in the BxPC-3 (human orthotopic pancreatic cancer cells) tumor model, Ab1-MC-VC-PAB-MMAE and C1-DL02-MMAE have similar efficacy at a low dose of 2 mg / kg, and both show very good tumor inhibition effect. And C1-DL02-MMAE has obvious dose dependence, and tumor regression is observed at a dose of 6 mg / kg.

[0661] Example 64 Anti-CDCPl antibody drug conjugate (camptothecin load) xenograft tumor growth inhibition experiment on EBC-1 cells (human lung adenocarcinoma cells) (5 mg / kg dose)

[0662] 1) EBC-1 cells were cultured in MEM medium containing 10% FBS, and maintained in a 37°C saturated humidity incubator containing 5% CO2.

[0663] 2) Collect the EBC-1 cells in the logarithmic growth phase, resuspend in MEM basal medium containing 50% Matrigel, and adjust the cell density to 1E8 / mL.

[0664] 3) Under sterile conditions, 0.1 mL of the cell suspension was inoculated subcutaneously on the right flank of the mice at a density of 1E7 cells / 0.1 mL / mouse.

[0665] 4) Seven days after inoculation, the animals with tumors that were too large, too small, or irregular were removed, and the animals with regular tumors with a volume of 100.72 mm 3 - 193.56 mm 3 were grouped, 7 animals per group, and the average tumor volume of each group of animals was 134.79 mm 3 around the average, the grouping day was recorded as Day 0, and the animals were dosed according to their body weight, on days 0 and 24.

[0666] 5) During the experiment, the body weight and tumor volume of the animals were measured twice a week, and the clinical symptoms of the animals were observed and recorded daily.

[0667] The results of the experiment (Figure 5) show that at a dose of 5 mg / kg, Ab1-DL13-dDxd has a significantly better inhibitory effect on tumors than Ab1-DL13-Dxd, and Ab1-DL13-dDxd shows better in vivo efficacy.

[0668] Example 65 Inhibition of EBC-1 cell (human lung adenocarcinoma cell) xenograft tumor growth by anti-CDCPl antibody drug conjugate (camptothecin-based load) (2.5 mg / kg dose)

[0669] 1) EBC-1 cells were cultured in MEM medium containing 10% FBS in a 37°C saturated humidity incubator containing 5% CO2.

[0670] 2) Logarithmic growth phase EBC-1 cells were collected and resuspended in MEM base medium containing 50% Matrigel, and the cell density was adjusted to 1E8 / mL.

[0671] 3) Under sterile conditions, 0.1 mL of the cell suspension was inoculated subcutaneously on the right flank of the mice at a density of 1E7 cells / 0.1 mL / mouse.

[0672] Seven days after inoculation, the animals with tumors that were too large, too small, or irregular were removed, and the animals with regular tumors with a volume of 123.27 mm 3 - 213.02 mm 3 were grouped, 7 animals per group, and the average tumor volume of each group of animals was 149.65 mm 3 around the average, the grouping day was recorded as Day 0, and the animals were dosed according to their body weight.

[0673] The body weight and tumor volume of the animals were measured twice a week during the experiment, and the clinical symptoms of the animals were observed and recorded daily.

[0674] The results of the experiment (Figure 6) show that at a dose of 2.5 mg / kg, the inhibitory effect of Ab1-DL13-dDXd on tumors is better than that of Ab1-DL01-Dxd.

[0675] Example 66 Toxicity tolerance test

[0676] 1) Quarantined ICR mice were weighed before grouping, and the body weight of the female animals ranged from about 26 to 31 grams.

[0677] 2) Randomly divided into the following dose groups according to body weight: Ab1-DL01-dDxd 600 mg / kg, Ab1-DL01-Dxd 600 mg / kg, 4 mice in each group, intravenous injection of the corresponding drug solution.

[0678] 3) Another batch of mice was taken as the control group, and 0.9% sodium chloride injection was given by the same volume of tail vein. The body weight was detected at 4, 7, 11, 14, and 17 days after administration.

[0679] The results of the experiment (Figure 7) show that under the conditions of this experiment, one mouse died at a dose of 600 mg / kg of Ab1-DL01-dDxd, and all mice died of Ab1-DL01-Dxd. The data show that Ab1-DL01-dDxd has a higher tolerance to mice than Ab1-DL01-Dxd, and dDxd as an ADC load has better safety characteristics in vivo than Dxd.

[0680] The results of the above examples show that the anti-CDCP1 antibody in the present application has high yield, good thermal stability and protein binding stability, excellent purity and cell binding activity, and better endocytosis effect, which shows broad application prospects in improving the accuracy and effectiveness of tumor treatment. The anti-CDCP1 antibody drug conjugate in the present application has high load delivery efficiency, high internalization efficiency, excellent inhibitory activity on tumor cells, good in vivo efficacy and safety, the deuterated load of the present application also greatly reduces the in vivo toxicity of the antibody drug conjugate, significantly improves the safety of the prepared antibody drug conjugate, and provides an innovative solution for the optimization of tumor treatment drugs.

[0681] The above only discloses some embodiments of the present application, and does not limit the present application in any form. Those skilled in the art can understand that the present application is not limited to the above specific embodiments. Without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

An antibody drug conjugate targeting CDCP1, characterized in that, The antibody drug conjugate comprises a targeting moiety targeting CDCP1, the targeting moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the heavy chain variable region (VH) comprises 3 CDRs of CDR-H1 of the sequence set forth in SEQ ID NO: 19, CDR-H2 of the sequence set forth in SEQ ID NO: 20, and CDR-H3 of the sequence set forth in SEQ ID NO: 21; and, the light chain variable region (VL) comprises 3 CDRs of CDR-L1 of the sequence set forth in SEQ ID NO: 22, CDR-L2 of the sequence set forth in SEQ ID NO: 23, and CDR-L3 of the sequence set forth in SEQ ID NO: 24; or (2) the heavy chain variable region (VH) comprises 3 CDRs of CDR-H1 of the sequence set forth in SEQ ID NO: 29, CDR-H2 of the sequence set forth in SEQ ID NO: 30, and CDR-H3 of the sequence set forth in SEQ ID NO: 31; and, the light chain variable region (VL) comprises 3 CDRs of CDR-L1 of the sequence set forth in SEQ ID NO: 32, CDR-L2 of the sequence set forth in SEQ ID NO: 33, and CDR-L3 of the sequence set forth in SEQ ID NO: 34; and the CDRs are defined according to IMGT numbering system. The antibody drug conjugate according to claim 1, wherein: (1) the heavy chain variable region (VH) comprises a sequence as set forth in SEQ ID NO: 25 or a sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 25, and the light chain variable region (VL) comprises a sequence as set forth in SEQ ID NO: 26 or a sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26; or (2) the heavy chain variable region (VH) comprises a sequence as set forth in SEQ ID NO: 35 or a sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35, and the light chain variable region (VL) comprises a sequence as set forth in SEQ ID NO: 36 or a sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

36. The targeting moiety is selected from an antibody or an antigen binding fragment. The antibody drug conjugate according to claim 1 or 2, characterized in that, The antibody or antigen binding fragment is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody or a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, a Fv fragment, a dAb, a Fd, a single chain antibody (scFv). The antibody drug conjugate according to any one of claims 1-3, characterized in that, The targeting moiety further comprises a constant region of an immunoglobulin, and further preferably, the immunoglobulin is selected from IgG1, IgG2, IgG3 or IgG4. The antibody drug conjugate according to claim 4, characterized in that, ​ The antibody drug conjugate according to any one of claims 1-5, characterized in that, The antibody moiety comprises a heavy chain and a light chain, wherein, (1) the heavy chain comprises a sequence as set forth in SEQ ID NO: 27 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27, and the light chain comprises a sequence as set forth in SEQ ID NO: 28 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28; or (2) the heavy chain comprises a sequence as set forth in SEQ ID NO: 37 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37, and the light chain comprises a sequence as set forth in SEQ ID NO: 38 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

38. The antibody drug conjugate according to any one of claims 1-6, characterized in that, The antibody drug conjugate has a general structure of Ab-U n wherein: The Ab represents a targeting moiety, the U represents a linker and a payload moiety; The n is an integer selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of Us attached to the Ab is 1, 2, 3, 4, 5, 6, 7, or 8, respectively; The Ab is covalently attached to the payload via a linker. The antibody drug conjugate according to claim 7, characterized in that, One, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the payloads are attached to the Ab via one or more of the linkers. The antibody drug conjugate according to claim 8, characterized in that, The load is selected from is the site of attachment of the payload and linker. The antibody drug conjugate according to any one of claims 7-9, characterized in that, The linker-payload moiety is represented by the following formula (I): wherein, L is an antibody-attaching linker selected from the following structures: Qi, Q2, Q3, Q4are each independently a single bond or selected from the group consisting of -(CH2CH2O)p-, -NH-(CH2OCH2)p-C(O)-, -(NHCH2C(O))-O-, -NH(CH2CH2O)p-CH2CH2C(O)-, -(CH2CH2O)p-CH2CH2C(O)-, -CH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -NHCH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -(CH2)-Y-, -NHCH2C(O)CH2C(O)-; Y is selected from C3-8 cycloalkyl or C3-8 cycloheteroalkyl or C3-8 cycloheteroalkenyl, the heteroalkyl comprising 1-3 atoms selected from N, O, or S, p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A1is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; A2is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; A3is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; S1, S3are independently selected from S2, S4are independently selected from -NH-CH2- or D1is a first payload compound, D2is a second payload compound; W is selected from the group consisting of m1is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m2is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m9 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; m10 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; the subscript n1 is 0 or 1. The antibody drug conjugate according to any one of claims 7-10, characterized in that, the load is independently selected from the following structures: is the site where the load and the linker are connected, wherein: R1, R2, R3, R4, R5are independently selected from hydrogen (H), deuterium (D), or tritium (T), at least one, two, three, or four of R1, R2, R3, R4, R5are D, R7is independently selected from -NH- or -NH-C(O)-CH2-O-. The antibody drug conjugate according to claim 11, characterized in that, said load is independently selected from the group consisting of: is the site where the load and the linker are connected, wherein: R1, R2, R3, R4, R5are independently selected from hydrogen (H), deuterium (D), or tritium (T), at least one, two, three, or four of R1, R2, R3, R4, R5are D, R7is independently selected from -NH- or -NH-C(O)-CH2-O-. The antibody drug conjugate according to any one of claims 7-12, characterized in that, said -L- is selected from the following structures: The antibody drug conjugate according to any one of claims 7-12, characterized in that, the A1, A2, A3are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val). The antibody drug conjugate according to claim 14, characterized in that, the A1, A2, A3are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), valine (Val). The antibody drug conjugate according to claim 15, characterized in that, the A1, A2, A3are independently selected from a single bond or a peptide comprising the following structures: -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly- Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; preferably, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Gly-, -Phe-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Val-Ala-, or -Ala-Ala-Ala-; preferably, the linker comprises the following structure: -Val-Ala-. The antibody drug conjugate according to any one of claims 7-16, characterized in that, Q1, Q2, Q3, Q4linking groups are independently selected from a single bond or the following structures: wherein, m11 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m12 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m13 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m14 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m15 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m16 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m17 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m18 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The antibody drug conjugate of claim 17, wherein Q1, Q2, Q3, Q4linking groups are independently selected from a single bond or the following structures: The antibody drug conjugate according to any one of claims 7-18, characterized in that, S1, S3 are independently selected from the following structures: wherein, m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The antibody drug conjugate of claim 19, wherein S1, S3 are independently selected from the following structures: The antibody drug conjugate according to any one of claims 7-20, characterized in that, S2, S4 are selected from -NH-CH2- or the following structures: wherein, m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The antibody drug conjugate of claim 21, wherein S2, S4 are selected from -NH-CH2- or the following structures: The antibody drug conjugate according to any one of claims 7-22, characterized in that, W is selected from the following structures: The antibody drug conjugate according to claim 7, characterized in that, The antibody drug conjugate is selected from the following structures: n8 is selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of linker-payload moieties attached to the Ab is 1, 2, 3, 4, 5, 6, 7, or 8; the antibody is linked to the linker-payload moieties via disulfide bonds on cysteines in the antibody. The antibody drug conjugate according to claim 7, characterized in that, The antibody drug conjugate is selected from the following structures: n2 is selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of linker-payload moieties attached to the Ab is 1, 2, 3, 4, 5, 6, 7, or 8; the antibody is linked to the linker-payload moieties via disulfide bonds on cysteines in the antibody. A treatment method for tumors, characterized in that, The method of treatment comprises administering to a patient an effective amount of the antibody drug conjugate of any one of claims 1-25. The method of treatment according to claim 26, wherein The tumor is CDCP-1 positive. The method of treatment according to claim 27, wherein The tumor is lung cancer or pancreatic cancer; further preferred, the tumor is lung cancer squamous carcinoma, lung adenocarcinoma, non-small cell lung cancer or pancreatic adenocarcinoma. An antibody or antigen-binding fragment thereof targeting CDCP1, characterized in that, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein, (1) the heavy chain variable region (VH) comprises the following 3 CDRs: CDR-H1 of the sequence set forth in SEQ ID NO: 19, CDR-H2 of the sequence set forth in SEQ ID NO: 20, and CDR-H3 of the sequence set forth in SEQ ID NO: 21; and, the light chain variable region (VL) comprises the following 3 CDRs: CDR-L1 of the sequence set forth in SEQ ID NO: 22, CDR-L2 of the sequence set forth in SEQ ID NO: 23, and CDR-L3 of the sequence set forth in SEQ ID NO: 24; or (2) the heavy chain variable region (VH) comprises the following 3 CDRs: CDR-H1 of the sequence set forth in SEQ ID NO: 29, CDR-H2 comprising the sequence set forth in SEQ ID NO: 30, and CDR-H3 of the sequence set forth in SEQ ID NO: 31; and, the light chain variable region (VL) comprises the following 3 CDRs: CDR-L1 of the sequence set forth in SEQ ID NO: 32, CDR-L2 of the sequence set forth in SEQ ID NO: 33, and CDR-L3 of the sequence set forth in SEQ ID NO: 34; The CDRs are defined according to the IMGT numbering system. The antibody or antigen-binding fragment thereof of claim 29, wherein The antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody, or a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, a Fv fragment, a dAb, a Fd, a single-chain antibody (scFv). The antibody or antigen-binding fragment thereof according to claim 29 or 30, further comprises a constant region of an immunoglobulin, wherein the immunoglobulin is selected from IgG1, IgG2, IgG3, or IgG4. The antibody or antigen-binding fragment thereof according to any one of claims 29-31, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups: (1) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 25, or a sequence having the same CDRs 1-3 as SEQ ID NO: 25 and having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25; and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 26, or a sequence having the same CDRs 1-3 as SEQ ID NO: 26 and having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26; or (2) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 35, or a sequence having the same CDRs 1-3 as SEQ ID NO: 35 and greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35; and the light chain variable region comprises a sequence as set forth in SEQ ID NO: 36, or a sequence having the same CDRs 1-3 as SEQ ID NO: 36 and greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

36. The antibody or antigen-binding fragment thereof according to claim 32, comprising a heavy chain and a light chain, wherein, (1) the heavy chain comprises a sequence as set forth in SEQ ID NO: 27 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27; and the light chain comprises a sequence as set forth in SEQ ID NO: 28 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28; or (2) the heavy chain comprises a sequence as set forth in SEQ ID NO: 37 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37; and the light chain comprises a sequence as set forth in SEQ ID NO: 38 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

38. A fusion protein characterized in that, The fusion protein comprises the antibody or antigen-binding fragment thereof of any one of claims 29-33. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 29-33 or the fusion protein of claim 34. A nucleic acid construct comprising the polynucleotide of claim 35. The nucleic acid construct of claim 36, wherein The nucleic acid construct is an expression vector, wherein the polynucleotide is operably linked to regulatory sequences permitting its expression of the encoded polypeptide in a host cell or a cell-free expression system. A host cell, characterized in that, The host cell comprises the polynucleotide of claim 35 or the nucleic acid construct of claim 36, 37; preferably, the host cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, an E. coli cell; more preferably, the host cell is selected from the group consisting of a CHO cell, a NS0 cell, a Sp2 / 0 cell, or a BHK cell. The method for producing the antibody or antigen-binding fragment thereof according to any one of claims 29 to 33 or the fusion protein according to claim 34, characterized in that, The method comprises culturing the host cell of claim 38 under conditions permitting expression of the nucleic acid construct of claim 36, and recovering the expressed protein produced from the culture. The antibody or antigen-binding fragment thereof according to claim 32, comprising a heavy chain and a light chain, wherein, (1) the heavy chain comprises a sequence as set forth in SEQ ID NO: 27 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27; and the light chain comprises a sequence as set forth in SEQ ID NO: 28 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28; or (2) the heavy chain comprises a sequence as set forth in SEQ ID NO: 37 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37; and the light chain comprises a sequence as set forth in SEQ ID NO: 38 or a sequence having greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

38. The fusion protein comprises the antibody or antigen-binding fragment thereof of any one of claims 29-33. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 29-33 or the fusion protein of claim 34. A nucleic acid construct comprising the polynucleotide of claim 35. The nucleic acid construct is an expression vector, wherein the polynucleotide is operably linked to regulatory sequences permitting its expression of the encoded polypeptide in a host cell or a cell-free expression system. The host cell comprises the polynucleotide of claim 35 or the nucleic acid construct of claim 36, 37; preferably, the host cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, an E. coli cell; more preferably, the host cell is selected from the group consisting of a CHO cell, a NS0 cell, a Sp2 / 0 cell, or a BHK cell. The method comprises culturing the host cell of claim 38 under conditions permitting expression of the nucleic acid construct of claim 36, and recovering the expressed protein produced from the culture. The use of the antibody or antigen-binding fragment thereof of any one of claims 29 to 33 or the fusion protein of claim 34 or the polynucleotide of claim 35 in the manufacture of a medicament. According to the use of claim 40, the medicament comprises a mono-specific antibody, a bi-specific antibody, a multi-specific antibody, or an antibody drug conjugate, an antibody radionuclide conjugate. A linker, such as shown in formula (II), wherein, L is an antibody-attaching linker selected from the following structures: wherein, Q1, Q2, Q3, Q4 are each independently a single bond or selected from the group consisting of -(CH2CH2O)p-, -NH-(CH2OCH2)p-C(O)-, -(NHCH2C(O))p-O-, -NH(CH2CH2O)p-CH2CH2C(O)-, -(CH2CH2O)p-CH2CH2C(O)-, -CH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -NHCH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -(CH2)-Y-, or -NHCH2C(O)CH2C(O)-; Y is selected from C3-8 cycloalkyl or C3-8 cycloheteroalkyl or C3-8 cyclo- unsaturated heteroalkyl, the heteroalkyl comprising 1-3 atoms selected from N, O, or S, p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A1 is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; A2 is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; A3 is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; S1, S3are independently selected from S2, S4are independently selected from -NH-CH2- or W is selected from m1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m9 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; m10 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; the subscript n3 is 0 or 1. The linker of claim 42, said L is selected from the following structures: According to the linker of claim 43, A1, A2, A3 are independently selected from a single bond or a peptide consisting of an amino acid selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), or citrulline (Cit). The linker according to claim 44, wherein said A1, A2, A3 are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val). The linker according to claim 45, wherein said A1, A2, A3 are independently selected from a single bond or a structure comprising: -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly- Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly- or -Ala-Ala-Ala-; preferably, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Gly-, -Phe-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Val-Ala- or -Ala-Ala-Ala-; more preferably, said linker comprises the structure: -Val-Ala-. wherein, The linker of any one of claims 42-46, the Q1, Q2, Q3, Q4linking groups are independently selected from a single bond or the following structures: m11 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m12 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m13 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m14 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m15 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m16 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m17 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m18 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. ​ The linker of claim 47, wherein Q1, Q2, Q3, Q4linking groups are independently selected from a single bond or the following structures: The linker of any one of claims 42-48, wherein S1, S3 is independently selected from the following structures: wherein m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The linker of claim 49, wherein S1, S3 are independently selected from the following structures: The linker of any one of claims 42-50, wherein S2, S4 is selected from -NH-CH2- or the following structures: wherein m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The linker of claim 51, S2, S4 is selected from -NH-CH2- or the following structures: The linker according to any one of claims 42-52, wherein W is selected from the following structures: The linker of claim 53, wherein W is selected from the following structures: The linker according to any one of claims 42-54, selected from the following structures: wherein, The associated with an antibody; said is linked to a drug unit. The linker of claim 55 selected from the following structures: wherein The associated with an antibody; said is linked to a payload compound. A linker-load, such as the structure shown in Formula (III): wherein, L is an antibody-attaching linker selected from the following structures: wherein, Q1, Q2, Q3, Q4 are each independently a single bond or selected from the following structures -(CH2CH2O)p-, -NH-(CH2OCH2)p-C(O)-, -(NHCH2C(O))p-O-, -NH(CH2CH2O)p-CH2CH2C(O)-, -(CH2CH2O)p-CH2CH2C(O)-, -CH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -NHCH2-Y-(CH2CH2O)p-CH2CH2C(O)-, -(CH2)-Y-, or -NHCH2C(O)CH2C(O)-; Y is selected from C3-8 cycloalkyl or C3-8 cycloheteroalkyl or C3-8 cyclo- unsaturated heteroalkyl, the heteroalkyl comprising 1-3 atoms selected from N, O, or S, p is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A1 is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; A2 is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; A3 is a single bond or a peptide consisting of 1, 2, 3, or 4 amino acid residues; S1, S3are independently selected from S2, S4are independently selected from -NH-CH2- or D1 is a first payload compound, D2 is a second payload compound, D1, D2 are independently selected from a compound as shown in formula (IV), or a stereoisomer, a pharmaceutically acceptable salt, or a solvate of the compound shown in formula (IV): wherein, R1, R2, R3, R4, R5 are independently selected from hydrogen (H), deuterium (D), or tritium (T), at least one, two, three, or four of R1, R2, R3, R4, R5 are D; R6 is selected from -NH2 or -NH-C(O)-CH2-OH; D1, D2 are linked to S1, S2, or S4 through either of the hydroxyl or amine groups present; W is selected from the group consisting of m1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m9 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; m10 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; the subscript n5 is 0 or 1. The linker-load of claim 57, wherein D1, D2 are independently selected from the following structures: Preferably, independently selected from: More preferably, D1, The structure of D2 is: The linker-payload according to claim 57 or 58, said -L is selected from the following structures: The linker-payload according to claim 59, wherein said A1, A2, A3 are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), or citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), or valine (Val). The linker-payload according to claim 60, wherein said A1, A2, A3 are independently selected from a single bond or a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys), or citrulline (Cit); preferably, a peptide comprising 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala), or valine (Val). The linker-payload according to claim 61, wherein said A1, A2, A3 are independently selected from a single bond or a structure comprising: -Gly-Ala-, -Ala-Gly-, -Val-Gly-, -Gly-Val-, -Val-Cit-, -Val-Ala-, -Gly-Phe-, -Phe-Gly-, -Gly-Gly-Ala-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Phe-Gly-, -Gly-Ala-Gly-Gly-, -Gly-Val-Gly-Gly-, -Gly-Phe-Gly-Gly-, -Gly-Gly-Lys-Gly-, -Gly-Gly-Ser-Gly-, -Gly-Gly-Glu-Gly-, -Gly-Lys-Gly-Gly-, -Gly-Ser-Gly-Gly-, -Gly-Glu-Gly-Gly-, -Gly-Gly-Val-Ala-, -Gly-Gly-Cit-Gly-, -Gly-Cit-Gly-Gly-, or -Ala-Ala-Ala-; preferably, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Gly-, -Phe-Gly-, -Gly-Gly-Val-Gly-, -Gly-Gly-Val-Ala-, or -Ala-Ala-Ala-; more preferably, said A1 is a single bond, and said A2 and A3 are independently -Val-Ala-. The linker-payload according to any one of claims 57-62, the Q1, Q2, Q3, Q4linker groups are independently selected from a single bond or the following structures: wherein, m11 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m12 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m13 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m14 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m15 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m16 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m17 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m18 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The linker-load of claim 63, the Q1, Q2, Q3, Q4linker groups are independently selected from a single bond or the following structures: The linker-payload according to any one of claims 57-64, wherein, S1, S3 are independently selected from the following structures: wherein m5 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m6 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m7 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The linker-load of claim 65, S1, S3 is independently selected from the following structures: The linker-load according to any one of claims 57-66, wherein, S2, S4 are selected from -NH-CH2- or the following structures: wherein m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The linker-load of claim 67, S2, S4 is selected from -NH-CH2- or the following structures: The linker-payload of any one of claims 57-68, said W is selected from the following structures: The linker-payload of claim 69, wherein W is selected from the following structures: The linker-load according to any one of claims 57-70, wherein, The linker-payload compound is selected from the following structures: The linker-load of claim 71, wherein The linker-payload compound is selected from the following structures: A linker-load, the linker-load is a stereoisomer, a pharmaceutically acceptable salt, or a solvate of the compound of any one of claims 57-72. A deuterated camptothecin compound of formula (V): wherein: R1, R2, R3, R4, R5are independently selected from hydrogen (H), deuterium (D), or tritium (T), at least one, two, three, or four of R1, R2, R3, R4, R5are D; R6is selected from -NH2or -NH-C(O)-CH2-OH. The deuterated camptothecin compound according to claim 74, the structure represented by formula (V) is selected from the group consisting of: Preferably, the structure of formula (V) is selected from the group consisting of: More preferably, formula (V) The structure shown is: The deuterated camptothecin compound according to claim 75, the structure represented by formula (V) is selected from the group consisting of: Preferably, the structure shown in formula (V) is: More preferably, formula (V) Optionally, the compound has the structure: A compound, the compound is a stereoisomer, a pharmaceutically acceptable salt, or a solvate of the compound of any one of claims 74-76. A compound of formula (IIIa): L2is a linker that attaches the antibody; W2is an optional group; A5is a peptide consisting of 1, 2, 3, or 4 amino acid residues; A6is a peptide consisting of 1, 2, 3, or 4 amino acid residues; S2, S4are independently selected from -NH-CH2- or D3is a first payload compound; D4is a second payload compound; m8 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X3is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X4is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X5is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The compound of claim 78, wherein The L2 includes the following structure: wherein R8is selected from N, O or S; X1is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X2is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The compound of claim 79, wherein The L2 includes the following structure: wherein R8is selected from N, O or S; X1is selected from 1, The compound of claim 80, wherein The L2 includes the following structure: 2, 3, 4, 5, 6, 7, 8, 9, or 10. The compound of any one of claims 78-81, wherein The L2 includes the following structure: wherein X2is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The compound of any one of claims 78-82, wherein The L2 includes the following structure: The compound of any one of claims 78-83, wherein The L2 structure is shown below: The compound of any one of any one of claims 78-84, wherein The W includes the following structure: wherein R8is selected from N, O or S. wherein M9 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; M10 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The compound of any one of claims 78-85, wherein W is selected from the following structures: The compound of any one of claims 78-86, wherein W is selected from the following structures: The compound according to any one of claims 78-87, characterized in that, A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val). The compound of claim 88, wherein A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val). The compound of claim 89, wherein A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val). A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val). A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val). A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val). A1, A2, A3 are independently selected from a single bond or a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), valine (Val), lysine (Lys), arginine (Arg), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), cysteine (Cys) or citrulline (Cit); preferably, a peptide consisting of 2-4 amino acid residues selected from phenylalanine (Phe), glycine (Gly), alanine (Ala) or valine (Val). A compound as described in claims 78-90 characterized in that, The first and second payload compounds are independently selected from the group consisting of cytotoxic molecules, immunopotentiators, and radioisotopes, the cytotoxic molecules including but not limited to microtubulin inhibitors or DNA damaging agents; further preferably, the microtubulin inhibitors include but are not limited to dolastatins and auristatin class cytotoxic molecules, maytansine class cytotoxic molecules; the DNA damaging agents include but are not limited to calicheamicin class, duocarmycin class, antrmycin class derivatives PBD, camptothecins and camptothecin class derivatives, SN-38, Dxd; further preferably, the auristatin class cytotoxic molecules include but are not limited to MMAE or MMAF or their derivatives, the maytansine class cytotoxic molecules include but are not limited to DM1, DM4 or their derivatives; further preferably, the camptothecin class derivatives are selected from the group consisting of the compounds of claim 65 or 66, which are attached to S2 or S4 after the loss of one hydrogen from either of the hydroxyl or amine groups present. The compound according to any one of claims 78-91, characterized in that, S2, S4 are selected from -NH-CH2- or the following structures: A compound as described in claims 78-92 characterized in that The compound is selected from: wherein D3 is a first payload compound; and D4 is a second payload compound. A compound as described in claim 93, wherein The compound is selected from: wherein D3 is a first payload compound; and D4 is a second payload compound. A compound characterized in that, The compound is a stereoisomer, a pharmaceutically acceptable salt, or a solvate of any one of the compounds of claims 74-94. Use of the linker of any one of claims 42-56 in the manufacture of an antibody drug conjugate. Use of the linker-payload of any one of claims 57-73 or 78-95 in the manufacture of an antibody drug conjugate. Use of the compound of any one of claims 74-77 in the manufacture of an antibody drug conjugate. An antibody-drug conjugate comprising a targeting unit, a linker, and a payload, the antibody-drug conjugate using a linker selected from the group consisting of the linker structures of any one of claims 42-56, and / or the antibody-drug conjugate using a payload selected from the group consisting of the compounds of any one of claims 74-77. The antibody-drug conjugate of claim 99, the targeting unit is selected from the group consisting of the antibodies or antigen binding fragments thereof of any one of claims 29-33. The antibody-drug conjugate of claim 99, the linker-payload is selected from the group consisting of the compounds of any one of claims 57-73 or 78-95. The antibody-drug conjugate according to any one of claims 99-101 is selected from the following structures: wherein, Ab is a targeting unit; n9 is selected from 1, 2, 3, 4, 5, 6, 7, or 8. The antibody drug conjugate of claim 96, selected from the following structures: wherein Ab is a targeting unit; n4 is selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of linker-payload moieties attached to the Ab is 1, 2, 3, 4, 5, 6, 7, or 8. Ab is a targeting unit; n4 is selected from 1, 2, 3, 4, 5, 6, 7, or 8, representing the number of linker-payload moieties attached to the Ab is 1, 2, 3, 4, 5, 6, 7, or 8. The antibody drug conjugate according to claim 99, wherein the structure of the antibody drug conjugate is ADC-1300a; preferably, the targeting unit Ab is an antibody or antigen binding fragment thereof targeting CDCP1; more preferably, the targeting unit Ab is the antibody or antigen binding fragment thereof according to any one of claims 29-34. A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 1-25 or 99-103 and / or the antibody or antigen binding fragment thereof according to any one of claims 29-34, and a pharmaceutically acceptable carrier. Use of the antibody or antigen binding fragment thereof according to any one of claims 29-34, the fusion protein according to claim 34, the polynucleotide according to claim 35, the nucleic acid construct according to claim 36 or 37, the antibody drug conjugate according to any one of claims 1-25 or 99-103, or the pharmaceutical composition according to claim 105, in the manufacture of a medicament for treating or preventing cancer. The use according to claim 106, wherein the cancer is a CDCP1 -positive cancer. The use according to claim 107, wherein the CDCP1 -positive cancer is lung cancer, or pancreatic cancer; further preferably, the CDCP1 -positive cancer is lung cancer squamous carcinoma, lung adenocarcinoma, non-small cell lung cancer or pancreatic adenocarcinoma. A recombinant protein comprising the antibody or antigen binding fragment according to any one of claims 29-34. The recombinant protein according to claim 109, wherein the recombinant protein is a bispecific antibody or a multispecific antibody. Use of the antibody or antigen binding fragment according to any one of claims 29-34 in the manufacture of a recombinant protein.

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